Temporal modifications in bone following spinal cord injury in rats

Introduction: The aim of this study was to investigate the temporal modifications in bone mass, bone biomechanical properties and bone morphology in spinal cord injured rats 2, 4 and 6 weeks after a transection. Material and methods: Control animals were randomly distributed into four groups (n = 10 each group): control group (CG) - control animals sacrificed immediately after surgery; spinal cord-injured 2 weeks (2W) - spinal cord-injured animals sacrificed 2 weeks after surgery; spinal cord-injured 4 weeks (4W) - spinal cord-injured animals sacrificed 4 weeks after surgery; spinal cord-injured 6 weeks (6W) - spinal cord-injured animals sacrificed 6 weeks after surgery. Results: Biomechanical properties of the right tibia were determined by a threepoint bending test and injured animals showed a statistically significant decrease in maximal load compared to control animals. The right femur was used for densitometric analysis and bone mineral content of the animals sacrificed 4 and 6 weeks after surgery was significantly higher compared to the control animals and animals sacrificed 2 weeks after surgery. Histopathological and morphological analysis of tibiae revealed intense resorptive areas in the group 2 weeks after injury only. Conclusions: The results of this study show that this rat model is a valuable tool to investigate bone remodeling processes specifically associated with SCI. Taken together, our results suggest that spinal cord injury induced bone loss within 2 weeks after injury in rats.

A Comparative Analysis between Ultrasonometry and Computer-Aided Tomography to Evaluate Bone Healing

An ultrasonometric and computed-tomographic study of bone healing was undertaken using a model of a transverse mid-shaft osteotomy of sheep tibiae fixed with a semi-flexible external fixator. Fourteen sheep were operated and divided into two groups of seven according to osteotomy type, either regular or by segmental resection. The animals were killed on the 90th postoperative day and the tibiae resected for the in vitro direct contact transverse and axial measurement of ultrasound propagation velocity (UV) followed by quantitative computer-aided tomography (callus density and volume) through the osteotomy site. The intact left tibiae were used for control, being examined in a symmetrical diaphyseal segment. Regular osteotomies healed with a smaller and more mature callus than resection osteotomies. Axial UV was consistently and significantly higher (p?=?0.01) than transverse UV and both transverse and axial UV were significantly higher for the regular than for the segmental resection osteotomy. Transverse UV did not differ significantly between the intact and operated tibiae (p?=?0.20 for regular osteotomy; p?=?0.02 for resection osteotomy), but axial UV was significantly higher for the intact tibiae. Tomographic callus density was significantly higher for the regular than for the resection osteotomy and higher than both for the intact tibiae, presenting a strong positive correlation with UV. Callus volume presented an opposite behavior, with a negative correlation with UV. We conclude that UV is at least as precise as quantitative tomography for providing information about the healing state of both regular and resection osteotomy. (C) 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:10761082, 2012

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. (C) 2011 Elsevier Inc. All rights reserved.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 mu g of rhBMP-2, (3) laser and 7 mu g of rhBMP-2, (4) 7 mu g of rhBMP-2/monoolein gel, (5) laser and 7 mu g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Diametral tensile strength and film thickness of an experimental dental luting agent derived from castor oil

The need to develop new dental luting agents in order to improve the success of treatments has greatly motivated research. Objective: The aim of this study was to evaluate the diametral tensile strength (DTS) and film thickness (FT) of an experimental dental luting agent derived from castor oil (COP) with or without addition of different quantities of filler (calcium carbonate - CaCO3). Material and Methods: Eighty specimens were manufactured (DTS N=40; FT N=40) and divided into 4 groups: Pure COP; COP 10%; COP 50% and zinc phosphate (control). The cements were mixed according to the manufacturers' recommendations and submitted to the tests. The DTS test was performed in the MTS 810 testing machine (10 KN, 0.5 mm/min). For FT test, the cements were sandwiched between two glass plates (2 cm(2)) and a load of 15 kg was applied vertically on the top of the specimen for 10 min. The data were analyzed by means of one-way ANOVA and Tukey's test (alpha=0.05). Results: The values of DTS (MPa) were: Pure COP- 10.94 +/- 1.30; COP 10%- 30.06 +/- 0.64; COP 50%- 29.87 +/- 0.27; zinc phosphate- 4.88 +/- 0.96. The values of FT (pm) were: Pure COP- 31.09 +/- 3.16; COP 10%- 17.05 +/- 4.83; COP 50%- 13.03 +/- 4.83; Zinc Phosphate- 20.00 +/- 0.12. One-way ANOVA showed statistically significant differences among the groups (DTS - p=1.01E-40; FT - p=2.4E-10). Conclusion: The experimental dental luting agent with 50% of filler showed the best diametral tensile strength and film thickness.

The consequences of growth hormone-releasing hormone receptor haploinsufficiency for bone quality and insulin resistance

Objective Growth hormone (GH)/insulin-like growth factor (IGF) axis and insulin are key determinants of bone remodelling. Homozygous mutations in the GH-releasing hormone receptor (GHRHR) gene (GHRHR) are a frequent cause of genetic isolated GH deficiency (IGHD). Heterozygosity for GHRHR mutation causes changes in body composition and possibly an increase in insulin sensitivity, but its effects on bone quality are still unknown. The objective of this study was to assess the bone quality and metabolism and its correlation with insulin sensitivity in subjects heterozygous for a null mutation in the GHRHR. Patients and methods A cross-sectional study was performed on 76 normal subjects (68.4% females) (N/N) and 64 individuals (64.1% females) heterozygous for a mutation in the GHRHR (MUT/N). Anthropometric features, quantitative ultrasound (QUS) of the heel, bone markers [osteocalcin (OC) and CrossLaps], IGF-I, glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMAIR) was calculated. Results There were no differences in age or height between the two groups, but weight (P = 0.007) and BMI (P = 0.001) were lower in MUT/N. There were no differences in serum levels of IGF-I, glucose, T-score or absolute values of stiffness and OC, but insulin (P = 0.01), HOMAIR (P = 0.01) and CrossLaps (P = 0.01) were lower in MUT/N. There was no correlation between OC and glucose, OC and HOMAIR in the 140 individuals as a whole or in the separate MUT/N or N/N groups. Conclusions This study suggests that one allele mutation in the GHRHR gene has a greater impact on energy metabolism than on bone quality.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

Immunohistochemical evaluation of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth

The aim of this study was to evaluate the immunoexpression of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth and to correlate the results with demographic, survival, clinical (TNM staging) and histopathological variables (tumor grade, perineural invasion, embolization and bone invasion). Data from medical records and diagnoses of 41 patients were reviewed. Histological sections were subjected to immunostaining using primary antibodies for human MMP-2, MMP-9 and CD31 and streptavidin-biotin-immunoperoxidase system. Histomorphometric analyses quantified positivity for MMPs (20 fields per slide, 100?points grade, ×200) and for CD31 (microvessels <50?µm in the area of the highest vascularization, 5 fields per slide, 100?points grade, ×400). Statistical design was composed by non-parametric Mann-Whitney U test (investigating the association between numerical variables and immunostainings), chi-square frequency test (in contingency tables), Fisher's exact test (when at least one expected frequency was less than 5 in 2×2 tables), Kaplan-Meier method (estimated probabilities of overall survival) and Iogrank test (comparison of survival curves), all with a significance level of 5%. There was a statistically significant correlation between immunostaining for MMP-2 and lymph node metastasis. Factors associated negatively with survival were N stage, histopathological grade, perineural invasion and immunostaining for MMP-9. There was no significant association between immunoexpression of CD31 and the other variables. The intensity of immunostaining for MMP-2 can be indicative of metastasis in lymph nodes and for MMP-9 of a lower probability of survival

Biochemical, bone and renal patterns in hyperparathyroidism associated with multiple endocrine neoplasia type 1

Primary hyperparathyroidism associated with multiple endocrine neoplasia type I (hyperparathyroidism/multiple endocrine neoplasia type 1) differs in many aspects from sporadic hyperparathyroidism, which is the most frequently occurring form of hyperparathyroidism. Bone mineral density has frequently been studied in sporadic hyperparathyroidism but it has very rarely been examined in cases of hyperparathyroidism/multiple endocrine neoplasia type 1. Cortical bone mineral density in hyperparathyroidism/multiple endocrine neoplasia type 1 cases has only recently been examined, and early, severe and frequent bone mineral losses have been documented at this site. Early bone mineral losses are highly prevalent in the trabecular bone of patients with hyperparathyroidism/multiple endocrine neoplasia type 1. In summary, bone mineral disease in multiple endocrine neoplasia type 1related hyperparathyroidism is an early, frequent and severe disturbance, occurring in both the cortical and trabecular bones. In addition, renal complications secondary to sporadic hyperparathyroidism are often studied, but very little work has been done on this issue in hyperparathyroidism/multiple endocrine neoplasia type 1. It has been recently verified that early, frequent, and severe renal lesions occur in patients with hyperparathyroidism/multiple endocrine neoplasia type 1, which may lead to increased morbidity and mortality. In this article we review the few available studies on bone mineral and renal disturbances in the setting of hyperparathyroidism/multiple endocrine neoplasia type 1. We performed a meta-analysis of the available data on bone mineral and renal disease in cases of multiple endocrine neoplasia type 1-related hyperparathyroidism.

Quantitative analysis of photodynamic therapy effects in rat mammary tumor vascular density using image-pro plus software

Photodynamic therapy (PDT) is a treatment modality that has advanced rapidly in recent years. It causes tissue and vascular damage with the interaction of a photosensitizing agent (PS), light of a proper wavelength, and molecular oxygen. Evaluation of vessel damage usually relies on histopathology evaluation. Results are often qualitative or at best semi-quantitative based on a subjective system. The aim of this study was to evaluate, using CD31 immunohistochem- istry and image analysis software, the vascular damage after PDT in a well-established rodent model of chemically induced mammary tumor. Fourteen Sprague-Dawley rats received a single dose of 7,12-dimethylbenz(a)anthraxcene (80 mg/kg by gavage), treatment efficacy was evaluated by comparing the vascular density of tumors after treatment with Photogem® as a PS, intraperitoneally, followed by interstitial fiber optic lighting, from a diode laser, at 200 mW/cm and light dose of 100 J/cm directed against his tumor (7 animals), with a control group (6 animals, no PDT). The animals were euthanized 30 hours after the lighting and mammary tumors were removed and samples from each lesion were formalin-fixed. Immunostained blood vessels were quantified by Image Pro-Plus version 7.0. The control group had an average of 3368.6 ± 4027.1 pixels per picture and the treated group had an average of 779 ± 1242.6 pixels per area (P < 0.01), indicating that PDT caused a significant decrease in vascular density of mammary tumors. The combined immu- nohistochemistry using CD31, with selection of representative areas by a trained pathology, followed by quantification of staining using Image Pro-Plus version 7.0 system was a practical and robust methodology for vessel damage evalua- tion, which probably could be used to assess other antiangiogenic treatments.

Muscular development and physical activity as major determinants of femoral bone mass acquisition during growth

[EN] OBJECTIVES: To investigate to what extent bone mass accrual is determined by physical activity and changes in lean, fat, and total body mass during growth. METHODS: Twenty six physically active and 16 age matched control boys were followed up for three years. All subjects were prepubertal at the start of the survey (mean (SEM) age 9.4 (0.3) years). The weekly physical activity of the active boys included compulsory physical education sessions (80-90 minutes a week), three hours a week of extracurricular sports participation, and occasional sports competitions at weekends. The physical activity of the control group was limited to the compulsory physical education curriculum. Bone mineral content (BMC) and areal density (BMD), lean mass, and fat mass were measured by dual energy x ray absorptiometry. RESULTS: The effect of sports participation on femoral bone mass accrual was remarkable. Femoral BMC and BMD increased twice as much in the active group as in the controls over the three year period (p < 0.05). The greatest correlation was found between the increment in femoral bone mass and the increment in lean mass (BMC r = 0.67 and BMD r = 0.69, both p < 0.001). Multiple regression analysis revealed enhancement in lean mass as the best predictor of the increment in femoral bone BMC (R = 0.65) and BMD (R = 0.69). CONCLUSIONS: Long term sports participation during early adolescence results in greater accrual of bone mass. Enhancement of lean mass seems to be the best predictor of this bone mass accumulation. However, for a given muscle mass, a greater level of physical activity is associated with greater bone mass and density in peripubertal boys.

Exercise and bone mass in adults

[EN] There is a substantial body of evidence indicating that exercise prior to the pubertal growth spurt stimulates bone growth and skeletal muscle hypertrophy to a greater degree than observed during growth in non-physically active children. Bone mass can be increased by some exercise programmes in adults and the elderly, and attenuate the losses in bone mass associated with aging. This review provides an overview of cross-sectional and longitudinal studies performed to date involving training and bone measurements. Cross-sectional studies show in general that exercise modalities requiring high forces and/or generating high impacts have the greatest osteogenic potential. Several training methods have been used to improve bone mineral density (BMD) and content in prospective studies. Not all exercise modalities have shown positive effects on bone mass. For example, unloaded exercise such as swimming has no impact on bone mass, while walking or running has limited positive effects.

Bone mass and the CAG and GGN androgen receptor polymorphisms in young men

[EN] BACKGROUND: To determine whether androgen receptor (AR) CAG (polyglutamine) and GGN (polyglycine) polymorphisms influence bone mineral density (BMD), osteocalcin and free serum testosterone concentration in young men. METHODOLOGY/PRINCIPAL FINDINGS: Whole body, lumbar spine and femoral bone mineral content (BMC) and BMD, Dual X-ray Absorptiometry (DXA), AR repeat polymorphisms (PCR), osteocalcin and free testosterone (ELISA) were determined in 282 healthy men (28.6+/-7.6 years). Individuals were grouped as CAG short (CAG(S)) if harboring repeat lengths of < or = 21 or CAG long (CAG(L)) if CAG > 21, and GGN was considered short (GGN(S)) or long (GGN(L)) if GGN < or = 23 or > 23. There was an inverse association between logarithm of CAG and GGN length and Ward's Triangle BMC (r = -0.15 and -0.15, P<0.05, age and height adjusted). No associations between CAG or GGN repeat length and regional BMC or BMD were observed after adjusting for age. Whole body and regional BMC and BMD values were similar in men harboring CAG(S), CAG(L), GGN(S) or GGN(L) AR repeat polymorphisms. Men harboring the combination CAG(L)+GGN(L) had 6.3 and 4.4% higher lumbar spine BMC and BMD than men with the haplotype CAG(S)+GGN(S) (both P<0.05). Femoral neck BMD was 4.8% higher in the CAG(S)+GGN(S) compared with the CAG(L)+GGN(S) men (P<0.05). CAG(S), CAG(L), GGN(S), GGN(L) men had similar osteocalcin concentration as well as the four CAG-GGN haplotypes studied. CONCLUSION: AR polymorphisms have an influence on BMC and BMD in healthy adult humans, which cannot be explained through effects in osteoblastic activity.

Human bone: the tissue characteristics determining its mechanical behaviour

The present thesis illustrates the research carried out during the PhD studies in Bioengineering. The research was aimed to characterise the human bone tissue, with particular regard to the differences between cortical and trabecular bone. The bone tissue characteristics that affect its mechanical properties were verified or identified, using an experimental approach, to corroborate or refute hypotheses based on the state of the art in bone tissue biomechanics. The studies presented in the present PhD thesis were designed to investigate aspects of bone tissue biomechanics, which were in need of a more in-depth examination since the data found in the literature was contradictory or scarce. In particular, the work was focalised on the characterisation of the basic structure of the bone tissue (groups of lamellae), its composition, its spatial organisation (trabecular bone microarchitecture) and their influence on the mechanical properties. In conclusion, the present thesis integrates eight different studies on the characterisation of bone tissue. A more in-depth examination of some of the aspects of bone tissue biomechanics where the data found in the literature was contradictory or scarce was performed. Bone tissue was investigated at several scales, from its composition up to its spatial organization, to determine which parameters influence the mechanical behaviour of the tissue. It was found that although the composition and real density of bone tissue are similar, the differences in structure at different levels cause differences between the two types of bone tissue (cortical and trabecular) in mechanical properties. However, the apparent density can still be considered a good predictor of the mechanical properties of both cortical and trabecular bone. Finally, it was found that the bone tissue characteristics might change when a pathology is present, as demonstrated for OA.

Reduced muscle mass and bone size in pediatric patients with inflammatory bowel disease

BACKGROUND: Decreased bone mineral density has been reported in children with inflammatory bowel disease (IBD). We used peripheral quantitative computed tomography (pQCT) to assess bone mineralization, geometry, and muscle cross-sectional area (CSA) in pediatric IBD. METHODS: In a cross-sectional study, pQCT of the forearm was applied in 143 IBD patients (mean age 13.9 +/- 3.5 years); 29% were newly diagnosed, 98 had Crohn's disease, and 45 had ulcerative colitis. Auxological data, cumulative glucocorticoid dose, disease activity indices, laboratory markers for inflammation, and bone metabolism were related to the results of pQCT. RESULTS: Patients were compromised in height (-0.82 +/- 1.1 SD), weight (-0.77 +/- 1.0 SD), muscle mass (-1.12 +/- 1.0 SD), and total bone cross-sectional area (-0.79 +/- 1.0 SD) compared to age- and sex-matched healthy controls (z-scores). In newly diagnosed patients, the ratio of bone mineral mass per muscle CSA was higher than in those with longer disease duration (1.00 versus 0.30, P = 0.007). Serum albumin level and disease activity correlated with muscle mass, accounting for 41.0% of variability in muscle mass (P < 0.01). The trabecular bone mineral density z-score was on average at the lower normal level (-0.40 +/- 1.3 SD, P < 0.05). CONCLUSIONS: Reduced bone geometry was explained only in part by reduced height. Bone disease in children with IBD seems to be secondary to muscle wasting, which is already present at diagnosis. With longer disease duration, bone adapts to the lower muscle CSA. Serum albumin concentration is a good marker for muscle wasting and abnormal bone development.