The mechanical properties of cancellous bone in the proximal tibia of ovariectomized rats.

The "mature rat model" is an effective and often-used surrogate for studying mechanisms and characteristics of estrogen-deficient osteopenia. The purpose of this study was to extend our understanding of this animal model to include the mechanical properties of cancellous bone in the proximal tibia. Female Sprague-Dawley rats were divided into two groups (n=13 each) at 14 weeks of age: an ovariectomized group (OVX) and a sham-operated control group (sham). The study terminated after a duration of 5 weeks. Specimens 2 mm long were cut from the proximal tibial metaphysis just below the growth plate and tested using two methods: (1) "whole-slice" compression, in which the entire specimen is loaded between two larger flat platens and (2) "reduced-platen" compression (RPC), which uses platens sized and aligned to load only the cancellous bone in the center of the sample. Three-point bending tests also were conducted on the femur. The short duration of estrogen deficiency yielded only minimal differences (< 10%) in femoral cortical bone but dramatic reductions (approximately 60%) in cancellous bone properties as determined by the RPC method. Ultimate stress was 7.23 MPa +/- 1.97 MPa for OVX versus 18.1 MPa +/- 5.21 MPa for sham; and elastic modulus was 252 MPa +/- 104 MPa for OVX versus 603 MPa +/- 180 MPa for sham. These changes in mechanical properties are similar in many respects to the dramatic effects reported in histomorphometric studies. For the whole-slice method, differences in mechanical properties between the two groups were not as large because the test directly loads both cancellous and cortical bone, and the latter is not affected as severely by estrogen deficiency. In this case, ultimate stress and elastic modulus were only 30% (or less) lower for the OVX group.

Alcohol's harmful effects on bone.

Long-term alcohol consumption can interfere with bone growth and replacement of bone tissue (i.e., remodeling), resulting in decreased bone density and increased risk of fracture. These effects may be exerted directly or indirectly through the many cell types, hormones, and growth factors that regulate bone metabolism. Alcohol consumption during adolescence reduces peak bone mass and can result in relatively weak adult bones that are more susceptible to fracture. In adults, alcohol consumption can disrupt the ongoing balance between the erosion and the remodeling of bone tissue, contributing to alcoholic bone disease. This imbalance results in part from alcohol-induced inhibition of osteoblasts, specialized cells that deposit new bone. Some evidence suggests that moderate drinking may decrease the risk of fracture in postmenopausal women.

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta.

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.

Spondyloarthropathy in progressive ankylosis (ank/ank) mice: morphological features.

Progressive ankylosis mice rapidly develop an ankylosing spondyloarthropathy that rapidly affects all of the articulations of the vertebral column. The disorder symmetrically affects the nonsynovial synchondroses and symphyses of the intervertebral spaces, the diarthrodial synovial apophyseal and costovertebral joints, and the sacroiliac joint. These joints present a clear progression from syndesmophyte formation through joint bridging to total fusion. The similarities and differences of the disorder identified in this mouse and human spondyloarthropathies are discussed.

Histopathology of the intervertebral disc of progressive ankylosis mice.

The intervertebral disks of progressive ankylosis mice were examined at successive periods in order to describe the pathologic lesions involved and to explore possible relationships with any known human disorder. The techniques employed in this study showed the presence of necrotic foci of calcification that ultimately obliterated the anulus fibrosus. Following the appearance of these lesions, peripheral proliferation of hyaline cartilage appeared that eventually bridged adjacent vertebral bodies, calcified, and was replaced with bone. The results of this study support the hypothesis that the progressive ankylosis mouse has a disorder affecting mineralization. Although it does not exactly resemble any known human disorder, it should be an important model for studying both crystal deposition and abnormal mineralization of hyaline cartilage and fibrocartilage since many vertebral ankylosis disorders, as well as the normal aging process, involve increased mineralization.

Intracellular calcium localization in stimulated and non-stimulated extraorbital lacrimal glands of rats.

Acinar cells of extraorbital lacrimal glands from control, pilocarpine-treated, atropine-treated and atropine + pilocarpine-treated rats were studied using a potassium pyroantimonate technique and X-ray microanalysis for calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalization of calcium and to elucidate any calcium translocation that might occur during the secretory process. Calcium-pyroantimonate complexes were identified in the mitochondria, plasma membrane and cytoplasmic vesicles of the untreated specimens and in the plasma membrane of atropine-treated specimens, these complexes decreased drastically in the actively-secreting cells. The function of calcium in lacrimal gland secretion and the action of pilocarpine and atropine on membrane calcium are discussed.

Magnesium levels in alcohol-treated rodents using different consumption paradigms.

To develop an animal model system that examines magnesium (Mg) deficiency associated with chronic alcohol consumption, we tried to reproduce a Mg-deficient state, caused by alcohol consumption, in rats using different alcohol consumption experimental designs. Serum and bone samples were collected from 2-day binge (high BACs achieved by intubating a 5% ethanol solution 2 consecutive days/week), 5-day binge, moderate drinking, and adolescent (4-week-old rats, equivalent to late teen/early adult humans) alcohol consumption projects. Mg content was measured using color spectrophotometry. Alcohol-fed animals consumed a liquid diet containing 0.38 g/kg/day ethanol in the moderate project and 35% ethanol-derived calories in the adolescent drinking project. Animals in the two binge drinking projects were intubated with 12 g/kg/day of ethanol in a 5% solution. When looked at acutely and chronically, no consistent deficiencies in Mg were seen. The lack of a chronic Mg deficiency in rats may indicate a different mechanism of action than observed in humans.

Alcohol consumption inhibits osteoblastic cell proliferation and activity in vivo.

To better understand the effect of alcohol consumption on the bone remodeling process in vivo, we used a rodent animal model system to compare osteoblast activity and number in alcohol-fed, pair-fed, and chow-fed animals. Adult, virgin female Sprague-Dawley rats were assigned to alcohol-fed, pair-fed, and chow groups based on weights. Alcohol animals were fed a liquid diet containing 35% ethanol-derived calories for 6 weeks. Pair-fed animals were matched to test animals on the basis of initial weight and fed an isocaloric diet equivalent to that consumed by the alcohol-matched animals on the previous day with alcohol replaced by maltose-dextrin. Right tibias were fixed and embedded in methyl methacrylate for sectioning. Sections (5 microm) were stained for cement lines and packets were measured using histomorphometric techniques on a BioQuant morphometric system. Alcohol-fed animals exhibited statistically significant decreases in the amount of bone surface containing active osteoblasts and a decrease in mean wall thickness. Osteocalcin values were significantly reduced from pair-fed levels and slightly, but not significantly, reduced from chow-fed animals.

Tissue calcium levels following spinal cord transection in rats.

Adult rats were subjected to midthoracic spinal cord transections. Three segments of spinal cord, each approximately 5 mm in length, were removed from each animal at intervals from 5 min to 48 h postlesion; one from the lesion site and one each immediately rostral and caudal to the transection. Total tissue calcium concentrations ([Ca]t) for each spinal cord segment were determined using atomic absorption spectrophotometry and compared to control segments from untransected animals. [Ca]t levels in the segment at the lesion site was significantly elevated above control values at 30 min post-lesion, but decreased to control levels by 1 h. All other segments remained at control levels for the duration of the postlesion period. The rapid rise and fall of [Ca]t at the lesion site differs from spinal cord contusion studies in which [Ca]t remains at elevated levels for extended periods. It is postulated that the "open" transection injury permits the rapid clearance of calcium from the injury site.

The effect of in ovo boron supplementation on bone mineralization of the vitamin D-deficient chicken embryo.

It has been hypothesized that boron (B) is an essential element for animals, but its action will vary greatly depending on the nutriture of the organism. One of the nutrients implicated as having an interaction with boron is cholecalciferol (Vit D3). This study was carried out to determine if such an interaction exists. The study was conducted utilizing vitamin D-deficient chicken embryos that were injected through the shell at 8 d of embryogenesis with carrier (NaCl and/or acetone), B (0.5 mg), B + Vit D3 (0.5 mg and 0.3 microgram, respectively), or Vit D3 (0.3 or 1.5 micrograms). The in ovo concomitant administration of boron and vitamin D enhanced (p less than 0.05) the hatchability of the vitamin D-deficient embryos. Furthermore, boron and/or vitamin D3 increased (p less than 0.05) the percent of bone ash and decreased (p less than 0.05) the exaggerated height of the proliferative zone of the epiphyseal growth plate normally observed in vitamin D deficiency, suggesting a more rapid bone formation. The results provide further evidence supporting the hypothesis that boron plays a role in bone mineralization through an interaction with vitamin D.

In ovo administration of boron alters bone mineralization of the chicken embryo.

It has been hypothesized that boron (B) is an essential element for animals, especially in bone metabolism. In this study, the influence of in ovo boron administration was assessed in the chicken. At 8 d of embryogenesis, carrier or B (0.1, 0.5, or 1.0 mg) was injected on to the chorioallantoic membrane of fertile eggs. At hatching, body weights were recorded and tissue samples collected. Although boron failed to alter bone mineralization, it decreased (p less than 0.05) dried bone weight, suggesting a reduction in the bone organic matrix. Furthermore, 1 mg boron decreased (p less than 0.05) hatchability and increased (p less than 0.05) the height of the proliferative zone in the growth plate, indicating an unfavorable effect on bone elongation of the developing chick.

The relationship of nutritional copper to the development of postmenopausal osteoporosis in rats.

Factors that influence tissue copper concentration include age, diet, hormones, and pregnancy. In this study we altered diet independently, hormone (estrogen) independently, and various combinations of diet and hormone in animals of the same age to study the effects of ovariectomy complicated with dietary copper deficiency; a deficiency that has been demonstrated to cause bone defects. Sprague-Dawley rats were placed on various combinations of copper deficient or enriched diets before and/or after ovariectomy to determine if copper deficiency aggravated osteoporosis and if return to a copper-adequate diet alleviated it. In this study, ovariectomy did induce an osteopenia that was characterized by decreased trabecular bone. This osteopenia was slightly more severe with copper deficiency, but was not necessarily alleviated by the return of normal copper levels to the diet.

Acute exposure to hymenoxon: electron microscopic study of the mouse liver.

Young, male mice (25 to 30 g) were given oral doses of hymenoxon, a sesquiterpene lactone, for 5, 10, or 20 days. Hymenoxon, isolated from Hymenoxys odorata DC, was dissolved in dimethyl sulfoxide (DMSO) and water (1:1, v/v) and was administered daily at a dosage level of 100 mg/kg of body weight for 5, 10, or 20 days. Two control groups were maintained; 1 group was given DMSO and water, and 1 group was given water only. Twenty-four hours after the last dosing, the mice were euthanatized and their livers were processed for transmission electron microscopy. Hepatocyte organelles of hymenoxon-treated mice appeared normal although the bile canaliculi contained a fine granular material and membranous structures, including myelin figures. The canaliculi of hymenoxon-treated mice were also markedly dilated, compared with bile canaliculi in the control groups. The mean area of bile canaliculi of the 5-, 10-, and 20-day mice was 14.08 microns2, 15.65 microns2, and 17.56 microns2, respectively, compared with 7.73 microns2 for the canaliculi of DMSO + water-treated mice. The P values were less than 0.001, 0.02, and 0.05 for the 5-, 10-, and 20-day hymenoxon-treated mice, respectively. Seemingly, the mouse was not a good model for studying hepatic ultrastructural changes produced by hymenoxon, using dosages less than or equal to 100 mg/kg/day for 20 days.

The vastus medialis oblique muscle and its relationship to patellofemoral joint deterioration in human cadavers.

Patellofemoral joint deterioration (PFJD) is frequently seen in physical therapy clinics and represents a significant problem for both patients and rehabilitation clinicians. The vastus medialis oblique (VMO) muscle is reported to be the primary stabilizer of the patella during knee extension. Most studies and treatment protocols emphasize strengthening of the VMO as the nonsurgical treatment of choice for patients with PFJD. The purpose of this study was to determine whether any relationship exists between the morphology of the VMO and the presence and severity of PFJD in human cadavers. Dissection of 374 vastus medialis (VM) muscles and patellofemoral joints was performed on 229 human cadaver lower limbs to determine what relationships exist between gender, VMO features, and PFJD. Patellofemoral joint deterioration was determined by direct visual observation and assigned a score based on severity of joint deterioration present. Two-way chi-square tests were performed to determine the relationships between cadaver gender, the presence of VMO features, and the presence and severity of PFJD. Linear regression was performed to determine whether any correlation existed between the VMO fiber angle and the severity of PFJD. A one-way analysis of variance was performed to determine whether any differences existed between the VMO fiber angle and the PFJD groups. No statistically significant relationships, correlation, or differences existed in any of the tests performed between cadaver gender, VMO features, and presence or severity of PFJD. The presence or severity of PFJD in human cadavers is not related to either gender or VMO morphologic features. The results of this study do not support the premise that a more distal insertion of the VMO onto the patella of the VMO will have any effect on the presence or severity of PFJD.

Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction.

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% N(2)) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury.

Ultrastructure of the mineralizing metacarpophalangeal joint of progressive ankylosis (ank/ank) mice.

The metacarpophalangeal joint of the progressive ankylosis mouse was examined at 4, 6 and 8 weeks of age by using electron microscopy and electron probe microanalysis. These studies describe the progression of the disorder, and they reveal three overlapping phases: pannus proliferation, chondrophyte formation and mineralization, and articular cartilage mineralization. The chondrophyte mineralized in a manner fairly similar to endochondrial bone, whereas articular cartilage mineralized by advancement of a calcification front across the cartilage. Synovial crystal phagocytosis was also observed. The progressive ankylosis mouse should be an important model for studying both crystal deposition and abnormal articular cartilage calcification.

Alcohol, osteoporosis, and bone regulating hormones.

The mechanism of the production of ethanol-associated osteopenia seems to be a direct effect of alcohol on bone cells and an indirect or modulating effect through mineral regulating hormones such as vitamin D metabolites, parathyroid hormone, and calcitonin. The modulating effects of these hormones on bone and mineral metabolism in acute and chronic alcohol consumption is discussed herein.

Effect of alcohol consumption on adult and aged bone: a histomorphometric study of the rat animal model.

The adult and aged skeleton exist in a time when osteoporosis and age-related bone loss is at a maximum, and it is modified by lifestyle factors such as alcohol. To determine the effect of life-long alcohol consumption on the adult and aged rat model, 4-week-old female Sprague-Dawley rats were divided into three diet groups. Alcohol-treated animals were fed a modified Lieber-DeCarli diet ad libitum containing 35% ethanol-derived calories, whereas the pair-fed animals (weight-matched to ethanol rats) received an isocaloric liquid diet in which maltose-dextrin substituted calories supplied by ethanol. Chow animals were fed a standard rat chow ad libitum. Proximal tibiae were removed and prepared for histomorphometric analysis after 3, 6, 9, 12, or 18 months on the diets. Previous studies, with young animals, showed that chronic alcohol consumption during the age of bone development reduced bone volume and trabecular number in cancellous bone. The present study demonstrates that these reductions last throughout life. The rate of bone formation is reduced in alcohol-fed animals, but most bone cell parameters are relative normal, except for wall thickness, indicating a reduced osteoblast activity.

Silicone bleed associated with double-lumen breast prostheses.

In an effort to reduce contact of tissue with silicone gel, it has become popular to use double-lumen prostheses in mammaplasty. We report a case of silicon incorporation by human tissue after insertion of a double-lumen implant.