Matthew J. Cielinski

The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption.

The toothless (tl) rat is an osteopetrotic mutation characterized by a generalized skeletal sclerosis, reduced bone resorption, few osteoclasts and a total absence of erupted teeth. This mutation is not cured by bone marrow transplants from normal littermates. It is known that the skeletal defects in tl rats are greatly improved after treatment with colony-stimulating factor-1 (CSF-1). This investigation concerns the effects of CSF-1 on the development and eruption of the dentition of tl rats. Untreated tl rats had no erupted teeth by 56 days after birth, and the roots of incisors and molars were severely distorted by compression against bone. The apex of the mandibular incisor did not extend past the first molar and continued growth of its apical end produced odontoma-like masses consisting of distorted dentine and enamel matrices. In addition, few osteoclasts were seen on alveolar bone surfaces surrounding the developing teeth. Mutants given CSF-1 were characterized by delayed eruption of all molars and sometimes incisors. The incidence of incisor eruption was related inversely to the age at which CSF-1 treatment began. Molars of treated tl rats had well-developed roots similar to those in normal rats. Treated mutants had numerous osteoclasts in alveolar bone and well-developed haemopoietic marrow spaces in the mandible. Histochemical staining for both tartrate-resistant acid phosphatase and tartrate-resistant acid ATPase was reduced or negligible in osteoclasts of untreated tl rats, heavy in normal osteoclasts and of intermediate intensity in CSF-1-treated mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental studies of osteopetrosis in laboratory animals

Osteopetrosis is a metabolic bone disease characterized by a systemic increase in skeletal mass. It results from a defect in the production or function of osteoclasts and is inherited in nine genetically distinct osteopetrotic animal mutations and man. Studies of these mutations have revealed that osteopetrosis is a complex, heterogeneous disorder in its expression, etiology, and response to treatment by bone marrow transplantation or by hormone/growth factor therapy. These animal mutations have been valuable tools for probing the pathogenesis and treatment of osteopetrosis, and information obtained from these studies has been used clinically for the treatment of humans with osteopetrosis. In addition, studies of these mutations have contributed significantly to understanding normal bone cell biology, including the origin of the osteoclast and the significance of colony-stimulating factor-1 in osteoclast development. The resistance of some of these mutations to cure by stem cell transplantation and hormone therapy, coupled with similar observations and experiences in the human condition, indicates that these animal mutations will continue to serve important roles in the development of alternative therapies to treat resistant forms of the disease. These studies are bound to improve the understanding of normal bone biology by providing additional insights into the regulation of osteoclasts by osteoblasts and their products or by other elements of the skeletal microenvironment.

The skeletal effects of colony-stimulating factor-1 in toothless (osteopetrotic) rats: persistent metaphyseal sclerosis and the failure to restore subepiphyseal osteoclasts

Toothless (tl), one of four osteopetrotic mutations in the rat, is characterized by few osteoclasts, undetectable bone resorption, and failure of correction by bone marrow transplantation. We recently reported that CSF-1 treatment improves these skeletal problems but that metaphyseal sclerosis persists. In the present study we demonstrate that optimal reduction of the skeletal sclerosis in tl rats following CSF-1 treatment has lower and upper dosage thresholds and that skeletal sclerosis returns after CSF-1 withdrawal. Osteoclasts increase significantly in tl rats after CSF-1 treatment, but compared to untreated normal littermates, histochemical staining for characteristic enzymes and osteoclast number is reduced and no osteoclasts appear in the subepiphyseal areas of long bones. These data are interpreted to mean that there are dosage limits to the beneficial skeletal effects of CSF-1, that persistent sclerosis is related to the failure to restore subepiphyseal osteoclasts, that osteoclast or progenitor populations may be deficient or differ in their responses to CSF-1, and that the defect in tl rats is not merely lack of a circulating, biologically active form of CSF-1.

Bone surface morphology reflects local skeletal metabolism

The metabolic activity of bone cells is faithfully reflected in the surface topography of mineralized bone surfaces, and this can be easily detected by scanning electron microscopy (SEM). Forming bone surfaces exhibit knobby projections which represent foci of mineralization, resorbing surfaces are scalloped, and resting surfaces undergoing neither activity are smooth, as shown by Boyde and Hobdell 25 years ago. These phenomena are illustrated in vivo by tooth eruption, a local activity in alveolar bone where resorption and formation are polarized around an erupting tooth, and osteopetrosis, a metabolic bone disease characterized by a congenital reduction or absence of bone resorption. The ability to analyze bone metabolism over large areas of the skeleton by SEM offers a convenient and powerful microscopic technique to assess regional and global bone cell activity in an era where the investigative focus is increasingly molecular.

The contrasting effects of colony-stimulating factor-1 and epidermal growth factor on tooth eruption in the rat

Both epidermal growth factor (EGF) and colony-stimulating factor-1 (CSF-1) have been shown to accelerate eruption of teeth in rodents. We compared the effects of neonatal injections of EGF (1 micrograms/g body weight) and CSF-1 (10(6) units) alone or together on the eruption of incisors and first molars. EGF accelerated the eruption of incisors with no significant effect on first molars. CSF-1, in contrast, accelerated molar eruption more than incisor eruption. CSF-1, but not EGF, increased the numbers of mononuclear cells in the dental follicle and osteoclasts on adjacent alveolar bone surfaces around the first molar and produced enhanced resorption of crypt surfaces as revealed by scanning electron microscopy. These data suggest that during eruption rodent incisors and molars may preferentially respond to different molecular regulators.

Bone metabolism in the osteopetrotic rat mutation microphthalmia blanc

We have examined parameters of bone metabolism in a new mutation, microphthalmia blanc (mib), in the rat exhibiting a skeletal sclerosis at birth that improves with age. There were no significant differences in the rate of bone formation during the first postnatal month except a temporary reduction in mutants at 3 weeks that coincided with compromised nutrition at weaning. At birth the ruffled border in mutant osteoclasts was absent or poorly developed and mRNA analyses of mutant bone compared to normal bone showed significant reductions in the messages for the osteoclast-specific genes carbonic andydrase II and tartrate-resistant ATPase. These distinctive ultrastructural and molecular differences were not present 1 month later. These data show that the transient osteopetrosis in mib rats results from a perinatal reduction in ultrastructural and enzymatic features of active osteoclasts and is not complicated by elevations in bone formation. The molecular basis for both the production and resolution of these abnormalities deserves further study.

The effects of colony-stimulating factor-1 on the number and ultrastructure of osteoclasts in toothless (tl) rats and osteopetrotic (op) mice

The role of colony-stimulating factor-1 (CSF-1 or M-CSF) in osteoclast development is illustrated by observations that administration of exogenous CSF-1 increases osteoclast number and improves the skeletal sclerosis of two osteopetrotic mutations, toothless (tl) in the rat and osteopetrotic (op) in the mouse. We examined the effects of CSF-1 treatment on the number and ultrastructure of osteoclasts in the tibial metaphysis of normal and mutant animals of both stocks to understand the similarities and differences between these two mutations. Osteoclasts from normal animals of both stocks were abundant and possessed the ultrastructural features of active cells. These included apical areas in contact with mineralized surfaces with tightly apposed clear zones, extensive ruffled borders, and a vacuolated cytoplasm with numerous mitochondria. In toothless rats osteoclasts were difficult to locate and those present had poorly defined ruffled borders, fewer cytoplasmic vacuoles, and a basal membrane with both smooth and ruffled areas. Large lipid accumulations were often found near tl osteoclasts. Osteoclasts in op mice were difficult to find, but more numerous than in tl rats. Unlike tl osteoclasts, those of op mice possessed very well developed ruffled borders, small clear zones, and large electron-dense cytoplasmic inclusions. These cells also had unusual basal membranes with both smooth and ruffled regions. CSF-1 treatment increased the number of osteoclasts in both mutant stocks, normalizing the numbers in op mice, but not tl rats. CSF-1 injections caused dramatic changes in the morphology of tl osteoclasts, including increased incidence and size of ruffled borders and cytoplasmic vacuolization. The growth factor had little effect on ruffled borders or clear zones in op mice. Interestingly, mutant osteoclasts of both stocks exhibited a ruffled basal membrane in response to CSF-1 treatment. This increase in membrane ruffling may reflect the ability of CSF-1 to promote rapid formation of osteoclasts from mononuclear precursors in a more permissive microenvironment. Our data indicate that CSF-1 is not required for the development of at least some osteoclasts. The differences in response to CSF-1 treatment which we report lead us to speculate that additional factors may be involved in osteoclastogenesis.

Dental abnormalities in the osteopetrotic rat mutation microphthalmia blanc

Dental manifestations of the mild, transient osteopetrosis in the rat mutation microphthalmia blanc (mib) were examined. Eruption of all teeth was delayed in mib rats compared to normal littermates. The delays ranged from 5 days for incisors to 3 and 2 days for the first and second molars. Normal rats had straight incisors in the sagittal plane that exhibited signs of wear, but in mib littermates the incisors were maloccluded, distorted, and showed no signs of wear. Radiographic and histological examination of the dentition of 1- and 4-week-old rats revealed that the apical end of incisors in mib rats failed to extend posteriorly to the third molar region as in normal siblings, but ended at the first molar. Histological examination of longitudinal sections of mandibles through the incisors of neonatal normal and mib rats showed that in 1-day-old mutants the incisor was closely surrounded by alveolar bone to which it was ankylosed. The incisor body in mib rats was also malformed, with an indented apical end. This ankylosis was temporary, being resolved by 3 days. These findings show that neonatal reductions in bone resorption cause incisor defects and delay the eruption of all teeth in mib rats. The malocclusion and distortion of incisors of mib rats are likely caused by temporary ankylosis of incisor matrices to alveolar bone. Taken together, these findings illustrate the concept that bone resorption is an essential and rate-limiting element of tooth eruption.