Clarisa Bozzini

Dexamethasone effects on mechanical, geometric and densitometric properties of rat femur diaphyses as described by peripheral quantitative computerized tomography and bending tests

In previous studies with cortisol, betamethasone and oxazacort we attributed glucocorticoid effects on bone biomechanics to changes in bone mass and geometry rather than to an action on bone material properties. In this experiment, groups of 7 rats each received subcutaneous doses of 15.6, 31.2, 62.5, 125, 250, 500 or 1000 micrograms/kg per day of dexamethasone (DMS) and an additional 14 animals were controlled untreated for 4 weeks. Their fresh femurs were then scanned by peripheral quantitative computerized tomography (pQCT; XCT-960, Stratec, Germany) at the midshaft and submitted to three-point bending tests. In consonance with our earlier investigations, a significant, log-dose-related reduction in bone load-bearing capacity was observed, associated with an impairment in bone geometric properties (cross-sectional area and moment of inertia) and in body weight gain. However, the pQCT-assessed volumetric mineral density of cortical bone (vCtBMD; regarded as a material quality indicator in terms of mineralization) was significantly reduced by DMS following a dose-response relationship. Furthermore, a direct association was detected between vCtBMD and diaphyseal load-bearing capacity and stiffness. In contrast with our previous approach, data suggests that, apart from changes in bone geometric properties, glucocorticoid effects on bone material quality--as assessed by vCtBMD changes in this study--seem also to play a significant role in the determination of their biomechanical consequences.

Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible

Morphological and biomechanical features of the mandible are negatively affected by protein-energy malnutrition, whose effects are apparently dependent on the time of life of application. The aim here was to investigate, in neonatal rats nursed by dams put on a protein-free diet to depress milk production and thus create a state of protein-energy malnutrition in the offspring, subsequent growth and long-term effects by analyzing mandibular dimensions and bone quality in adulthood. Pregnant Wistar rats were fed a 20% protein diet (control) or a protein-free diet (malnourished) to obtain normal or subnormal milk production, respectively. After weaning, the offspring (males) were fed a 20% protein diet for 70 days. The dimensions of their excised mandibles were measured directly between anatomical points; the geometry and material quality of mandibular bone were assessed by peripheral quantitative computed tomography. Pups suckling from malnourished dams weighed 49.4% of those suckling from control dams at weaning; the actual difference between control and malnourished pups was 25.1g, which persisted until day 91 of age, indicating the absence of catch-up growth. As with body size, the mandibular base length, height and area (an index of mandibular size) were significantly smaller in malnourished than control rats at the end of the study. The mandibular cortical area, volumetric cortical bone mineral content and volumetric cortical bone mineral density assessed by peripheral quantitative computed tomography were similar in both groups of rats at the end of the observation period, but there was a significant reduction in the cortical axial moment of inertia in malnourished rats at this time of postnatal life. These findings suggest that catch-up growth was incomplete in rats malnourished during the suckling period and that the adaptation of mandibular bone architecture to body growth was apparently insufficient to attain normal values, thus not allowing complete compensation in mechanical competence at the end of the study because of an inadequate spatial distribution of resistive material through its cross-section rather than qualitative or quantitative impairment of cortical bone.

Bone status in an animal model of chronic sub-optimal nutrition: a morphometric, densitometric and mechanical study.

In children, inappropriate eating habits can induce a disease known as nutritional dwarfing (ND). Due to the link between nutritional condition and bone growth, the effects induced by a 20 % reduction of food intake on bone competence were assessed in an animal model of ND. Bone status during catch-up growth was also analysed. Male Wistar rats were divided into control (C) and ND groups. C rats were fed ad libitum. ND received 80 % of the diet consumed by C for 4 weeks (T4); thereafter, they were fed ad libitum for 8 weeks. Results, expressed as mean (SEM) for ND v. C, were as follows. At T4, body weight (g) and length (cm) and femur weight (g) and length (mm) were 97.35 (SEM 5.89) v. 199.07 (SEM 9.24), 16.91 (SEM 0.41) v. 20.26 (SEM 0.31), 0.30 (SEM 0.01) v. 0.46 (SEM 0.01) and 23.09 (SEM 0.29) v. 26.98 (SEM 0.26), respectively (P<0.001); bone mineral content (g) and density (g/cm(2)) were 0.014 (SEM 0.002) v. 0.030 (SEM 0.002) and 0.061 (SEM 0.004) v. 0.080 (SEM 0.003), respectively (P<0.001); load-bearing capacity (N), yielding load (N) and elastic stiffness (N/mm) were 25.06 (SEM 1.24) v. 50.34 (SEM 2.94), 23.72 (SEM 1.02) v. 46.97 (SEM 1.75) and 65.98 (SEM 4.42) v. 115.07 (SEM 3.85), respectively (P<0.001); cross-sectional area (mm(2)) and moment of inertia (mm(4)) were 2.86 (SEM 0.19) v. 4.54 (SEM 0.17) and 1.27 (SEM 0.08) v. 3.03 (SEM 0.16), respectively (P<0.001). Significant effects were not evident in material properties. Parameters assessed normalized during re-feeding. These results suggest that the impaired mechanical femur competence in ND rats could be due to an altered bone mass and architectural distribution rather than to intrinsic quality. Re-feeding caused a reversal of the effects of food restriction on growth and bone parameters in ND rats.

Erythropoietin formation during hypoxia in mice with impaired responsiveness to erythropoietin induced by irradiation or 5-fluorouracil injection

Plasma erythropoietin levels during continuous exposure to hypobaric hypoxia in mice with marrow aplasia induced by whole body X-irradiation or 5-fluorouracil injection were higher than in control mice similarly exposed. These findings give support to the hypothesis that a relationship exists between erythropoietin production rate and erythroid responsiveness to the hormone.

The Concentration of Dietary Casein Required for Normal Mandibular Growth in the Rat

To determine a suitable casein concentration for normal, undeformed mandibular growth, we placed weanling male rats on diets containing graded levels of casein between 0% and 30% for 19 days. Some weanlings were killed so that initial values could be established. Ten linear dimensions corresponding to the six skeletal units of the mandible were evaluated so that their growth rates at the end of the experimental period could be established. Other dimensions were also evaluated for study of the growth rate of the bone as a whole. The macroscopic growth of the mandible showed a sigmoidal relationship with dietary casein concentration, most of the measurements reaching a plateau at 20% casein. Within the skeletal units, four dimensions corresponding to the alveolar and symphyseal regions did not change with age and were not affected by the casein content of the diet. The remaining six dimensions-corresponding to condylar, coronoid, angular, and basal regions of the mandible-increased with age and were related positively to dietary casein concentration. Their growth patterns were not uniform, although all of them reached maximal values when the diet contained 20% casein. Therefore, deformation of the mandible appears to occur in rats fed diets with a casein concentration lower than 20%. It appears that a dietary casein concentration of 20% is required for normal, undeformed mandibular growth.

Plasma disappearance of exogenous erythropoietin in mice under different experimental conditions

Erythropoietin (EPO) is a glycoprotein hormone produced primarily in the kidneys and to a lesser extent in the liver that regulates red cell production. Most of the studies conducted in experimental animals to assess the role of EPO in the regulation of erythropoiesis were performed in mouse models. However, little is known about the in vivo metabolism of the hormone in this species. The present study was thus undertaken to measure the plasma t 1/2 of radiolabeled recombinant human EPO (rh-EPO) in normal mice as well as in mice with altered erythrocyte production rates (EPR), plasma EPO (pEPO) titer, marrow responsiveness, red cell volume, or liver function. Adult CF-1 mice of both sexes were used throughout. For the EPO life-span studies, 30 mice in each experiment were intravenously injected with 600,000 cpm of 125I-rh-EPO and bled by cardiac puncture in groups of five every hour for 6 h. Trichloroacetic acid (TCA) was added to each plasma sample and the radioactivity in the precipitate measured in a γ-counter. EPO, pEPO, marrow responsiveness, or red cell volume were altered by either injections of rh-EPO, 5-fluorouracil, or phenylhydrazine, or by bleeding, or red cell transfusion. Liver function was altered by Cl4C administration. In the normal groups of mice, the estimated t 1/2 was 182.75 ± 14.4 (SEM) min. The estimated t 1/2 of the other experimental groups was not significantly different from normal. These results, therefore, strongly suggest that the clearance rate of EPO in mice is not subjected to physiologic regulation and that pEPO titer can be really taken as the reflection of the EPO production rate, at least in the experimental conditions reported here.

Growth-dependent effects of dietary protein concentration and quality on the biomechanical properties of the diaphyseal rat femur☆

OBJECTIVES This study compares the effects of feeding growing rats with increasing concentrations of casein (C) and wheat gluten (G), proteins that show different biological qualities, on the morphometrical and biomechanical properties of the femoral diaphysis. MATERIALS AND METHODS Female rats were fed with one of ten diets containing different concentrations (5-30%) of C and G between the 30th and 90th days of life (Control=C-20%). Biomechanical structural properties of the right femur middiaphysis were estimated using a 3-point bending mechanical test with calculation of some indicators of bone material properties. RESULTS Body weight and length were affected by treatments, values being highest in rats fed the C-20% diet. G diets affected negatively both parameters. Changes in cross-sectional geometry (mid-diaphyseal cross-sectional and cortical areas, femoral volume, and rectangular moment of inertia) were positively related to the C content of the diet, while they were severely and negatively affected by G diets. Similar behaviors were observed in the bone structural properties (fracture load, yielding load, diaphyseal stiffness and elastic energy absorption). When values of strength and stiffness were normalized for body weight, the differences disappeared. The bone material quality indicators (elastic modulus, yielding stress, elastic energy absorption/volume) did not differ significantly among all studied groups. Femoral calcium concentration in ashes was not significantly different among groups. CONCLUSION The clear differences in strength and stiffness of bone beams induced by dietary protein concentration and quality seemed to be the result of an induced subnormal gain in bone structural properties as a consequence of a correlative subnormal gain in bone growth and mass, yet not in bone material properties.

Alveolar bone loss associated to periodontal disease in lead intoxicated rats under environmental hypoxia

Previously reported studies from this laboratory revealed that rats chronically intoxicated with lead (Pb) under hypoxic conditions (HX) impaired growth parameters and induced damages on femoral and mandibular bones predisposing to fractures. We also described periodontal inflammatory processes under such experimental conditions. Periodontitis is characterised by inflammation of supporting tissues of the teeth that result in alveolar bone loss. The existence of populations living at high altitudes and exposed to lead contamination aimed us to establish the macroscopic, biochemical and histological parameters consistent with a periodontal disease in the same rat model with or without experimental periodontitis (EP). Sixty female rats were divided into: Control; Pb (1000ppm of lead acetate in drinking water); HX (506mbar) and PbHX (both treatments simultaneously). EP was induced by placing ligatures around the molars of half of the rats during the 14 days previous to the autopsy. Hemi-mandibles were extracted to evaluate bone loss by histomorphometrical techniques. TNFα plasmatic concentration was greater (p<0.01) in Pb and HX animals. TBA-RS content was significantly higher in gums of rats with or without EP only by means of Pb. The SMG PGE2 content increased by Pb or HX was higher in PbHX rats (p<0.01). Pb and HX increased EP induced alveolar bone loss, while Pb showed spontaneous bone loss also. In conclusion, these results show that lead intoxication under hypoxic environment enhanced not only alveolar bone loss but also systemic and oral tissues inflammatory parameters, which could aggravate the physiopathological alterations produced by periodontal disease.

Biomechanical properties of the mandible, as assessed by bending test, in rats fed a low-quality protein

OBJECTIVE The present study describes the effects of feeding growing rats with diets containing increasing concentrations of wheat gluten (a low quality protein, G) on both the morphometrical and the biomechanical properties of the mandible. DESIGN Female rats were fed one of six diets containing different concentrations (5-30%) of G between the 30th and 90th days of life. Control rats were fed a diet containing 20% casein (C), which allows a normal growth and development of the bone. Mandibular growth was estimated directly on excised and cleaned bones by taking measurements between anatomical points. Mechanical properties of the right hemimandibles were determined by using a three-point bending mechanical test to obtain a load/deformation curve and estimate the structural properties of the bone. Bone material properties were calculated from structural and geometric properties. The left hemimandibles were ashed and the ash weight obtained. Calcium content was determined by atomic energy absorption. Results were summarised as means±SEM. Comparisons between parameters were performed by ANOVA and post-test. RESULTS None of the G-fed groups could achieve a normal growth performance as compared to the C-fed control group. Like body size, age-related increments in mandibular weight, length, height and area (index of mandibular size) were negatively affected by the G diets, as was the posterior part of the bone (posterior to molar III). The cross-sectional geometry of the mandible (cross-sectional area and rectangular moment of inertia) as well as its structural properties (yielding load, fracture load, and stiffness) were also severely affected by the G diets. However, material properties (Youngs modulus and maximum elastic stress) and calcium concentration in ashes and the degree of mineralisation were unaffected. CONCLUSIONS The differences in strength and stiffness between treated and control rats seemed to be the result of an induced loss of gain in bone growth and mass, in the absence of changes in the quality of the bone mineralised material.

Androgens and Erythropoiesis. Induction of Erythropoietin- Hypersecretory State and Effect of Finasteride on Erythropoietin Secretion

The major activity of androgens in promoting erythropoiesis is mediated by the ability of these steroids to increase erythropoietin (EPO) production. Testosterone exerts a nephrotrophic action which is evidenced by an increased renal mass following chronic administration of this steroid. The increased renal mass, in turn, may be associated with an increased capacity for production of EPO by the organ. The present study was thus designed to test the hypothesis that chronic administration of testosterone induces an EPO-hypersecretory state (EPO-HS) defined as a condition of higher than normal EPO synthesis and secretion in response to well-known erythropoietic stimuli, such as hypoxia, anaemia or cobalt. In addition, since 5α-dehydrotestosterone (5α-DHT) was found to be more active than testosterone in stimulating EPO production, the effect of inhibition of the 5α-reductase by finasteride on both constitutive and stimulated EPO secretion was also estimated. Female adult mice receiving 2 mg testosterone propionate three times a week for 3.5 weeks increased their erythrocyte production rates at day 4 and at the time of each sampling points thereafter during the injection period. Average renal weight was about 1.8 times higher in testosterone-treated than in vehicle-treated mice. When mice were hypertransfused at the end of the injection period and exposed to hypobaric air, both RBC-59Fe uptake and plasma EPO concentration (pEPO) were several times greater in androgen-treated than in vehicle-injected mice. No changes were observed in both parameters under normobaric conditions Testosterone treatment altered neither the plasma disappearance (t1/2) of radiolabelled rh-EPO nor the erythropoietic response to exogenously administered rh-EPO. pEPO was similar into normoxic male and female mice and neither orchidectomy nor finasteride (10 mg/kg/day for 12 days) modified these levels. Significant differences were seen in pEPO levels between male and female mice under hypoxic conditions. Orchidectomy and finasteride both lowered the hypoxia-dependent increment in pEPO in male mice to levels that were not significantly different from those of hypoxic female mice. In summary, an EPO-HS followed chronic administration of testosterone in mice, which may or may not be related to the nephrotrophic effect of this androgen. Since finasteride adversely affected stimulated-EPO production in mice, it is suggested that the testosterone effect on stimulated-EPO secretion is mediated by DHT. In contrast, the findings of unchanged baseline pEPO in either orchidectomised or finasteride-treated mice suggest that constitutional EPO production is not influenced by testosterone.