Dental Abnormalities Associated with Failure of Tooth Eruption in src Knockout and op/op Mice

c-src knockout and op/op mice develop osteopetrosis as a result of defective osteoclast function and osteoclast formation, respectively. The mutant mice can be distinguished readily from their wild-type littermates around 10–12 days after birth because their incisors do not erupt, but the morphology of their teeth and surrounding bone has not been reported previously in detail. Histologic examination of jaws of src-mutant mice reveals unerupted, abnormal incisors within their bony crypts. The tooth roots are distorted by foci of haphazard proliferation of odontogenic epithelium associated with primitive tooth structures that strongly resemble the tumor-like lesions in humans, known as odontomas. The crowns of the incisors are fused to the adjacent bone, and the developing periodontal ligament is disordered and hypocellular. Osteoclasts are present in the bone surrounding the distorted teeth, but as in other bones in these mice they lack ruffled borders and thus do not resorb effectively. Similar odontogenic proliferation is present around unerupted incisors in op/op mice which form very few osteoclasts, but the amount is significantly less than in src mutant mice. Molars fail to erupt in both types of mutant mice, but they are not accompanied by aberrant odontogenic proliferation. These findings and previous reports of similar abnormalities in jaws from op/op rats suggest that failure of incisor eruption and associated proliferation of odontogenic epithelium in osteopetrotic rodents are a direct result of defective osteoclastic bone resorption. Received: 6 August 1998 / Accepted: 23 December 1998     

Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents

We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.     

Administration of colony stimulating factor-1 corrects some macrophage, dental, and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat.

The toothless (tl/tl) mutation in the rat results in a paucity of osteoclasts and osteopetrosis that cannot be corrected by bone marrow transplantation. In the present study we demonstrate that tl/tl rats also have profound deficiencies of femoral, peritoneal, and pleural cavity macrophages. Furthermore, the macrophage colony stimulating activity of post-endotoxin sera from tl/tl rats is substantially reduced, suggesting that, as in the case of the op mutation in mice, the basis of the tl mutation is a deficiency of the macrophage growth factor, colony stimulating factor-1 (CSF-1). Consistent with this suggestion, treatment of tl/tl rats from birth for up to six weeks with CSF-1 reduced the osteopetrosis, increased body weight, and permitted tooth eruption. In addition, CSF-1 treatment induced large numbers of osteoclasts in tl/tl bones and macrophages in the peritoneal cavity and bone marrow. Persistence of metaphyseal sclerosis, however, indicated that the disease was not totally corrected by this treatment. These studies indicate that the basis of the tl mutation is most likely another CSF-1 deficiency, and further emphasize the role of this growth factor in osteoclast differentiation.     

Skeletal consequences of RANKL-blocking antibody (IK22-5) injections during growth: Mouse strain disparities and synergic effect with zoledronic acid

High doses of bone resorption inhibitors are currently under evaluation in pediatric oncology. Previous works have evidenced transient arrest in long bone and skull bone growth and tooth eruption blockage when mice were treated with zoledronic acid (ZOL). The question of potential similar effects with a RANKL-blocking antibody (IK22.5) was raised. Sensitivity disparities in these inhibitors between mouse strains and synergic effects of zoledronic acid and a RANKL-blocking antibody were subsidiary questions. In order to answer these questions, newborn C57BL/6J and CD1 mice were injected every two or three days (4 injections in total so 7 or 10 days of treatment length) with high doses of a RANKL-blocking antibody. The consequences on the tibia, craniofacial bones and teeth were analyzed by μCT and histology at the end of the treatment and one, two and three months later. The results obtained showed that RANKL-blocking antibody injections induced a transient arrest of tibia and skull bone growth and an irreversible blockage of tooth eruption in C57BL/6J mice. In CD1 mice, tooth eruption defects were also present but only at much higher doses. Similar mouse strain differences were obtained with zoledronic acid. Finally, a synergic effect of the two inhibitors was evidenced. In conclusion as previously observed for bisphosphonates (ZOL), a RANKL-blocking antibody induced a transient arrest in long bone and skull bone growth and a blockage of tooth eruption with however disparities between mouse strains with regard to this last effect. A synergic effect of both bone resorption inhibitors was also demonstrated.     

Histochemical examination of osteoblastic activity in op/op mice with or without injection of recombinant M-CSF

Osteopetrotic (op/op) mice do not exhibit bone remodeling because of defective osteoclast formation caused by the depletion of macrophage colony-stimulating factor (M-CSF). In the present study, we investigated tibial bones of op/op mice with or without prior injections of M-CSF to determine whether osteoclast formation and subsequent bone resorption could activate osteoblasts, which is known as a "coupling" phenomenon. In op/op mice, no osteoclasts were present, but the metaphyseal osteoblasts adjacent to the growth plate cartilage seemed to be active, revealing an intense alkaline phosphatase (ALPase) immunoreactivity. Consequently, primary trabecular bones were extended continuously to the diaphysis, indicating that bone modeling is well achieved in op/op mice. In contrast with the metaphysis, most of the diaphyseal osteoblasts were flattened and showed weak ALPase activity, and, as a result, they seemed to be less active. Osteopontin (OPN) was localized slightly at the interface between bone and cartilage matrices of the primary trabeculae. In contrast, in op/op mice injected with M-CSF, tartrate-resistant acid phosphatase-positive osteoclasts appeared, resorbing trabecular bones of the diaphyseal region. The diaphyseal osteoblasts in the vicinity of the active osteoclasts were cuboidal and exhibited strong ALPase immunoreactivity. OPN was observed not only at the bone-cartilage interface, but also significantly on the resorption lacunae beneath the bone-resorbing osteoclasts. These observations indicate that the activation of diaphyseal osteoblasts appears to be coupled with osteoclast formation and subsequent osteoclastic bone resorption. Alternatively, the metaphyseal osteoblasts at the chondro-osseous junction seemed to be less affected by osteoclastic activity.     

Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects

CSF-1, a key regulator of mononuclear phagocyte production, is highly expressed in the skeleton by osteoblasts/osteocytes and in a number of nonskeletal tissues such as uterus, kidney and brain. The spontaneous mutant op/op mouse has been the conventional model of CSF-1 deficiency and exhibits a pleiotropic phenotype characterized by osteopetrosis, and defects in hematopoiesis, fertility and neural function. Studies to further delineate the biologic effect of CSF-1 within various tissues have been hampered by the lack of suitable models. To address this issue, we generated CSF-1 floxed/floxed mice and demonstrate that Cre-mediated recombination using Meox2Cre, a Cre line expressed in epiblast during early embryogenesis, results in mice with ubiquitous CSF-1 deficiency (CSF-1KO). Homozygous CSF-1KO mice lacked CSF-1 in all tissues and displayed, in part, a similar phenotype to op/op mice that included: failure of tooth eruption, osteopetrosis, reduced macrophage densities in reproductive and other organs and altered hematopoiesis with decreased marrow cellularity, circulating monocytes and B cell lymphopoiesis. In contrast to op/op mice, CSF-1KO mice showed elevated circulating and splenic T cells. A striking feature in CSF-1KO mice was defective osteocyte maturation, bone mineralization and osteocyte-lacunar system that was associated with reduced dentin matrix protein 1 (DMP1) expression in osteocytes. CSF-1KO mice also showed a dramatic reduction in osteomacs along the endosteal surface that may have contributed to the hematopoietic and cortical bone defects. Thus, our findings show that ubiquitous CSF-1 gene deletion using a Cre-based system recapitulates the expected osteopetrotic phenotype. Moreover, results point to a novel link between CSF-1 and osteocyte survival/function that is essential for maintaining bone mass and strength during skeletal development.     

Administration of high-dose macrophage colony-stimulating factor increases bone turnover and trabecular volume fraction

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that plays a critical role in early osteoclastogenesis. To characterize the skeletal effects of M-CSF, we administered soluble M-CSF to mice. It was hypothesized that M-CSF would stimulate bone formation through coupled activity of osteoclasts and osteoblasts. Twenty-four male C57BL/6 J mice (n = 12/group, aged 7 weeks) received subcutaneous injections of human M-CSF [5 mg/(kg day)] or inert vehicle (VEH) for 21 days. M-CSF increased serum bone turnover markers (+57% TRAP-5b and +44% osteocalcin). Microcomputed tomography revealed an anabolic effect on tibial trabecular bone, with higher bone volume fraction (+35%), connectivity density (+79%), and number (+18%), as well as lower trabecular separation (−18%). M-CSF had no significant effect on cortical bone mineral content, geometry, or strength. There was no change in quantitative histomorphometry parameters of femoral cortical bone. These results reveal the complex, site-specific effects of M-CSF. In particular, we have demonstrated an anabolic effect of M-CSF on trabecular bone achieved through coupled activation of osteoblasts. However, in contrast to previous studies, M-CSF was found to have no effect on cortical bone. M-CSF was demonstrated to significantly influence both bone modeling and remodeling in relatively young animals.     

The osteopetrotic rabbit: general and skeletal features of a new outbred stock

Stock of the lethal osteopetrotic mutation in the rabbit has been outbred to robust New Zealand White stock. Features of this new stock include skeletal sclerosis and failure of tooth eruption characteristic of osteopetrosis, together with hypocalcemia, hypophosphatemia, and osteoclasts of reduced numbers and abnormal cytology. No abnormalities of carbonic anhydrase II were detectable by electrophoresis. The commercial availability of mutants of this new stock makes them a potentially important source of information about both osteopetrosis and osteoclast biology.     

Committed osteoclast precursors colonize the bone and improve the phenotype of a mouse model of autosomal recessive osteopetrosis

Osteopetrosis is a genetic disease characterized by defective osteoclasts. Autosomal recessive osteopetrosis is fatal within the first years of life. Hematopoietic stem cell transplantation (HSCT) cures fewer than 50% of cases but often leaves severe neurologic damages and other dysfunctions. Osteoclast appearance after HSCT is a slow process, during which disease progression continues. We hypothesize that a support osteoclast precursor therapy may contribute to improve the osteopetrotic phenotype. To this end, we established a procedure to obtain the best yield of osteoclast precursors from human peripheral blood or mouse bone marrow mononuclear cells. These cells were injected in vivo in animal models, testing different cell injection protocols, as well as in association with CD117+ stem cells. Injected cells showed the ability to form multinucleated osteoclasts and to improve the phenotype of oc/oc osteopetrotic mice. In the best working protocol, animals presented with longer survival, improved weight and longitudinal growth, increased tibial length, tooth eruption, decreased bone volume, reduced bone marrow fibrosis, and improved hematopoiesis compared with sham‐treated mice. These results provide first‐hand information on the feasibility of a support osteoclast precursor therapy in osteopetrosis. © 2010 American Society for Bone and Mineral Research     

CD4+ T cell recognition of a single discordant HLA-A2-transgenic molecule through the indirect antigen presentation pathway induces acute rejection of murine cardiac allografts.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.     

Developmentally regulated monocyte recruitment and bone resorption are modulated by functional deletion of the monocytic chemoattractant protein-1 gene

Tooth eruption involves the movement of a tooth from its site of development within the alveolar bone to its functional position in the oral cavity. Because this process is dependent upon monocytes and formation of osteoclasts, it represents an excellent model for examination of these processes under developmental regulation. We investigated the functional role of monocyte chemoattractant protein-1 (MCP-1) in monocyte recruitment and its impact on bone resorption by examining each parameter in MCP-1(-/-) mice as compared with wild-type controls during tooth eruption. The peak number of monocytes occurred on day 5 in the MCP-1(-/-) mice and on day 9 in the wild-type mice. The peak number of osteoclasts followed the same pattern, occurring sooner in the MCP-1(-/-) (day 5) than in wild-type mice (day 9). Consistent with this, MCP-1(-/-) mice had an accelerated rate of tooth eruption in the early phase when the teeth first entered the oral cavity as compared with the wild-type mice. However, there was accelerated eruption in the wild-type group in the later phase of tooth eruption. When examined at the molecular level, inducible nitric oxide synthase (iNOS) and interleukin-11 and -6 were expressed at considerably higher levels in the experimental group with accelerated tooth eruption. This is the first report identifying these factors as potential modulators of bone resorption that can accelerate the rate of tooth eruption. We conclude that, at early timepoints, monocyte recruitment occurs by MCP-1-independent mechanisms. However, at a later timepoint, MCP-1 may play a contributory role in the recruitment of monocytic cells, allowing the wild-type animals to catch up.     

Immunohistochemical identification of proteoglycan form of macrophage colony-stimulating factor on bone surface

Several studies using op/op mice have shown that macrophage colony-stimulating factor (M-CSF) was necessary for osteoclast formation in vivo. Previously we reported that osteoblastic cells produced two molecular forms of M-CSF; one is an 85-kDa M-CSF, and the other is a proteoglycan form of M-CSF (PG-M-CSF) which has a binding affinity for bone-derived collagens and is extractable from human bone. In this study, we performed immunostaining of human bone using a newly established anti-PG-M-CSF antibody, and showed positive staining PG-M-CSF, probably produced by bone lining cells, on the bone surface. This observation suggests that the bone surface is suitable for osteoclast formation because of the presence of PG-M-CSF.     

Establishment and characterization of new osteoclast progenitor cell lines derived from osteopetrotic and wild type mice

Malignant infantile osteopetrosis is a rare and lethal disease characterized by the absence of bone resorption due to inactive osteoclasts (OCLs). Among the murine models of osteopetrosis, the Tcirg1oc/oc mouse is the most resembling to the human pathology. In the majority of patients as in Tcirg1oc/oc mouse, the gene involved is the Tcirg1 gene, encoding the a3 subunit of the vacuolar proton pump. However, to date, no osteoclastic cell lines from osteopetrotic mice are available to facilitate the study of either OCL differentiation in osteopetrosis or the factors involved in the control of Tcirg1 gene expression. Heterozygotes Tcirg1+/oc mice were crossed with p53+/- mice to obtain homozygotes p53-/-Tcirg1oc/oc and p53-/-Tcirg1+/+ animals. The p53-/-Tcirg1oc/oc mice display the same bone and hematological phenotype as the original Tcirg1oc/oc mice. From the bone marrow of these mice, we have derived cell lines named POC-MGoc/oc and POC-MG+/+. These cell lines express standard osteoclastogenic markers and differentiate into OCLs in the presence of RANK-L and M-CSF. Furthermore, both cell lines can be transduced by a lentiviral vector with a high efficiency and without alteration of their OCL differentiation potential. Therefore, these cell lines provide valuable new tools to study the differentiation and function of osteoclasts in normal and resorption defective conditions.     

Tumor necrosis factor modulates apoptosis of monocytes in areas of developmentally regulated bone remodeling.

Tooth eruption is characterized by spatially segregated bone resorption along the path of eruption and bone formation in the opposite direction. Monocyte recruitment occurs in two distinct peaks in both areas of resorption and formation. Without such recruitment tooth eruption does not occur. The signals that regulate this recruitment are thought to involve the expression of cytokines and chemokines. One such cytokine is tumor necrosis factor (TNF), which can affect monocyte recruitment through the induction of chemokines and adhesion molecules and increase their lifespan by acting as antiapoptotic cell survival signals. We examined the latter by studying mice with targeted deletions of TNF receptors p55 and p75 (TNFRp55/p75). The results indicate that mice that lack functional TNF receptors have a significantly reduced number of monocytes in the apical area associated with bone formation. The reduced number of monocytes in this area can be accounted for by an increase in apoptosis in TNFRp55-/-/p75-/-. In contrast, the number of monocytes, the rate of monocyte apoptosis, and the formation of osteoclasts in the occlusal area associated with bone resorption occurred independently of TNF activity. These results suggest that TNF receptor signaling can affect tooth eruption by acting as a monocyte survival signal in some but not all areas of bone undergoing developmentally regulated remodeling.     

Murine ameloblasts are immunonegative for Tcirg1, the v-H-ATPase subunit essential for the osteoclast plasma proton pump

Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator 1 (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification.     

Limited rescue of osteoclast-poor osteopetrosis after successful engraftment by cord blood from an unrelated donor.

We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation. INTRODUCTION: Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted. MATERIALS AND METHODS: Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. RESULTS: Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [alphavbeta3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis. CONCLUSIONS: This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.     

Accelerated bone formation and increased osteoblast number contribute to the abnormal tooth germ development in parathyroid hormone-related protein knockout mice

Our previous study showed that tooth germs at late embryonic stage [later than embryonic day 17.5 (E17.5)] and neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are compressed or penetrated by the surrounding alveolar bone tissue. In vivo and in vitro studies have shown that the development of the tooth germ proper is not disturbed, but insufficient alveolar bone resorption, due to the decreased number and hypofunction of osteoclasts, is the main cause of this abnormality. In addition to the insufficient alveolar bone resorption, progressive bone formation toward tooth germs was observed in homozygous mice, suggesting that accelerated bone formation also contributes to this abnormality. To further investigate this, homozygous mice at E14.0 and E15.5, when alveolar bone is forming, were used for histochemical and bone histomorphometric analyses. In contrast to the late embryonic stage, the alveolar bone did not yet compress developing tooth germs in homozygous mice on E14.0, but a larger amount of bone tissue was seen compared to wild-type littermates. Histomorphometric analysis of bone at E14.0 revealed that the osteoblast numbers and surfaces in the mandibles and in the bone collar of femora of homozygous mice were significantly higher than those of wild-type mice. However, unlike our previous study showing the osteoclast surface on E18.5 in homozygous mice to be significantly lower than that of wild-type mice, this study at E14.0 showed no significant difference between the two genotypes. To evaluate the amount of calcification around tooth germs, 3D images of mandibles were reconstructed from the calcein-labeled sections of the wild-type and mutant mice. Labeling was performed at E14.0, and the mice were sacrificed 1 h after the calcein injection to minimize the effect of bone resorption. Comparison of the 3D images revealed that the labeled surface was larger around developing tooth germs in homozygous mouse than in wild-type mouse. On day E15.5, osteoblasts approached the enamel organ of homozygous mice but this was not observed in wild-type mice. In this study, we report a systemic increase in osteoblast number and accelerated bone formation in homozygous PTHrP-knockout mice, both of which contribute to the abnormal tooth development.