Insulin Therapy on Bone Macroscopic, Microarchitecture, and Mechanical Properties of Tibia in Diabetic Rats.

BACKGROUND: This study evaluated tibia's macroscopic structure, mechanical properties, and bone microarchitecture in rats with type 1 diabetes mellitus (T1DM). METHODS: Eighteen animals were divided into three groups (n=6): non-diabetic (ND), diabetic (D), and diabetic+insulin (DI). T1DM was induced by streptozotocin; insulin was administered daily (4IU). The animals were euthanized 35 days after induction. The tibiae were removed and analyzed using macroscopic, micro-computed tomography (micro-CT) and three-point bending. The macroscopic analysis measured proximal-distal length (PD), antero-posterior thickness (AP) of proximal (AP-P) and distal (AP-D) epiphysis, and lateral-medial thickness (LM) of proximal (LM-P) and distal (LM-D) epiphysis. Micro-CT analysis closed porosity, tissue mineral density, and cortical thickness. The three-point bending test measured maximum strength, energy, and stiffness. RESULTS: The macroscopic analysis showed that D presented smaller measures of length and thickness (AP and AP-P) than ND and DI. More extensive measurements were observed of LM and AP-D thickness in DI than in D. In micro-CT, DI showed larger cortical thickness than D. Mechanical analysis showed lower strength in D than in other groups. CONCLUSIONS: T1DM reduces bone growth and mechanical strength. Insulin therapy in diabetic rats improved bone growth and fracture resistance, making diabetic bone similar to normoglycemic animals.

Automated bone healing evaluation: New approach to histomorphometric analysis.

This study aimed to assess different approaches for bone healing evaluation on histological images and to introduce a new automatic evaluation method based on segmentation with distinct thresholds. We evaluated the hyperbaric oxygen therapy (HBO) effects on bone repair in type 1 diabetes mellitus rats. Twelve animals were divided into four groups (n = 3): non-diabetic, non-diabetic + HBO, diabetic, and diabetic + HBO. Diabetes was induced by intravenous administration of streptozotocin (50 mg/kg). Bone defects were created in femurs and HBO was immediately started at one session/day. After 7 days, the animals were euthanized, femurs were removed, demineralized, and embedded in paraffin. Histological sections were stained with hematoxylin and eosin (HE) and Mallory's trichrome (MT), and evaluated using three approaches: (1) conventional histomorphometric analysis (HE images) using a 144-point grid to quantify the bone matrix; (2) a semi-automatic method based on bone matrix segmentation to assess the bone matrix percentage (MT images); and (3) automatic approach, with the creation of a plug-in for ImageJ software. The time required to perform the analysis in each method was measured and subjected to Bland-Altman statistical analysis. All three methods were satisfactory for measuring bone formation and were not statistically different. The automatic approach reduced the working time compared to visual grid and semi-automated method (p < .01). Although histological evaluation of bone healing was performed successfully using all three methods, the novel automatic approach significantly shortened the time required for analysis and had high accuracy.

Type I Diabetes Mellitus and Insulin Therapy on Bone Microarchitecture, Composition and Mechanical Properties.

BACKGROUND: The aim of this study was to evaluate the microarchitecture, composition and mechanical properties of cortical bone of rats with type I diabetes mellitus (TIDM) and submitted to insulin therapy (IT). METHODS: Thirty rats were divided into three groups (n=10): non-diabetic, diabetic and diabetic+insulin. TIDM was induced by intravenous injection of streptozotocin. In diabetic+insulin group, 4IU insulin was administered twice per day (1I U at 7 am and 3I U at 7 pm). The animals were euthanized five weeks after TIDM induction; the tibiae were removed and submitted to microcomputed tomography (micro-CT, 8 µm), fourier transform infrared spectroscopy (FTIR) and dynamic microhardness indentation. RESULTS: Micro-CT analysis showed that diabetic group had lower bone surface/tissue volume ratio (BS/BV) (p=0.018), cortical thickness (Ct.Th) (p<0.001) and degree of anisotropy (Ct.DA) (p=0.034) values compared to non-diabetic group. The diabetic group showed lower Ct.Th than diabetic + insulin group (p=0.018). The non-diabetic group had lower fractal dimension (Ct.FD) values compared to diabetic groups (p<0.001). The ATR-FTIR analyses showed lower values for all measured parameters in the diabetic group than the non-diabetic group (amide I ratio: p=0.046; crystallinity index: p=0.038; matrix:mineral ratios - M:MI: p=0.006; M:MIII: p=0.028). The diabetic+ insulin group showed a lower crystallinity index (p=0.022) and M:MI ratio (p=0.002) than nondiabetic and diabetic groups, respectively. The diabetic group showed lower Vickers hardness values than non-diabetic (p<0.001) and diabetic+insulin (p=0.003) groups. CONCLUSION: TIDM negatively affects bone microarchitecture, collagen maturation, mineralization and bone microhardness. Moreover, insulin minimized the effect of TIDM on cortical thickness and organic/mineral matrix.

Efeito do diabetes mellitus tipo I na organização espacial das trabéculas ósseas: análise por meio do processo de esqueletonização / Effects of type I diabetes mellitus on the spatial organization of bone trabeculae: analysis by skeletonization

Introdução: Diabetes mellitus é uma doença metabólica que afeta vários órgãos-alvo, incluindo os ossos. Objetivo: Avaliar pelo método de esqueletonização o efeito do Diabetes mellitus tipo I (DM1) na microarquitetura de osso esponjoso. Material e métodos: Quatorze ratos Wistar foram divididos em: Saudável (S, n=7) e Diabético (D, n=7). O DM1 foi induzido por meio de injeção endovenosa de estreptozotocina no grupo D, sendo a confirmação da condição realizada por checagem do nível glicêmico. Os animais foram sacrificados após 35 dias da indução no grupo D, juntamente com os do grupo S. As epífises femorais foram seccionadas, removidas, desmineralizadas e incluídas em parafina. Dois cortes (5 µm) foram obtidos, corados em Hematoxilina e Eosina, e analisados ao Microscópio de Luz. Foi realizada a delimitação interativa das trabéculas ósseas, seguido pelo processo de binarização utilizando threshold global, feita por dois operadores distintos. Depois, foi realizado o processo de esqueletonização para acesso às características das trabéculas e da rede de interconexão entre elas. Os parâmetros avaliados foram: Área óssea em micrômetros quadrados (B.Ar, seguido pela proporção em porcentagem BV/TV), Índice de Modelo estrutural (SMI), Dimensão Fractal (FD), Número de trabéculas (Tb.N), Número de ramos (B.N), Número total de junções (Junc.N), Média de pontos terminais (End.p), Média de extensão de cada ramo (R.Le) e Número de junções triplas (Triple.points.N). Resultados: Houve diferença significante apenas no parâmetro SMI para os diferentes operadores (p<0,0001), sendo o mesmo retirado da análise entre diabetes vs saudável. Houve diferença significante na quantidade óssea, sendo maior no grupo S (0,46±0,09) comparado ao grupo D (0,41±0,07) (p=0,0082). Os demais parâmetros não mostraram diferença significante. Conclusão: Conclui-se que a área óssea no grupo saudável é maior em comparação ao DM1. Dentro das limitações deste estudo, parece que a distribuição espacial das trabéculas e suas características de interconexão não são alteradas no diabetes.

Introduction: Diabetes is a metabolic disease that affects several target-organs, including bone. Objective: Analyze the effects of Diabetes Mellitus Type 1 (DM1) on the trabecular bone microarchitecture by using the skeletonization process. Material and methods: Fourteen Wistar rats were divided in two groups: Health (S, n=7) and Diabetic (D, n=7). DM1 was induced with streptozotocin in D group, and glycemic levels were tested on peripheral blood samples. After 35 days, the animals were euthanized and had their femurs removed. The epiphysis were decalcified and embedded in paraffin. Five microns sections were stained in Hematoxylin and Eosin, and analyzed at the light microscope. Bone trabeculae were manually delimited, and then the binarization process with a global threshold was performed for each image. The whole process were conducted by two operators separately. Skeletonization was applied to binary images in order to evaluate the trabeculae characteristics and their network. Bone area (B.Ar), Bone proportion (BV/TV) Strucutre Model Index (SMI), Fractal Dimension (FD), Trabeculae number (Tb.N), Mean branches (B.N), Mean junction points (Junc.N), Mean End-points (End.p), Mean branches length (B.Le), and Mean triple points (Triple.points.N) were evaluated. Results: There was a significant difference only for SMI between different operators (p<0.0001), being this parameter excluded for the evaluation between health and diabetic groups. There was a significant difference between S and D for bone area, with S (0.46±0.09) higher than D (0.41±0.07) (p=0.0082). The other parameters analyzed were not significantly different. Conclusion: Bone trabecular area was higher in health compared with diabetes. Within the limitations of this study, one could suggest that there are no alterations of the spatial distribution of the trabeculae with their network and their inner structural characteristics.

Morphometric evaluation and planning of anticurvature filing in roots of maxillary and mandibular molars.

This study aimed to guide the planning of anticurvature filing using pre-determined anatomical points on teeth to establish directions for proper implementation of the technique. Two hundred digital periapical radiographs of human molar teeth were selected and divided into two groups (n = 100): MX (maxillary) and MD (mandibular) molars. Mesiobuccal roots were considered for the MX group and mesial roots for the MD group. Pre-determined anatomical points required for planning the anticurvature filing on the root canal path were located, and the distances between these points obtained. The anticurvature filing was simulated in two different protocols for each group, and the region of dentin removal and the remaining dentin thickness were measured in the safety and danger zones of the root canals. Statistical analysis was carried out at a significance level of 5%. The distances between the anatomical points and the thickness of remaining dentin showed significant differences when the two groups were compared (p < 0.001). No significant differences were found between the two experimental groups regarding the area of dentin removal at the root region, but differences were detected in comparison with dentin removal at the crown (p < 0.001). In terms of wear produced after simulation of both anticurvature filing protocols, significant differences were verified for all regions, except for the dentin remaining at the danger zone. The radiographic location of anatomical points allows for planning and implementation of controlled and efficient anticurvature filing and can be performed in the same manner for maxillary and mandibular molars.

Morphometric evaluation and planning of anticurvature filing in roots of maxillary and mandibular molars

This study aimed to guide the planning of anticurvature filing using pre-determined anatomical points on teeth to establish directions for proper implementation of the technique. Two hundred digital periapical radiographs of human molar teeth were selected and divided into two groups (n = 100): MX (maxillary) and MD (mandibular) molars. Mesiobuccal roots were considered for the MX group and mesial roots for the MD group. Pre-determined anatomical points required for planning the anticurvature filing on the root canal path were located, and the distances between these points obtained. The anticurvature filing was simulated in two different protocols for each group, and the region of dentin removal and the remaining dentin thickness were measured in the safety and danger zones of the root canals. Statistical analysis was carried out at a significance level of 5%. The distances between the anatomical points and the thickness of remaining dentin showed significant differences when the two groups were compared (p < 0.001). No significant differences were found between the two experimental groups regarding the area of dentin removal at the root region, but differences were detected in comparison with dentin removal at the crown (p < 0.001). In terms of wear produced after simulation of both anticurvature filing protocols, significant differences were verified for all regions, except for the dentin remaining at the danger zone. The radiographic location of anatomical points allows for planning and implementation of controlled and efficient anticurvature filing and can be performed in the same manner for maxillary and mandibular molars.

Critical instrumentation area: influence of root canal anatomy on the endodontic preparation.

The aim of this study was to evaluate the root canal anatomy of mandibular incisors before and after endodontic instrumentation, identifying regions inaccessible to the action of files (Critical instrumentation Area - CA) in a three-dimensional perspective. Thirty human mandibular central incisors were selected, assigned to two groups (n=15) and instrumented using ProTaper Universal rotary files. In the RX group, longitudinal digital radiographic images were obtained in the buccolingual (BL) and mesiodistal (MD) views. In the CT group, cross-sectional micro-computed tomography (µCT) images were obtained at 3, 9 and 15 mm from the apex. The canal area of the specimens was evaluated before and after instrumentation using digital images from each group. Data were analyzed using t-test, one-way ANOVA with subdivided parcels and Tukey's test (α=0.05). The canal area found in the MD radiographs was larger than in the BL radiographs, which was also confirmed in the transversal images (p<0.01). The CA was only detected in the MD radiographs and µCT scans. On the root canal configuration, a continuous reduction in the canal conicity was observed in BL radiographs, while in MD view there was a constriction at the cervical third and subsequent increase at the middle third (p<0.01). The conical shape of the root canal was observed only in the BL view. The canal enlargement in BL radiographs was not indicative of homogeneous instrumentation, since unprepared areas (CA) were also verified on the buccal and lingual walls in different images.

Critical Instrumentation Area: Influence of Root Canal Anatomy on the Endodontic Preparation

The aim of this study was to evaluate the root canal anatomy of mandibular incisors before and after endodontic instrumentation, identifying regions inaccessible to the action of files (Critical instrumentation Area - CA) in a three-dimensional perspective. Thirty human mandibular central incisors were selected, assigned to two groups (n=15) and instrumented using ProTaper Universal rotary files. In the RX group, longitudinal digital radiographic images were obtained in the buccolingual (BL) and mesiodistal (MD) views. In the CT group, cross-sectional micro-computed tomography (µCT) images were obtained at 3, 9 and 15 mm from the apex. The canal area of the specimens was evaluated before and after instrumentation using digital images from each group. Data were analyzed using t-test, one-way ANOVA with subdivided parcels and Tukey's test (α=0.05). The canal area found in the MD radiographs was larger than in the BL radiographs, which was also confirmed in the transversal images (p<0.01). The CA was only detected in the MD radiographs and µCT scans. On the root canal configuration, a continuous reduction in the canal conicity was observed in BL radiographs, while in MD view there was a constriction at the cervical third and subsequent increase at the middle third (p<0.01). The conical shape of the root canal was observed only in the BL view. The canal enlargement in BL radiographs was not indicative of homogeneous instrumentation, since unprepared areas (CA) were also verified on the buccal and lingual walls in different images.

Este estudo avaliou a anatomia do canal radicular de incisivos inferiores, antes e após a instrumentação endodôntica, identificando regiões inacessíveis à ação das limas (Área Crítica de instrumentação - AC), em uma perspectiva tridimensional. Trinta incisivos centrais inferiores humanos foram selecionados, divididos em dois grupos (n=15), e instrumentados usando limas rotatórias ProTaper Universal. No grupo RX, imagens longitudinais foram obtidas em duas incidências, vestíbulo-lingual (VL) e mésio-distal (MD), por meio de radiografias digitais. No grupo CT, imagens transversais foram obtidas por meio de microtomografia computadorizada (μTC), em secções localizadas a 3 mm, 9 mm, e 15 mm do ápice. A área do canal das amostras foi avaliada antes e após a instrumentação usando as imagens digitais de cada grupo. Os dados foram analisados por meio do teste t, ANOVA a um critério com parcela subdividida e ao teste de Tukey (α=0,05). A área de canal encontrada nas radiografias MD foi maior do que nas radiografias VL, o que também foi confirmado nas imagens transversais (p<0,01). A Área Crítica de instrumentação só foi detectada nas radiografias MD e nas seções de μTC. Na configuração de canal, a redução contínua na conicidade do canal foi observada nas radiografias VL, enquanto na incidência MD, houve uma constrição no terço cervical e um subsequente aumento no terço médio (p<0,01). A forma cônica do canal radicular foi observada somente na visão VL. O alargamento do canal verificado nas radiografias VL não foi indicativo de instrumentação homogênea, uma vez que áreas não instrumentadas (AC) foram observadas nas paredes vestibular e lingual em visões distintas.