Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72. h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20 compression, 1. Hz) over 3. days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24. h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24. h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.

Validation of mechanical, electrical and thermal nociceptive stimulation methods in horses

To validate a model for investigating the effects of analgesic drugs on mechanical, thermal and electrical stimulation testing. To investigate repeatability, sensitivity and specificity of nociceptive tests. Randomised experiment with 2 observers in 2 phases. Mechanical (M), thermal (TL) and electrical (E) stimuli were applied to the dorsal metacarpus (M-left and TL-right) and coronary band of the left thoracic limb (E) and a thoracic thermal stimulus (TT) was applied caudal to the withers in 8 horses (405 ± 43 kg). Stimuli intensities were increased until a clear avoidance response was detected without exceeding 20 N (M), 60°C (TL and TT) and 15 V (E). For each set of tests, 3 real stimuli and one sham stimulus were applied (32 per animal) using a blinded, randomised, crossover design repeated after 6 months. A distribution frequency and, for each stimulus, Chi-square and McNemar tests compared both the proportion of positive responses detected by 2 observers and the 2 study phases. The κ coefficients estimated interobserver agreement in determining endpoints. Sensitivity (384 tests) and specificity (128 tests) were evaluated for each nociceptive stimulus to assess the evaluators' accuracy in detecting real and sham stimuli. Nociceptive thresholds were 3.1 ± 2 N (M), 8.1 ± 3.8 V (E), 51.4 ± 5.5°C (TL) and 55.2 ± 5.3°C (TT). The level of agreement after all tests, M, E, TL and TT, was 90, 100, 84, 98 and 75%, respectively. Sensitivity was 89, 100, 89, 98 and 70% and specificity 92, 97, 88, 91 and 94%, respectively. The high interobserver agreement, sensitivity and specificity suggest that M, E and TL tests are valid for pain studies in horses and are suitable tools for investigating antinociceptive effects of analgesics in horses.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Morphology of osteocyte cells in normal and hyper-gravity

Osteocyte cells are the most abundant cells in human bone tissue. Due to their unique morphology and location, osteocyte cells are thought to act as regulators in the bone remodelling process, and are believed to play an important role in astronauts’ bone mass loss after long-term space missions. There is increasing evidence showing that an osteocyte’s functions are highly affected by its morphology. However, changes in an osteocyte’s morphology under an altered gravity environment are still not well documented. Several in vitro studies have been recently conducted to investigate the morphological response of osteocyte cells to the microgravity environment, where osteocyte cells were cultured on a two-dimensional flat surface for at least 24 hours before microgravity experiments. Morphology changes of osteocyte cells in microgravity were then studied by comparing the cell area to 1g control cells. However, osteocyte cells found in vivo are with a more 3D morphology, and both cell body and dendritic processes are found sensitive to mechanical loadings. A round shape osteocyte’s cells support a less stiff cytoskeleton and are more sensitive to mechanical stimulations compared with flat cellular morphology. Thus, the relative flat and spread shape of isolated osteocytes in 2D culture may greatly hamper their sensitivity to a mechanical stimulus, and the lack of knowledge on the osteocyte’s morphological characteristics in culture may lead to subjective and noncomprehensive conclusions of how altered gravity impacts on an osteocyte’s morphology. Through this work empirical models were developed to quantitatively predicate the changes of morphology in osteocyte cell lines (MLO-Y4) in culture, and the response of osteocyte cells, which are relatively round in shape, to hyper-gravity stimulation has also been investigated. The morphology changes of MLO-Y4 cells in culture were quantified by measuring cell area and three dimensionless shape features including aspect ratio, circularity and solidity by using widely accepted image analysis software (ImageJTM). MLO-Y4 cells were cultured at low density (5×103 per well) and the changes in morphology were recorded over 10 hours. Based on the data obtained from the imaging analysis, empirical models were developed using the non-linear regression method. The developed empirical models accurately predict the morphology of MLO-Y4 cells for different culture times and can, therefore, be used as a reference model for analysing MLO-Y4 cell morphology changes within various biological/mechanical studies, as necessary. The morphological response of MLO-Y4 cells with a relatively round morphology to hyper-gravity environment has been investigated using a centrifuge. After 2 hours culture, MLO-Y4 cells were exposed to 20g for 30mins. Changes in the morphology of MLO-Y4 cells are quantitatively analysed by measuring the average value of cell area and dimensionless shape factors such as aspect ratio, solidity and circularity. In this study, no significant morphology changes were detected in MLO-Y4 cells under a hyper-gravity environment (20g for 30 mins) compared with 1g control cells.

Acceleration Measurements in Reduced Gravity

Thesis (Master's)--University of Washington, 2013

Translation of biomechanical concepts in bone tissue engineering: from animal study to revision knee arthroplasty.

Bone defects in revision knee arthroplasty are often located in load-bearing regions. The goal of this study was to determine whether a physiologic load could be used as an in situ osteogenic signal to the scaffolds filling the bone defects. In order to answer this question, we proposed a novel translation procedure having four steps: (1) determining the mechanical stimulus using finite element method, (2) designing an animal study to measure bone formation spatially and temporally using micro-CT imaging in the scaffold subjected to the estimated mechanical stimulus, (3) identifying bone formation parameters for the loaded and non-loaded cases appearing in a recently developed mathematical model for bone formation in the scaffold and (4) estimating the stiffness and the bone formation in the bone-scaffold construct. With this procedure, we estimated that after 3 years mechanical stimulation increases the bone volume fraction and the stiffness of scaffold by 1.5- and 2.7-fold, respectively, compared to a non-loaded situation.

Análise da influência do ultrassom de baixa intensidade na região de reparo ósseo em ratos sob ausência de carga

Há evidências de que o ultrassom (US) de baixa intensidade pode acelerar a regeneração óssea. Este trabalho objetivou verificar a ação do US no defeito ósseo, criado experimentalmente em tíbias de ratos sob ausência de carga. Vinte Rattus novergicus albinus, Wistar adultos, divididos em: G1 (n=10), grupo experimental de 15 dias sem suspensão, e G2 (n=10), grupo experimental de 15 dias suspenso pela cauda, foram submetidos à osteotomia em ambas as tíbias e à aplicação do US, frequência de 1,5 MHz, ciclo de trabalho 1:4, 30 mW/cm², nas tíbias direitas por 12 sessões de 20 minutos. Após o sacrifício, as tíbias foram submetidas à análise da Densidade Mineral Óssea (DMO). Os resultados demonstraram DMO de 0,139±0,018 g/cm² para tíbia tratada; 0,131±0,009 g/cm² para tíbia controle no G1; e no G2 registrou-se 0,120±0,009 g/cm² para tíbia tratada e 0,106±0,017 g/cm² para tíbia controle. Houve diferença significante entre os grupos nos quais o G2 apresentou menor DMO, o que demonstra que a suspensão prejudica a manutenção das propriedades ósseas, e entre as tíbias tratadas e controles do G2, demonstrando que o US acelerou o processo de reparo, concluindo que a impossibilidade do estímulo mecânico causada pela não deambulação em um processo de reparo ósseo pode ser minimizada pela ação do US. No G1, a aplicação do US não teve influência significante no aumento da DMO, talvez pelo fato dos animais já terem estímulo mecânico suficiente à formação óssea.

Injeção epidural preventiva de xilazina ou amitraz, em eqüinos: efeito antinociceptivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Absence of promoting potential of bracken fern (Pteridium aquilinum) in rat urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine and uracil

Conventional studies on bracken fern (Pteridium aquilinum; PA) carcinogenicity have used high dietary concentrations (around 30%) and long-term exposure (up to 52-70 weeks) without consideration of the multistep character of the chemical carcinogenesis process. The present study evaluated specifically the promoting potential of 3-5% dietary crude PA in the rat urinary bladder mucosa in a 32-week-long initiation-promotion assay for chemical carcinogenesis. Initiation of urothelial carcinogenesis was accomplished with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Uracil (U) was provided through the diet in order to expand the population of initiated cells. Seven groups (C) of male Wistar rats were submitted to the following treatments: G1 = BBN (n = 8); G2 = U (n = 10); G3 = BBN-U (n = 9); G4 = BBN-PA-U-PA (n = 16); G5 = PA (n = 8); G6 = BBN-PA (n = 10); G7 = PA-U-PA (n = 12). At the end of the experiment rats presenting epithelial papillary or nodular hyperplasia (PNH), papillomas (PAP), or simultaneous PNH plus PAP numbered, respectively G1: 2-0-1; G2: 0-0-0; G3: 3-0-2; G4: 4-3-2; G5: 1-0-1; G6: 8-0-0; and G7: 0-0-0, with no significant differences in the incidence of lesions among the groups. More frequent and more severe lesions occurred in BBN-initiated animals, predominantly in those also exposed to uracil (G3 and G4). Low-dose crude bracken fern in the diet does not promote rat urinary bladder carcinogenesis after a 32-week period of exposure, even when the initiated urothelial cell population has been expanded through a mechanical stimulus. (C) 1995 Wiley-Liss, Inc.

Hidroterapia e eletroterapia em ratos com denervação do nervo isquiático

Pós-graduação em Ciência Animal - FMVA

O efeito da carga oclusal no processo de reparo do ligamento periodontal após reimplante imediato em molar de ratos: avaliação histológica e imunoistoquímica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antinociceptive effects of methadone combined with detomidine or acepromazine in horses

To investigate two protocols to provide antinociception in horses. To evaluate the antinociceptive effects of intravenous methadone combined with detomidine or acepromazine in adult horses. Randomised, blinded, crossover study. Mechanical, thermal and electrical stimuli were applied to the dorsal left and right metacarpus and coronary band of the left thoracic limb, respectively. A thermal stimulus was applied caudal to the withers. The horses were treated with saline (C), a combination of methadone (0.2 mg/kg bwt) and detomidine (10 μg/kg bwt) (MD) or methadone (0.2 mg/kg bwt) and acepromazine (0.05 mg/kg bwt) (MA) at 1 week intervals. Nociceptive thresholds were measured before and at 15 min intervals until 150 min after treatment. Wilcoxon rank-sum and Wilcoxon signed rank tests were used to compare data between groups at each time point and over time within each group, followed by the Bonferroni method to adjust the P value. The mechanical stimulus was the most sensitive test to differentiate the antinociceptive effects of the treatments. Mechanical thresholds were greater after MD than MA between 15 and 30 min and with both MD and MA these thresholds were greater than C from 15 to 60 min. Electrical and thermal limb thresholds were greater after MD than C at 15 and 45 min and at 15, 30, 45, 75 and 105 min, respectively. Thermal limb thresholds were greater with MA than C at 30 min. Thoracic thermal threshold in MD and MA were higher than C at 45, 75, 90 and 120 min and from 30 to 75 min, respectively. Methadone and acepromazine produced less pronounced mechanical antinociception than MD.

Effects of Er:YAG and Er,Cr:YSGG lasers on dentine hypersensitivity. Short-term clinical evaluation

Dentine hypersensitivity (DH) is a painful condition and is a clinical challenge due to the different treatment strategies available. High-intensity lasers have been studied as a possible option. The aim of this randomized, controlled, double-blind clinical study was to evaluate the effects of Er:YAG and Er,Cr:YSGG lasers on DH. The study group comprised 28 subjects who met the inclusion criteria. A visual analogue scale was used to quantify sensitivity before treatment as baseline, immediately before and immediately after treatment, and 1 week and 1 month after treatment. Teeth were assigned to four groups: group 1 control (no treatment), group 2 Er:YAG laser treatment (2 Hz/32.4 mJ/5.9 J/cm(2)), group 3 Er,Cr:YSGG laser treatment (0.25 W/4.4 J/cm(2)), and group 4 Er,Cr:YSGG laser treatment (0.50 W/ 8.9 J/cm(2)). Data were collected and submitted to statistical analysis for both evaporative (air) and mechanical (probe) stimulation. For both the air and probe stimulation no differences were observed between the pretreatment sensitivities. With the evaporative stimulus, the pain level immediately after treatment was reduced; however, after this the values remained stable. Irradiation with the Er:YAG laser was associated with the lowest level of pain. With the mechanical stimulus, group 4 showed the most pronounced decrease in pain immediately after treatment; however, by the end of the study, pain levels had increased. Groups 1, 2 and 3 showed a reduction in pain that was significantly different from that in group 4 after the 4 weeks of clinical follow up. Based on the results and within the limits of this study, it can be concluded that none of the laser treatments studied was capable of completely eliminating pain, but the Er:YAG and Er,Cr:YSGG lasers are suitable for the treatment of DH.