Remodeling of the sagittal suture in osteopetrotic (op/op) mice associated with cranial flat bone growth.

It is well known that cranial flat bone experiences growth and development at the sutural interface, which is regarded as a neutral zone to control mechanical stimuli. In osteopetrotic (op/op) mice, meanwhile, cranial deformation is produced by the deficiency of osteoclasts and the subsequent defect of bone resorption. It would be a reasonable assumption that such disturbance in bone remodeling affects sutural modification and the relevant cranial flat bone development. The present study was thus conducted to examine histological features of the sagittal sutures in op/op mice, with special reference to the relevant bone remodeling. The sagittal sutures in 10-, 15-, 30-, and 60-day-old normal and op/op mice were observed microscopically. Furthermore, osteoclastic activity was evaluated on the sections stained with tartrate-resistant acid phosphatase (TRAP). The sutures of 15-day-old op/op mice showed stenosis and synostosis, and less-developed collagen fibers associated with an irregular arrangement of fibroblasts, whereas these changes were rarely found in normal mice. Osteoclasts were hardly detected in the parietal bones around the sutures of op/op mice, although the number was numerous in normal mice. These results emphasize that congenital deficiency in osteoclast produces unbalanced bone remodeling at the sutural interface and on the surfaces of the cranial bones, which is assumed to be closely related to cranial bone deformity in op/op mice.     

Morphological change of the nasopremaxillary suture in growing "toothless" osteopetrotic (op/op) mice.

Osteopetrotic (op/op) mice are known to commonly show a failure of tooth eruption. It is also well understood that masticatory function is highly associated with the craniofacial morphology of the growing mouse; however, the effects on sutural growth have not been studied. The present study was conducted to examine, in detail, the morphological and histological changes of the nasopremaxillary suture in these mutant mice and to assess a role of mechanical stress from mastication in the sutural growth. The width of the nasopremaxillary suture was measured on the section for the superior (P1), middle (P2), and inferior (P3) levels. The width of the nasopremaxillary suture for the P1 level in the normal mice fed a solid diet was significantly smaller in 30-day-old mice than in 15-day-old mice, whereas the width for the level P3 was significantly greater in the 30-day-old mice than in the 15-day-old mice. These changes in the sutural space were more prominent in the normal mice fed a solid diet than in the normal mice fed a granular diet. The sutural widths for all the levels became smaller in the 30-day-old op/op mice than in the 10-day-old op/op mice. The endocranial area of the nasopremaxillary suture showed synostosis in 30-day-old op/op mice. In both the normal and op/op mice, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells was greatest at the age of 15 days. Moreover, the TRAP-positive cell number was smaller in the op/op mice than in the normal mice for all the experimental stages. Since, in general, mastication begins in mice after tooth eruption, i.e. from 15 to 30 days after birth, the present findings suggest that failure of tooth eruption and the reduced masticatory function restrict sutural modification.     

Influences of osteoclast deficiency on craniofacial growth in osteopetrotic (op/op) mice

It is well known that the defect in bone resorption in osteopetrotic (op/op) mice brings about deformation of the cranium and failure of tooth eruption. However, the influences on longitudinal growth of the craniofacial skeleton have not been elucidated. This study was thus conducted to examine craniofacial morphology and longitudinal changes in the op/op mice by means of morphometric analysis with lateral cephalograms. Lateral cephalograms, taken every 10 days from 10- to 90-day-old mice, were analyzed on a personal computer for 11 measurement items. For the nasal bone region, the most prominent differences were found between the op/op and normal mice. The anterior cranial base and occipital bone height presented almost equivalent growth changes in both the op/op and normal mice. The size of mandible, meanwhile, was significantly smaller in the op/op mice than in the normal controls. The gonial angle was also significantly larger in the op/op mice than in the normal mice throughout the experimental period. Thus, substantial differences in craniofacial growth were demonstrated in various areas of the craniofacial complex, which are assumed essentially due to the lack of osteoclastic bone resorption during growing period. Since the difference became more prominent in the anatomic regions relevant to the masticatory functions, it would be a reasonable assumption that reduced masticatory function is also a key determinant for the less-developed craniofacial skeleton in the op/op mouse.     

Facial skeletal growth in growing "toothless" osteopetrotic (op/op) mice: radiographic findings

The defective bone resorption in the osteopetrotic (op/op) mouse brings about failure of tooth eruption. Furthermore, the op/op mouse has been studied as a "toothless" mouse in recent morphological and physiological investigations of the relationship between mastication and masseter muscle development. The present study was conducted to examine in detail the nasal bone and the premaxillary bone in this mutant mouse and to assess the roles of incisor growth and the mechanical stress of mastication in nasal bone and premaxillary bone growth. The forms of the nasal bone and the premaxillary bone were observed using roentgenography in both toothless op/op and normal (control) mice. In the op/op mouse, the nasal bone and the premaxillary bone show remarkable deformity. In contrast, the normal mouse appears well developed. This suggests that growth of the incisor root is important to normal upper jaw growth in the mouse. Furthermore, it is proposed that the upper facial phenotype seen in the op/op mice results from not only decreased bone resorption, but also from absence of the mechanical stress provided by normal mastication.     

Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse.

Osteopetrosis (op/op) is a new mutation in the mouse that is transmitted as an autosomal recessive linked with variant waddler (Va) on chromosome 12. Compared with normal littermates, young op/op mice have excessive accumulations of bone without marrow cavities, increases in bone matrix formation and concentrations of parafollicular cells of the thyroid, and are hypophosphatemic. Osteoclasts from op/op mice are small, few in number and have an abnormal cytoplasmic distribution of the lysosomal enzyme acid phosphatase. In contrast to the three other mutations that transmit osteopetrosis in mice, the skeletal signs of the disease slowly disappear in op/op animals after bone matrix formation declines about 6 weeks after birth from 145 percent to 20 percent of that in normal siblings. The main skeletal defect in op/op mice appears to be a severe restriction in bone remodeling that is capable of slowly removing the excessive skeletal mass characteristic of the disease only after bone formation has declined to one-fifth that of normal littermates.     

Tibial dimensions before and during the recovery phase in the osteopetrotic mutant mouse.

Osteopetrosis is a genetic bone disease characterized by excessive bone mass and 'clubbing' of long bones. In the osteopetrotic (op) mouse, remission of the disease begins at 45 days of age. This study attempted to describe changes in the op tibia before and during remission. Osteopetrotic and normal littermates were killed at intervals from 10 to 120 days of age. Left tibiae were processed for transmission electron microscopy. Microradiographs of right tibiae were projected and drawn. Bone dimensions were compared between mutants and controls by ANOVA and bones were viewed in a scanning electron microscope. Differences between mutants and controls were: at all ages mutant tibiae were shorter than those of controls; 10-day distal shafts of mutant tibiae were significantly narrower; 30-day proximal shafts of mutant tibiae were wider, distal shafts were narrower, and there was no bone resorption along the external proximal metaphysis. At 48 days, resorption was seen along the proximal metaphyses of the mutant tibiae and by 60 days, extensive resorption areas were evident. However, proximal shafts of mutant tibiae were still significantly wider than those of controls. These results indicated that before remission there was an unequal deposition of bone on the mutant tibia. After remission, resorption occurred along the external proximal shaft, but was not enough to remove significant amounts of bone from the proximal metaphyses of mutant tibiae by 120 days of age.     

Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.     

Midpalatal suture of osteopetrotic (op/op) mice exhibits immature fusion.

The midpalatal suture was observed histologically in both toothless osteopetrotic (op/op) and normal (control) mice. The normal mice had a mature sutural structure, which consists of a well-developed cartilage cell zone and palatal bone. In contrast, the thickness of the cartilage cell zone was substantially greater in the op/op mice than that in the controls. Moreover, the cartilage cells in the op/op mice were frequently found in the palatal bone as well as in the sutural space, exhibiting an imperfect fusion. It seems that immature fusion at the sutural interface in the op/op mice is related to a decrease in biting or masticatory force accompanied by the failure of tooth eruption in addition to an essential defect in osteoclast differentiation, which is a congenital symptom in op/op mice.     

Osteoclast biology in the osteopetrotic (op) rat

Osteopetrosis is a metabolic bone disease characterized by reduced bone resorption. From experimental studies of various osteopetrotic mutations has emerged the hypothesis that each is unique with respect to mechanisms whereby osteoclast development and/or function are reduced. The osteopetrotic (op) mutation in the rat was discovered in Fatty/ORL stock over a decade ago. The paucity of data about osteoclast biology in this mutation prompted this study of cytological, cytochemical, and ultrastructural features of osteoclasts. In op rats, osteoclasts are significantly reduced in number, but are larger and more vacuolated than in normal littermates. Mutant osteoclasts can form ruffled borders and clear zones, but their ability to fragment and excavate bone surfaces is greatly impaired. Cytoplasmic vacuoles in op osteoclasts are randomly distributed and greatly enlarged, and they stain weakly for two cytochemical characteristics of osteoclasts, tartrate-resistant acid phosphatase and acid ATPase. These findings suggest that an abnormality in the lysosomal/vacuolar system, an important component of the resorptive mechanism, may be involved in the interception of osteoclast function in this mutation.     

Analysis of histochemically and morphometrically in the anterior belly digastric muscle of osteopetrotic (op/op) mice.

The fibers of the anterior belly digastric muscle of mice, fed a granulated diet for various periods, have been studied histochemically and morphometrically. The diameters of the anterior belly digastric fibers in normal mice fed only a granulated diet were smaller than those in mice fed a solid diet. Differences in the succinate dehydrogenase (SDH) activity of muscle fibers between op/op and normal mice gradually appeared in the anterior belly digastric muscle and, by the age of 90 days, under-development of muscle fibers was observed in the mild-belly region of the anterior belly digastric muscle of op/op mice fed a granulated diet. These results indicate mechanical stress in mastication plays an important role in the development of the anterior belly digastric muscle structures.     

A characterization of vitamin D-independent intestinal calcium absorption in the osteopetrotic (op/op) mouse.

Previous work in our laboratory showed that the osteopetrotic (op/op) mouse possesses a vitamin D-independent mechanism of intestinal calcium absorption. This study was performed in an effort to further characterize the mechanism. The vitamin D-deficient op/op mouse absorbed calcium faster than either a vitamin D-deficient or 1, 25-dihydroxyvitamin D(3)-supplemented wild-type mouse. This increased rate of absorption was not found at concentrations of calcium that result in diffusional calcium absorption. Thus, vitamin D-deficient op/op mice had intestinal calcium absorption similar to that of vitamin D-deficient wild-type littermates when increasing levels of calcium were administered. Also, mRNA and protein levels of calbindin-D9k were similar in vitamin D-deficient wild-type and op/op mice as well as in wild-type and op/op mice treated with 1, 25-dihydroxyvitamin D(3). Therefore, the mechanism of vitamin D-independent intestinal calcium absorption in the op/op mouse is distinct from vitamin D-dependent intestinal calcium absorption.     

A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility.

Correlative evidence suggests that maternal production of the mononuclear phagocyte growth factor colony stimulating factor-1 (CSF-1) regulates placental development. In order to study the role of CSF-1 in pregnancy the fertility of CSF-1-less osteopetrotic (op/op) mutant mice was investigated. Homozygous mutant crosses (op/op x op/op) were consistently infertile. As expected, op/op males were almost completely fertile when crossed with heterozygous females. Surprisingly, op/op females when mated to heterozygote males were fertile, although at a rate that was 46% of the rate for +/op females x op/op males. These data suggest that CSF-1 is required for pregnancy. However, a maternal CSF-1 source is not absolutely necessary in that pregnancies involving +/op fathers were partially rescued, suggesting that +/op fetuses and/or +/op seminal fluid provides CSF-1 or CSF-1-induced factors which compensate for the absence of maternally produced CSF-1. Despite the complete absence of CSF-1 in the uterus and placenta of op/op mice placental weights were normal, suggesting that proliferation of decidual cells and trophoblasts, both of which express the CSF-1 receptor, may not be solely regulated by CSF-1. Histochemical staining for F4/80 antigen was used to identify macrophages in the uterus and placenta. Uterine macrophages could not be detected in virgin op/op mice although they were abundant in +/op uteri. Interestingly, macrophages could be detected in op/op uteri as uncharacteristically rounded cells in early gestation, however, they were not maintained and no macrophages were apparent beyond Day 14 of pregnancy in op/op mice. Further studies in the osteopetrotic mouse will be useful in delineating those functions required for pregnancy that are regulated by CSF-1.     

Congenital osteoclast deficiency in osteopetrotic (op/op) mice is improved by ovariectomy and orchiectomy.

We examined the changes in the appearance of osteoclasts in the femora of ovariectomized (OVX) or orchiectomized (ORX) op/op mice. Osteoclasts on the trabecular bone surface of the OVX or ORX op/op mice significantly increased in number seven or eight times in comparison with sham-operated op/op mice. Furthermore, TRAP-positive cells increased about four times in 100-week-old females and males, compared with sham-operated groups. These results have indicated that a sex hormone reduction due to OVX or ORX induces prominent recruitment of osteoclasts in op/op mice.     

Changes in the Architecture of Cancellous Bone in the Femora of Developing Mice as a Result of Short Duration Hypergravity

SYNOPSIS. Mice were used as a model system to demonstrate theeffects of increased loading on the trabecular bone in the developingproximal femur. A two-dimensional free body diagram revealedthat because the femur is normally held in a horizontal positionduring support, functional loading resulted in a net posteriorbending moment and a muscle generated axial compressive loadthat was 7.7 times the normal support weight for that limb.Experimental treatment consisted of four times normal gravityfor 10 min, six times per day for 30 days in addition to unrestrainednormal exercise. Unrestrained normal exercise served as thecontrol. An animal whose foot was amputated by his mother shortlyafter birth was used as a less than normal load example forcomparison with the hypergravity and control groups. All groupswere treated for 30 days. Two areas of trabecular bone wereexamined: a proximal central webbing area that may be underpredominantly tensile loading much of the time, and a more distalarea near the third trochanter that presumably experiences bendingstresses when the animal is exposed to normal activity or hypergravity.Results indicate that: 1) The central webbing area appears tohave a similar mass of bone regardless of loading conditions;however, with increased loading the trabeculae become orientedin directions that approximate directions of presumed principaltensile stresses. 2) There is no genetically predetermined amountof bony tissue in the third trochanter area; when the limb isrelatively unloaded no trabeculae are present, as the loadingincreases trabecular bone is formed. In the hypergravity group,trabeculae in the area of the third trochanter increased inlength and complexity and became more centrally positioned inthe lumen of the femur, a position that presumably would allowthe femur to better resist bending moments. These results demonstratethat short duration, high intensity functional loading can stimulategrowth of trabecular bone, and further, specific types of functionalloading can be correlated with specific architectural changes.     

Op/op mice defective in production of functional colony-stimulating factor-1 lack macrophages in muscularis externa of the small intestine

The osteopetrotic (op/op) mutant mouse possesses an inactivating mutation in the colony-stimulating factor-1 (CSF-1) gene, which results in the absence of certain macrophages and in osteopetrosis, following a lack of osteoclasts. Studies of the op/op mouse indicate that CSF-1-dependent tissue macrophages may belong to a trophic and/or scavenger subpopulation, which through their effect on other cell types can significantly affect tissue functions, and that cells which are CSF-1 independent have antigen presentation and immunological functions.We have previously identified a cell system of regularly distributed macrophages in the muscularis externa of the small intestine and wanted to extend these studies to the op/op mouse.The present investigations with light- and electron-microscopic methods using fluorescent dextran, methylene blue and immunohistochemistry (F4/80, anti-kit receptor, anti-CD3, anti-CD45R/B220) show that macrophages are absent from the muscle layers, with only an occasional macrophage present in the subserosa. In the lamina propria and submucosa, macrophage numbers are reduced. In all other respects the muscularis externa appears normal, including normal organization and number of interstitial cells of Cajal. Control and op/op mice both lack cells expressing CD3 (T lymphocytes), CD45R/B220 (B lymphocytes) and mast cells in the muscularis externa. This leaves the muscularis externa macrophages as the most likely source of local cytokine production under such conditions as postoperative ileus and intussusception in infants, where the muscularis externa appears to be one target of cytokines. We conclude that the lack of macrophages, combined with the preservation of otherwise normal structure, will make the op/op mouse a valuable model by which to assess the functions and relative importance of the muscularis externa macrophages in relation to intestinal motility under normal and pathological conditions.     

Macrophage differentiation and granulomatous inflammation in osteopetrotic mice (op/op) defective in the production of CSF-1

Since the osteopetrotic (op/op) mouse was demonstrated to have a mutation within the coding region of the CSF-1 gene itself, it serves as a model for investigating the differentiation mechanism of macrophage populations in the absence of functional CSF-1. The op/op mice were severely monocytopenic and showed marked reduction and abnormal differentiation of tissue macrophages. Osteoclasts as well as marginal metallophilic macrophages and marginal zone macrophages in the spleen were absent. Most of the tissue macrophages were reduced in number and ultrastructurally immature. However, the degree of reduction in numbers of macrophages in the mutant mice was variable among tissues, suggesting that the heterogeneity of macrophages was generated by their different dependency on CSF-1. After daily CSF-1 injection, the numbers of monocytes, tissue macrophages, and osteoclasts were remarkably increased, and the macrophages showed morphological maturation. However, the numbers of macrophages in the ovary, uterus, and synovial membrane were not increased. In the bone marrow, macrophage precursors detected by monoclonal antibody ER-MP58 proliferated and differentiated into preosteoclasts and osteoclasts. In the spleen, marginal metallophilic macrophages and marginal zone macrophages developed slowly. In this manner, CSF-1 plays an important role in the development, proliferation, and differentiation of certain tissue macrophage populations and osteoclasts. In the op/op mice, Kupffer cells proliferated, transformed into epithelioid cells and multinucleated giant cells, and participated in glucan-induced granuloma formation. In CSF-1-treated op/op mice, the process of granuloma formation was similar to that in normal littermates due to increased monocytopoiesis and monocyte influx into the granulomas. These results indicate that CSF-1 is a potent inducer of the development and differentiation of CSF-1-dependent monocyte/macrophages, and that CSF-1-independent macrophages also play an important role in granuloma formation. Mol Reprod Dev 46:85–91, 1997. © 1997 Wiley Liss, Inc.     

Effects of Nutritional Deficiency of Boron on the Bones of the Appendicular Skeleton of Mice

Scientific evidence has shown the nutritional importance of boron (B) in the remodeling and repair of cancellous bone tissue. However, the effects of the nutritional deficiency of B on the cortical bone tissue of the appendicular skeleton have not yet been described. Thus, a study was performed to histomorphometrically evaluate the density of osteocyte lacunae of cortical bone of mouse femora under conditions of nutritional deficiency of B and to analyze the effects of the deficiency on the biomechanical properties of mouse tibiae. Weaning, 21-day-old male Swiss mice were assigned to the following two groups: controls (B+; n = 10) and experimental (B−; n = 10). Control mice were fed a basal diet containing 3 mg B/kg, whereas experimental mice were fed a B-deficient diet containing 0.07 mg B/kg for 9 weeks. The histological and histomorphometric evaluations of the mice fed a B-deficient diet showed a decrease in the density of osteocyte lacunae in the femoral cortical bone tissue and the evaluation of biomechanical properties showed lower bone rigidity in the tibia.

     

Susceptibility dynamics in neonatal BALB/c mice infected with Cryptosporidium parvum.

BALB/c Mice were infected as neonates and at different ages to study the susceptibility dynamics in this animal model to Cryptosporidium parvum. When 4-day-old animals were infected with 10(5) C. parvum oocysts, parasites were detected in the terminal ileum when the mice became 14-25 days old (10-21 days post-infection [PI]). The percentage of animals positive for parasites was 100% up to the age of 19 days (15 days PI) but decreased immediately thereafter until no parasites were detected in 26-day-old (22 days PI) or older mice. Parasite load also decreased in these animals from 184.7 parasites per high power field in 14-day-old animals (10 days PI) to 0.22 in 25-day-old (21 days PI) mice. In a second study, some neonatal mice became resistant to C. parvum when infection was attempted at day-10 of age (day-15 of age at sacrifice). The susceptibility to C. parvum decreased until 14 days of age (19 days of age at sacrifice) when mice could no longer be infected. Parasite load also decreased in infected mice from 235.6 parasites per high power field (9 days of age at sacrifice) to 0.25 (18 days of age at sacrifice).     

Effects of prolactin and bromocryptine on the regulation of testicular luteinizing hormone receptors in mice

The binding of luteinizing hormone (LH) to testicular homogenates increased gradually in mice from 15 to 60 days of age, while the level at 90 days was almost the same as that at 60 days. The plasma concentration of prolactin (PRL) increased significantly from 15 to 40 days and thereafter remained constant. In order to ascertain the influence of PRL on testicular receptors for LH, bromocryptine was injected subcutaneously for 10 days into immature (20-day-old) and adult (90-day-old) mice. In 20-day-old mice, treatment with bromocryptine significantly reduced the plasma levels of PRL but had no significant effects on the binding of LH to receptors in 30-day-old mice. However, in 90-day-old mice, treatment with bromocryptine led to a significant reduction in numbers of receptors for LH 10 days later. There was no difference in dissociation constants (Kd) between groups of oil-injected (Kd = 6.5 x 10(-10) M) and bromocryptine-injected (Kd = 4.6 x 10(-10) M) mice. The reduction in binding of LH per testis of 100-day-old mice after treatment with bromocryptine was eliminated by the simultaneous administration of ovine (o) PRL. The plasma level of follicle-stimulating hormone (FSH) in 100-day-old mice, which tended to be decreased as a result of treatment with bromocryptine, was markedly increased by treatment with oPRL. There were no distinct changes in binding of FSH in any of the groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical properties in long bones of rat osteopetrotic mutations.

Osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. Experimental studies with rat osteopetrotic mutations have shown increased bone density and decreased bone strength. However, it is not known if this reduction in bone strength is only due to changes in structure and geometry or if the tissue properties of bone material itself are changed as well. We have evaluated bone tissue properties with nanoindentation in three osteopetrotic mutations in the rat (incisors-absent ia/ia, osteopetrosis op/op and toothless tl/tl) to test the hypothesis that reduced bone resorption in these mutations results in reduced tissue properties of bone material. No significant differences in elastic modulus or hardness were found between osteopetrotic mutants and their normal littermates (NLMs) in any of the three stocks. This indicates that the tissue properties of bone material are not changed significantly in osteopetrosis, even if the mechanical strength is decreased at the macroscopic level.