Beth M. Bowman

AVAILABLE ANIMAL MODELS OF OSTEOPENIA - SMALL AND LARGE

Animal models of osteopenia are reviewed. Endocrine excess or deficiency conditions include ovariectomy, orchidectomy, glucocorticoid excess and other endocrine states. Seasonal and reproductive cycles are usually transient and include pregnancy and lactation, egg-laying, antler formation and hibernation. Dietary conditions include calcium deficiencies, phosphate excess and vitamin C and D deficiencies. Mechanical usage effects include skeletal underloading models. Aging is also associated with osteopenia in many species.

Effects of dihydrotestosterone alone and combined with estrogen on bone mineral density, bone growth, and formation rates in ovariectomized rats

Androgens are associated with the greater skeletal mass and size in men compared with women and have been used as anabolic agents promoting skeletal growth and mineral accretion in both sexes, but specific effects on growth and bone formation in the female skeleton are not well understood. The effects of 5 alpha-dihydrotestosterone (DHT) alone, and in combination with 17 beta-estradiol on bone and bone growth were studied in female ovariectomized (OVX) rats with established osteopenia. Eight weeks after OVX, rats were given 0.1 mg 17 beta-estradiol and/or 2.5 mg or 10 mg DHT administered by controlled-release pellets for 2 months. Body weights decreased with estrogen treatment but increased with DHT. Bone mineral density increased with the highest dose of DHT relative to OVX controls and the estrogen treated group. Dry and ashed bone weights and ash/dry weight ratios increased in the estrogen and DHT treated animals compared to the baseline OVX controls. Total bone calcium was greater with DHT and estrogen combined with DHT. The percent of calcium in the ash increased in all DHT treated groups. When normalized to final body weight, the total femur calcium content was significantly increased in the estrogen and estrogen with DHT groups, but not in the DHT groups compared with the baseline OVX and OVX control groups. The periosteal bone formation rates were increased with the high dose DHT alone and combined with estrogen. OVX rats had increased endochondral bone elongation rates relative to controls but this was decreased with estrogen treatment. DHT combined with estrogen increased endochondral growth rates relative to the estrogen treated group. Trabecular bone volume was decreased in all OVX groups relative to the base line group, but there were no significant effects observed with any treatments. Cancellous bone formation rates were suppressed with estrogen treatment but were partially reversed when combined with DHT. DHT treatments also increased most cancellous bone formation indices over OVX controls. While estrogen is known to preserve skeletal mass by reducing bone turnover, DHT increased skeletal mass by promoting bone growth and formation with concomitant increases in total body mass. DHT had greater effects on cortical bone and partially mitigated the suppressive effects of estrogen on bone growth and formation in the female skeleton.

Medullary bone osteogenesis following estrogen administration to mature male Japanese quail

Abstract The osteogenesis of medullary bone on endosteal bone surfaces of mature male Japanese quail was induced by estradiol valerate (EV) and the sequential changes were characterized by histology, autoradiography, microradiography, and electron microscopy. In untreated controls, endosteal surfaces of the femoral diaphysis were generally inactive and lined by low-density populations of flat bone-lining cells. Within 24 hr of EV administration the surfaces were lined by plump cells with abundant polyribosomes and with large oval to round nuclei. There was an increase in the concentration of [ 3 H]proline near some endosteal surfaces at this time. By 36 hr the developing osteoblasts had extensive rough endoplasmic reticulum (RER) and Golgi complexes. Extracellular matrix with isotropically arranged collagenous fibers was evident. By 48 hr small trabeculae had formed and some of the matrix was beginning to mineralize. Osteoblasts contained abundant dilated RER, many had numerous cell processes and some were becoming surrounded by bone matrix forming osteocytes. From 72 to 120 hr the developing bone grew rapidly from endosteal surfaces into the marrow space. Medullary bone development was accompanied by rapid and dramatic increases in total plasma calcium levels. This study demonstrates a well-defined rapid sequence of induced osteogenesis in vivo and suggests that the bone-lining cell in the postfetal organism may have osteogenic potential.

Greatly increased cancellous bone formation with rapid improvements in bone structure in the rat maternal skeleton after lactation.

There is a decrease in cancellous bone mass and strength during lactation but these are partially or completely reconstituted in the postlactational period. The purpose of this study was to determine changes in cancellous bone structure and formation after lactation in established breeder rats. For this, rats were taken at the end of the second pregnancy (Preg‐2) and second lactation (Lac‐2) and 2, 4, and 6 weeks after weaning. Nulliparous (NP) groups were included for comparisons. Bone structure was measured using morphometric methods and bone dynamics by histomorphometry. Tibial metaphyseal cancellous bone was lost during the first reproductive cycle, as expected, and again depleted during the Lac‐2. Bone formation indices were elevated at the end of Lac‐2, compared with those at the end of the second pregnancy or in the nulliparous animals. Within 2 weeks after the second weaning, the amount of double‐labeled surface (dLS) increased ∼800%, the mineralizing surface (MS) increased >400% with similar increases in bone formation rates (BFRs), compared with already elevated bone formation measured at the end of Lac‐2. From 2 to 4 weeks after lactation, there were commensurate increases in cancellous bone mass and structural indices with essentially complete restoration of cancellous bone volume and structure compared with that measured at the end of Preg‐2. The results show rapid and substantial increases in bone formation with reconstitution of cancellous bone mass and structure after lactation in rats. The skeletal changes that occur during the postlactational period may serve to prepare and protect the maternal skeleton for subsequent reproductive cycles.

Comparison of Bone Loss During Normal Lactation With Estrogen Deficiency Osteopenia and Immobilization Osteopenia in the Rat

Substantial changes in mineral and skeletal metabolism occur during pregnancy and lactation. The purpose of this study was to compare three contrasting osteopenic states in the rat: (1) physiological (lactation), (2) endocrine‐deficiency (ovariectomy), and (3) lack of mechanical usage (immobilization).

Rapid inactivation and apoptosis of osteoclasts in the maternal skeleton during the bone remodeling reversal at the end of lactation

There is a rapid reversal in maternal skeletal metabolism and bone remodeling from accelerated bone resorption during lactation to skeletal rebuilding after lactation. The purpose was to determine the changes that occur in maternal osteoclasts during the transition from lactation to postlactation. Skeletal samples were taken from female rats on days 10 and 19 of lactation and 1 and 7 days after lactation. The pups were weaned on day 20. There was a rapid change in the osteoclast population after weaning, resulting in less resorption surface. Osteoclasts detached from bone surfaces, lost their ruffled borders, and became fragmented with immunocytochemical evidence of apoptosis within 24 hr after lactation. Concomitant with the rapid regression in the osteoclast population was an over fivefold increase in maternal calcitonin (CT) levels at 24 hr after weaning. Serum calcium and estrogen (E2) increased, but prolactin (PRL) and PTH decreased after weaning. The hormone changes, particularly that of CT, are consistent with the rapid regression of the osteoclast population at the end of lactation. These changes are similar to a reversal phase of a bone remodeling cycle where bone formation commences when resorption ceases on bone surfaces and suggests that the fate of osteoclasts during bone remodeling is programmed cell death. These results also suggest that bone remodeling is well synchronized prior to, during, and after lactation to accommodate the mineral requirements of the offspring as well as the mother. Anat Rec, 290:65–73, 2007.

Identification and characterization of mononuclear phagocytes isolated from rat testicular interstitial tissues

Rat testicular interstitial tissues contain numerous mononuclear phagocytelike cells. Dispersed testicular tissues are allowed to settle onto a glass substrate and the strongly adherent cells are identified as the same cells that have been described in vivo, based on the retention of an in vivo marker (plutonium). These putative testicular intersitial tissue macrophages (TIMs) have adhesion and cell surface features characteristic of mononuclear phagocytes, as determined by scanning electron microscopy. Immediately after isolation from testicular tissues. TIMs show a strong staining reaction for nonspecific esterase. These cells have Fc and complement receptors and some express la cell‐ surface antigens. About two thirds of the TIMs phagocytose sheep red blood cells and most of the cells are capable of nonspecific phagocytosis of polystyrene beads. This study demonstrates that the phagocytic cells found in testicular interstitial tissues have morphological, histochemical, phagocytic, and immunological properties characteristic of functionally active mononuclear phagocytes. These results suggest that these resident mononuclear phagocytes may play a role in immune‐related functions in the testis.

Weaning Initiates a Rapid and Powerful Anabolic Phase in the Rat Maternal Skeleton

Abstract Maternal skeletal mineral lost during lactation is rapidly restored after weaning. The purposes of this study were to determine when increases of bone formation occur after weaning, whether the expanding osteoblast population is derived from proliferating progenitors, and to relate these skeletal changes to known endocrine events at weaning. Female rats were allowed to complete one reproductive cycle. Half of these rats were mated a second time and allowed to lactate for 20 days. The other half served as an age-matched, normal estrus cycling comparison group. One day after weaning, the dams and their comparison group were given four injections of bromodeoxyuridine (BrdU) at 8-h intervals. Indices of bone formation and the kinetics of BrdU-labeled cells were measured in lumbar vertebral cancellous bone. At 2 days after weaning, cancellous bone formation rates were substantially greater than those in the nonmated rats. Indices of bone formation more than doubled from the second to seventh day after weaning. At 25 h after the first BrdU injection in the postweaned rats, considerable numbers of labeled cells were observed on or near the bone surface, with about 17% of the osteoblast population labeled. Labeled osteoblasts peaked at 20%–24% compared with 4% in the normal estrus cycling group. Immediately following weaning, there is a profound increase in the osteoblast population in maternal cancellous bone. Many, if not most of these newly formed osteoblasts were derived from proliferating progenitors. It is possible that the endocrine milieu of lactation expands or primes the osteoprogenitor pool for this rapid anabolic phase.

Peptide decoration of nanovehicles to achieve active targeting and pathology-responsive cellular uptake for bone metastasis chemotherapy

To improve bone metastases chemotherapy, a peptide-conjugated diblock copolymer consisting of chimeric peptide, poly(ethylene glycol) and poly(trimethylene carbonate) (Pep-b-PEG-b-PTMC) is fabricated as a drug carrier capable of bone-seeking targeting as well as pathology-responsive charge reversal to ensure effective cellular uptake at the lesion sites. The chimeric peptide CKGHPGGPQAsp8 consists of an osteotropic anionic Asp8, a cathepsin K (CTSK)-cleavable substrate (HPGGPQ) and cationic residue tethered to polymer chain. Pep-b-PEG-b-PTMC can spontaneously self-assemble into negatively charged nanomicelles (~75 nm). As to the model drug of doxorubicin, Pep-b-PEG-b-PTM shows 30.0 ± 1 % and 90.1 ± 2 % for loading content and loading efficiency, respectively. High bone binding capability is demonstrated with that 66 % of Pep-b-PEG-b-PTMC micelles are able to bind to hydroxyl apatite, whereas less than 15 % is for Pep-free micelles. The nanomicelles exhibit a negative-to-positive charge conversion from -18.5 ± 1.9 mV to 15.2 ± 1.8 mV upon exposure to CTSK, an enzyme overexpressed in bone metastatic microenvironments. Such a pathology-responsive transition would lead to remarkably enhanced cellular uptake of the nanomicelles upon reaching lesion sites, thus improving the drug efficacy as verified by the in vitro cytotoxicity assay and the in vivo study in myeloma-bearing 5TGM1 mice model.

Effects of thermal injury on skeletal metabolism in two strains of mice

Long-term growth retardation occurs in children and osteopenias occur among children and adults who have been burned or suffer other injuries that result in a systemic inflammatory response. The purpose of this study was to define some of the growth, and to determine cancellous and cortical bone changes that occur following thermal injury in several contrasting strains of mice. Male C3H/HeN and Balb/c mice were given about a 20% total body thermal injury and 10 days later skeletal tissues were collected. The bone ash weights of the burned animals from both strains were less than their sham controls. In both strains, cancellous bone volumes were less in the burned animals than in their respective sham or baseline control groups. The loss of bone was particularly evident in the secondary spongiosa regions and also included a decrease in trabecular thickness and connectivity and an increase in trabecular separation. Longitudinal (endochondral) growth was suppressed in the burn animals. In the burned animals, indices of cancellous bone formation were substantially reduced whereas those in cortical bone were essentially nonexistent. The numbers of osteoclasts were increased in cancellous bone, and endocortical eroded surface was increased in the burn animals. These data show that there are rapid and profound changes in skeletal growth and metabolism in an experimental model of thermal injury. In general, a greater relative response was observed in the Balb/c vs. the C3H/HeN strain. Thermal injury resulted in a rapid and dramatic suppression of bone formation and endochondral growth with increased bone resorption in both cancellous and cortical bone.