Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.     

A comparison of the anabolic effects of rat and bovine parathyroid hormone (1-34) in ovariectomized rats

The current study was designed to compare the skeletal effects of comparable doses of rat parathyroid hormone 1-34 (rPTH) and bovine parathyroid hormone 1-34 (bPTH) in ovariectomized (OVX) rats. Female Sprague-Dawley rats were OVX or sham-operated at 6 months of age and maintained untreated for 28 days after surgery. Baseline control and OVX rats were sacrificed at the beginning of treatment. Beginning 28 days post-OVX, the remaining rats were subcutaneously injected daily with rPTH or bPTH at 0, 5, 25, or 50 microg/kg/d for 28 days. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of the distal femoral metaphyses were determined ex vivo using dual energy X-ray absorptiometry. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal tibia from each rat. Baseline OVX rats exhibited osteopenia as demonstrated by their significantly reduced femoral BMD and proximal tibia cancellous bone volume compared with those of baseline sham controls. Both rPTH and bPTH restored bone in OVX rats by markedly stimulating bone formation in a dose-dependent manner. However, a difference in potency between the two forms of PTH was evident. The percentage increases of BMC, BMD, cancellous bone volume, trabecular thickness, mineralizing surface, and bone formation rate in the OVX rats treated with bPTH at 5 microg/kg/d were the same as or above those treated with rPTH at the 25 microg/kg/d dose level. A relative potency analysis showed that bPTH was approximately 4- to 6-fold relatively more potent than rPTH in increasing distal femoral BMD as well as cancellous bone volume, mineralizing surface, and bone formation rate of proximal tibial metaphyses at comparable dose levels and a given time. These results may serve as a reference for in vivo study design when rPTH or bPTH are to be the agents for studies on bone anabolism.     

Cloning the full-length cDNA for rat connective tissue growth factor: implications for skeletal development

The mammalian osteopetroses represent a pathogenetically diverse group of skeletal disorders characterized by excess bone mass resulting from reduced osteoclastic bone resorption. Abnormalities involving osteoblast function and skeletal development have also been reported in many forms of the disease. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between op mutants and their normal littermates. RNA isolated from calvaria and long bones was used as a template for mRNA-differential display. Sequence information for one of the many cDNA that were selectively expressed in either normal or mutant bone suggested that it is the rat homologue of connective tissue growth factor (CTGF) previously cloned in the human, mouse, and other species. A consensus sequence was assembled from overlapping 5'-RACE clones and used to confirm the rat CTGF cDNA protein coding region. Northern blot analysis confirmed that this message was highly (8- to 10-fold) over-expressed in op versus normal bone; it was also upregulated in op kidney but none of the other tissues (brain, liver, spleen, thymus) examined. In primary rat osteoblast cultures, the CTGF message exhibits a temporal pattern of expression dependent on their state of differentiation. Furthermore, CTGF expression is regulated by prostaglandin E(2), a factor known to modulate osteoblast differentiation. Since members of the CTGF family regulate the expression of specific genes, such as collagen and fibronectin, we propose that CTGF may play a previously unreported role in normal skeletal modeling/remodeling. Its dramatic over-expression in the op mutant skeleton may be secondary to the uncoupling of bone resorption and bone formation resulting in dysregulation of osteoblast gene expression and function.     

Resveratrol Treatment Delays Growth Plate Fusion and Improves Bone Growth in Female Rabbits

Trans-resveratrol (RES), naturally produced by many plants, has a structure similar to synthetic estrogen diethylstilbestrol, but any effect on bone growth has not yet been clarified. Pre-pubertal ovary-intact New Zealand white rabbits received daily oral administration of either vehicle (control) or RES (200 mg/kg) until growth plate fusion occurred. Bone growth and growth plate size were longitudinally monitored by X-ray imaging, while at the endpoint, bone length was assessed by a digital caliper. In addition, pubertal ovariectomized (OVX) rabbits were treated with vehicle, RES or estradiol cypionate (positive control) for 7 or 10 weeks and fetal rat metatarsal bones were cultured in vitro with RES (0.03 µM–50 µM) and followed for up to 19 days. In ovary-intact rabbits, sixteen-week treatment with RES increased tibiae and vertebrae bone growth and subsequently improved final length. In OVX rabbits, RES delayed fusion of the distal tibia, distal femur and proximal tibia epiphyses and femur length and vertebral bone growth increased when compared with controls. Histomorphometrical analysis showed that RES-treated OVX rabbits had a wider distal femur growth plate, enlarged resting zone, increased number/size of hypertrophic chondrocytes, increased height of the hypertrophic zone, and suppressed chondrocyte expression of VEGF and laminin. In cultured fetal rat metatarsal bones, RES stimulated growth at 0.3 µM while at higher concentrations (10 μM and 50 μM) growth was inhibited. We conclude that RES has the potential to improve longitudinal bone growth. The effect was associated with a delay of growth plate fusion resulting in increased final length. These effects were accompanied by a profound suppression of VEGF and laminin expression suggesting that impairment of growth plate vascularization might be an underlying mechanism.     

Differential response of rat limb bones to strenuous exercise

We examined the influence of a strenuous exercise regimen on tibial and metatarsal bones to show not only how the geometric, histological, and mechanical properties of immature bone respond to strenuous exercise but also how long bones within the same limb may respond differentially to exercise. Female Sprague-Dawley rats (8 wk old) were divided randomly into two groups: a sedentary control (n = 15) and an exercised group (n = 15). The exercise intensity was 80-90% of maximum oxygen capacity 5 days/wk for 10 wk. Mechanical properties of tibia and second metatarsus (MT) were determined with three-point bending, and contralateral bones were used for geometric and histological analyses. Length and middiaphyseal cross-sectional geometry of the exercised tibiae were significantly less than controls, but material properties were not different. The exercised tibiae had significantly lower structural properties (e.g., loads at the proportional limit and maximum and energy at failure load). The middiaphyseal dorsal cortex of exercised MT was significantly thicker than controls, but tensile stress at the proportional limit and elastic modulus of exercised MT were significantly less than controls. The average number of osteons and osteocytes per unit area of the tibial middiaphysis was significantly greater in the exercised group--especially in lateral and posterior cortices. The number of osteons and osteocytes per unit area in the MT, however, was significantly less in the exercised group. The differential effects of strenuous exercise on tibia and MT suggest that local loading and bone-specific responses have important roles in modulating the response of immature bone to strenuous exercise.     

Greater efficacy of alfacalcidol in the red than in the yellow marrow skeletal sites in adult female rats

The present study compared the bone anabolic effects of graded doses of alfacalcidol in proximal femurs (hematopoietic, red marrow skeletal site) and distal tibiae (fatty, yellow marrow skeletal site). One group of 8.5-month-old female Sprague-Dawley rats were killed at baseline and 4 groups were treated 5 days on/2 days off/week for 12 weeks with 0, 0.025, 0.05 and 0.1 microg alfacalcidol/kg by oral gavage. The proximal femur, bone site with hematopoietic marrow, as well as the distal tibia bone site with fatty marrow, were processed undecalcified for quantitative bone histomorphometry. In the red marrow site of the proximal femoral metaphysis (PFM), 0.1 microg alfacalcidol/kg induced increased cancellous bone mass, improved architecture (decreased trabecular separation, increased connectivity), and stimulated local bone formation of bone 'boutons' (localized bone formation) on trabecular surfaces. There was an imbalance in bone resorption and formation, in which the magnitude of depressed bone resorption greater than depressed bone formation resulted in a positive bone balance. In addition, bone 'bouton' formation contributed to an increase in bone mass. In contrast, the yellow marrow site of the distal tibial metaphysis (DTM), the 0.1 microg alfacalcidol/kg dose induced a non-significant increased cancellous bone mass. The treatment decreased bone resorption equal to the magnitude of decreased bone formation. No bone 'bouton' formation was observed. These findings indicate that the highest dose of 0.1 microg alfacalcidol/kg for 12 weeks increased bone mass (anabolic effect) at the skeletal site with hematopoietic marrow of the proximal femoral metaphysis, but the increased bone mass was greatly attenuated at the fatty marrow site of the distal tibial metaphysis. In addition, the magnitude of the bone gain induced by alfacalcidol treatment in red marrow cancellous bone sites of the proximal femoral metaphysis was half that of the lumbar vertebral body. The latter data were from a previous report from the same animal and protocol. These findings indicated that alfacalcidol as an osteoporosis therapy is less efficacious as a positive bone balance agent that increased trabecular bone mass in a non-vertebral skeletal site where bone marrow is less hematopoietic.     

Skeletal Development of Mice Lacking Bone Sialoprotein (BSP) - Impairment of Long Bone Growth and Progressive Establishment of High Trabecular Bone Mass

Adult Ibsp-knockout mice (BSP−/−) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP−/− mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP−/− newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP−/− mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP−/− than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP−/− mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP−/− mice, while impairing primary mineralization.     

Growth plate abnormalities in a new dwarf mouse model: tich

Growth plate cartilage calcification has been examined in a recently described mouse mutant, tich, which is co-isogenic with the A.TL strain. Long bones were studied from 1-day-old and 1-month-old mice which carried a homozygous recessive gene mutation making them short limbed and dumpy. Specimens were studied by routine histology, scanning electron microscopy and radiography. In 1-day-old tich mice the front of calcified cartilage was recessed behind the advancing periosteum and bone. No similar recess was seen in control mice. At 1 month of age, a number of the long bone growth plates were irregularly thickened, particularly in the central area. This produced a central tongue of non-calcified cartilage (particularly prominent in the proximal tibia) which gave rise to a corresponding pit in the calcified cartilage layer, in macerated specimens. This was accompanied by poor resorption of calcified cartilage. At both ages the presence of the respective defects was radiographically confirmed. At present it is not known whether this is primarily a defect of calcification or resorption but its presence, apparently from a single mutation in a genetically defined mouse strain, makes it a potentially valuable model.     

Protective actions of green tea polyphenols and alfacalcidol on bone microstructure in female rats with chronic inflammation

This study investigated the effects of green tea polyphenols (GTP) and alfacalcidol on bone microstructure and strength along with possible mechanisms in rats with chronic inflammation. A 12-week study using a 2 (no GTP vs. 0.5%, w/v GTP in drinking water)×2 (no alfacalcidol vs. 0.05 μg/kg alfacalcidol orally, 5×/week) factorial design was employed in lipopolysaccharide (LPS)-administered female rats. A group receiving placebo administration was used to compare with a group receiving LPS administration only to evaluate the effect of LPS. Changes in tibial and femoral microarchitecture and strength of femur were evaluated. Difference in expression of tumor necrosis factor-α (TNF-α) in proximal tibia using immunohistochemistry was examined. Compared to the placebo group, the LPS-administered-only group had significantly lower femoral mass, trabecular volume, thickness and number in proximal tibia and femur, and lower periosteal bone formation rate in tibial shafts but had significantly higher trabecular separation and osteoclast number in proximal tibia and eroded surface in endocortical tibial shafts. Both GTP and alfacalcidol reversed these LPS-induced detrimental changes in femur, proximal tibia and endocortical tibial shaft. Both GTP and alfacalcidol also significantly improved femoral strength, while significantly suppressed TNF-α expression in proximal tibia. There were significant interactions in femoral mass and strength, trabecular separation, osteoclast number and TNF-α expression in proximal tibia. A combination of both showed to sustain bone microarchitecture and strength. We conclude that a protective impact of GTP and alfacalcidol in bone microarchitecture during chronic inflammation may be due to a suppression of TNF-α.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are characteristics of the osteosclerotic mutation which suggest that it is a unique osteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Ovariectomy of 12-month-old rats: effects on osteoprogenitor numbers in bone cell populations isolated from femur and on histomorphometric parameters of bone turnover in corresponding tibia

Ovariectomy (OVX) in rats results in increased bone turnover and decreased bone volume and bone mineral density when measured in the metaphyses of long bones. We have investigated the effects of OVX on changes in the number of progenitors in cell populations derived from the metaphyseal bone of femurs of ovariectomized rats at 12 months of age, by using colony assays, bone nodule assays, and limiting dilution analysis at 1.5 and 9 months post-OVX. We have also measured histomorphometric parameters of bone formation and resorption in the corresponding tibia at the same time-points. A significant increase, as shown by bone nodule assays and limiting dilution analysis, occurs in the number of progesterone- and dexamethasone-responsive osteoprogenitors in cell populations isolated from ovariectomized rats at the 9-month post-OVX time-point. Progesterone-responsive osteoprogenitors are also increased at 1.5 months post-OVX. The number of fibroblast colony-forming units does not change. Histomorphometry has shown that OVX causes an increase in osteoblast surfaces, mineralizing surfaces, and bone formation rate at both 1.5 and 9 months post-OVX. The mineral apposition rate is increased at 1.5 months post-OVX. OVX also increases parameters of bone resorption at both time-points, the net result being a decrease in bone mineral density and cancellous bone volume at 9 months post-OVX. Thus, OVX in rats at 12 months of age is associated with an increase in the number of both progesterone- and dexamethasone-responsive osteoprogenitors 9 months post-OVX; this corresponds with increases in the histomorphometric parameters of bone formation.     

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.     

Evolution of hindlimb bone dimensions and muscle masses in house mice selectively bred for high voluntary wheel‐running behavior

We have used selective breeding with house mice to study coadaptation of morphology and physiology with the evolution of high daily levels of voluntary exercise. Here, we compared hindlimb bones and muscle masses from the 11th generation of four replicate High Runner (HR) lines of house mice bred for high levels of voluntary wheel running with four non‐selected control (C) lines. Mass, length, diameter, and depth of the femur, tibia‐fibula, and metatarsal bones, as well as masses of gastrocnemius and quadriceps muscles, were compared by analysis of covariance with body mass or body length as the covariate. Mice from HR lines had relatively wider distal femora and deeper proximal tibiae, suggesting larger knee surface areas, and larger femoral heads. Sex differences in bone dimensions were also evident, with males having thicker and shorter hindlimb bones when compared with females. Several interactions between sex, linetype, and/or body mass were observed, and analyses split by sex revealed several cases of sex‐specific responses to selection. A subset of the HR mice in two of the four HR lines expressed the mini‐muscle phenotype, characterized mainly by an ～50% reduction in hindlimb muscle mass, caused by a Mendelian recessive mutation, and known to have been under positive selection in the HR lines. Mini‐muscle individuals had elongated distal elements, lighter and thinner hindlimb bones, altered 3rd trochanter muscle insertion positions, and thicker tibia‐fibula distal widths. Finally, several differences in levels of directional or fluctuating asymmetry in bone dimensions were observed between HR and C, mini‐ and normal‐muscled mice, and the sexes. This study demonstrates that skeletal dimensions and muscle masses can evolve rapidly in response to directional selection on locomotor behavior.     

Structural study of spongiosa tissue in growing sheep.

A study was made of 160 long bones taken from 40 native Merino sheep of both sexes. These animals, which represented uniform growth (mean growth-curve values), were divided into four groups which were slaughtered consecutively at 0, 45, 105 and 270 days old (0, 6.4, 15 and 38.5 weeks, respectively). The following bones were studied; humerus, femur, tibia and os coxae. Thin lamellae taken from the metaphyses of the bones obtained were fixed, decalcified and stained with hematoxylin-eosin to assess the development of the various components of growing bony tissue. The bones studied followed the same maturation pattern; the os coxae proved to be the best histological indicator in differentiating the age of the animals studied.     

The effects of low laser irradiation on angiogenesis in injured rat tibiae

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm(-2) were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm(-2), but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.     

An in vitro assay of bone development using fetal long bones of mice: morphological studies

The purpose of this study was to examine the morphological changes in an in vitro system in which the two elements of bone modelling, formation and resorption, could be studied simultaneously. Pregnant mice were killed on days 15, 16 and 17 of gestation, the fetuses were removed and the radii and ulnae dissected free of soft tissue. The bones were cultured for 6 days in media (BGJ) supplemented with 20% fetal calf serum and 150 micrograms/ml vitamin C. Growth and mineralization were estimated by measuring the total length of the bone, and diaphysis, and by light and transmission electron microscopy (TEM). The results of this study indicate that there is a continuous measurable increase in the total length of fetal mouse long bones over the 6 days of culture. These bones show a continuous growth of periosteal bone, with mesenchymal tissue penetrating into the diaphyseal shaft, and development of bone marrow like tissue. TEM examination showed differentiation of mesenchymal cells to osteoblasts, formation of new bone matrix and bone mineralization similar to that found in developmentally matched controls. In the cartilagenous epiphyses, however, many hydroxyapatite crystals were not associated with matrix vesicles. In addition, some of the chondrocytes of the hypertrophic zone appeared to be dedifferentiating into mesenchymal cells with osteoblast-like features. In spite of the lack of osteoclasts in the 15- and 16-day explants, osteoclasts appeared in the diaphysis after 2 and 4 days in culture. Our results suggest that this system can serve as a good model for the study of bone formation and resorption as they occur, simultaneously, during bone modelling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid loss of bone mass and strength in mice after abdominal irradiation

Localized irradiation is a common treatment modality for malignancies in the pelvic-abdominal cavity. We report here on the changes in bone mass and strength in mice 7-14 days after abdominal irradiation. Male C57BL/6 mice of 10-12 weeks of age were given a single-dose (0, 5, 10, 15 or 20 Gy) or fractionated (3 Gy × 2 per day × 7.5 days) X rays to the abdomen and monitored daily for up to 14 days. A decrease in the serum bone formation marker and ex vivo osteoblast differentiation was detected 7 days after a single dose of radiation, with little change in the serum bone resorption marker and ex vivo osteoclast formation. A single dose of radiation elicited a loss of bone mineral density (BMD) within 14 days of irradiation. The BMD loss was up to 4.1% in the whole skeleton, 7.3% in tibia, and 7.7% in the femur. Fractionated abdominal irradiation induced similar extents of BMD loss 10 days after the last fraction: 6.2% in the whole skeleton, 5.1% in tibia, and 13.8% in the femur. The loss of BMD was dependent on radiation dose and was more profound in the trabecula-rich regions of the long bones. Moreover, BMD loss in the total skeleton and the femurs progressed with time. Peak load and stiffness in the mid-shaft tibia from irradiated mice were 11.2-14.2% and 11.5-25.0% lower, respectively, than sham controls tested 7 days after a single-dose abdominal irradiation. Our data demonstrate that abdominal irradiation induces a rapid loss of BMD in the mouse skeleton. These effects are bone type- and region-specific but are independent of radiation fractionation. The radiation-induced abscopal damage to the skeleton is manifested by the deterioration of biomechanical properties of the affected bone.     

Effect of growth hormone and pamidronate on bone blood flow, bone mineral and IGF-I levels in the rat

So far it is not known whether the growth hormone (GH) has an effect on the local blood circulation in bones. Using male rats we studied the local blood circulation in the tibia and the distal end of the femur (by means of the uptake of 85Sr-microspheres), the density and ash weight of the tibia, the urinary excretion of pyridinoline (PD) and deoxypyridinoline (DPD) as an indicator of bone resorption and the blood levels of IGF-I after the administration of human GH (4 mg/kg s.c. daily for 4 weeks) and/or bisphosphonate pamidronate (Aredia, CIBA-Geigy, administered in the dose of 3 mg/kg i.p. on day 1, 2, 9 and 10). The rats were divided into four groups: 1. controls, 2. GH, 3. pamidronate, 4. GH plus pamidronate. After the administration of GH, we observed a significant increase in bone blood flow (and in the uptake of 85Sr-microspheres), a decrease in the density and ash weight of the tibia and increased urinary excretion of PD and DPD; IGF-I levels in the blood were non-significantly elevated. Simultaneously administered pamidronate inhibited all significant effects of GH and it also decreased the IGF-I levels in rats treated with GH. After the administration of pamidronate itself the bone density and ash weight of the tibia were increased and urinary DPD excretion was decreased. In view of the known vascular effects of IGF-I, we assume that the increase in bone blood flow after the administration of GH and its reduction after simultaneous administration of pamidronate could be mediated by the changes of IGF-I blood levels, although the effect of pamidronate on IGF-I is still not clear. Regarding the role of blood circulation in rat bones, we consider that our present results are further evidence for the relationship between the blood circulation in bones and bone resorption, although these results do not show how active is bone blood circulation in the regulation of bone tissue metabolism.     

Quantification of tartrate resistant acid phosphatase distribution in mouse tibiae using image analysis.

Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm2 region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p > 0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r2 = 0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.     

The osteopetrotic rabbit: skeletal cytology and ultrastructure

The lethal, autosomal recessive osteopetrotic mutation in the rabbit, osteosclerosis (os/os), has recently been made available for experimental investigation. We have examined the cytology and ultrastructure of skeletal cells in mutants and report abnormalities in osteoblasts, osteocytes, and osteoclasts. Mutant osteoclasts lack a well-defined ruffled border and show few morphological signs of bone resorption. Osteoblasts in mutants form bone in neonatal life but show signs of degeneration by 2 weeks after birth. Mutant osteoblasts and osteocytes contain large, electron-dense cytoplasmic inclusions. External surfaces of mutant long bones show no evidence of bone resorption by scanning electron microscopy, and fibrosis of intertrabecular spaces is a prominent feature in mutants. These data, considered with recent evidence that the functions of osteoblasts and osteoclasts are interrelated, suggest that reduced bone resorption, a characteristic feature of osteopetrosis, may be related to osteoblast incompetence in this mutation.