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Osteopetrosis is a rare inherited disease caused by osteoclast failure, resulting in increasing bone density in humans. Patients with osteopetrosis possess several dental and cranial complications. Since carbonic anhydrase II (CA-II) deficiency is a major cause of osteopetrosis, CA-II activators might be an attractive potential treatment option for osteopetrosis patients. We conducted comprehensive label-free quantitative proteomics analysis on Fluconazole-treated Dental Pulp Mesenchymal Stem/Stromal Cells from CA-II-Deficient Osteopetrosis Patients. We identified 251 distinct differentially expressed proteins between healthy subjects, as well as untreated and azole-treated derived cells from osteopetrosis patients. Twenty-six (26) of these proteins were closely associated with osteogenesis and osteopetrosis disease. Among them are ATP1A2, CPOX, Ap2 alpha, RAP1B and some members of the RAB protein family. Others include AnnexinA1, 5, PYGL, OSTF1 and PGAM4, all interacting with OSTM1 in the catalytic reactions of HCO3 and the Cl- channel via CAII regulation. In addition, the pro-inflammatory/osteoclast regulatory proteins RACK1, MTSE, STING1, S100A13, ECE1 and TRIM10 are involved. We have identified proteins involved in osteogenic and immune metabolic pathways, including ERK 1/2, phosphatase and ATPase, which opens the door for some CA activators to be used as an alternative drug therapy for osteopetrosis patients. These findings propose that fluconazole might be a potential treatment agent for CAII- deficient OP patients. Altogether, our findings provide a basis for further work to elucidate the clinical utility of azole, a CA activator, as a therapeutic for OP.

Autosomal Dominant Osteopetrosis type II (ADO2) is a bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We created mouse models of ADO2 by introducing a knock-in (p.G213R) mutation in the Clcn7 gene, which is analogous to one of the common mutations (G215R) found in humans. The mutation leads to severe osteopetrosis and lethality in homozygous mice but produces substantial phenotypic variability in heterozygous mice on different genetic backgrounds that phenocopy the human disease of ADO2. ADO2 is an osteoclast-intrinsic disease, and lysosomal enzymes and proteins are critical for osteoclast activity. Chloroquine (CQ) is known to affect lysosomal trafficking, intracellular signaling and the lysosomal and vesicular pH, suggesting it might improve ADO2 osteoclast function. We tested this hypothesis in cell culture studies using osteoclasts derived from wild-type (WT or ADO2 +/+ ) and ADO2 heterozygous (ADO2 +/- ) mice and found that CQ and its metabolite desethylchloroquine (DCQ), significantly increased ADO2 +/- osteoclasts bone resorption activity in vitro, whereas bone resorption of ADO2 +/+ osteoclasts was increased only by DCQ. In addition, we exploited our unique animal model of ADO2 on 129 background to identify the effect of CQ for the treatment of ADO2. Female ADO2 mice at 8 weeks of age were treated with 5 doses of CQ (1, 2.5, 5, 7.5 and 10 mg/kg BW/day) via drinking water for 6 months. Bone mineral density and bone micro-architecture were analyzed by longitudinal in vivo DXA and micro-CT at baseline, 3 and 6 months. Serum bone biomarkers (CTX, TRAP and P1NP) were also analyzed at these time points. CQ treatment at the doses tested failed to produce any significant changes of aBMD, BMC (whole body, femur and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group (water only). Further, levels of bone biomarkers were not significantly changed due to CQ treatment in these mice. Our findings indicate that while CQ increased osteoclast activity in vitro, it did not improve the osteopetrotic bone phenotypes in ADO2 heterozygous mice., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bone structure and function are maintained by well-regulated bone metabolism and remodeling. Although the underlying molecular and cellular mechanisms are now being understood, physiological and pathological states of bone are still difficult to predict due to the complexity of intercellular signaling. We have now developed a novel in silico experimental platform, V-Bone, to integratively explore bone remodeling by linking complex microscopic molecular/cellular interactions to macroscopic tissue/organ adaptations. Mechano-biochemical couplings modeled in V-Bone relate bone adaptation to mechanical loading and reproduce metabolic bone diseases such as osteoporosis and osteopetrosis. V-Bone also enables in silico perturbation on a specific signaling molecule to observe bone metabolic dynamics over time. We also demonstrate that this platform provides a powerful way to predict in silico therapeutic effects of drugs against metabolic bone diseases. We anticipate that these in silico experiments will substantially accelerate research into bone metabolism and remodeling., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

In autosomal dominant osteopetrosis type 2 (ADO2) CLCN7 mutations cause impaired osteoclast function. Severe consequences include skeletal fragility despite high bone mass, osteomyelitis, osteonecrosis, bone marrow failure, and severe cranial nerve impingement. There is no effective medical treatment for ADO2. We recruited subjects with ADO2 into a 14-week, open-label, pilot clinical trial of interferon gamma-1b. Doses were titrated based on tolerability and if fasting serum C-telopeptide (CTX) was <25% above baseline at week 8, targeting doses of 100 µg/m 2 three times a week. The primary outcomes were change from baseline in CTX and N-telopeptide/creatinine ratio (NTX/Cr) at week 14. Secondary outcomes included changes in urine calcium/creatinine ratio, bone formation markers and tolerability. Nine adults and three children were recruited. Severe manifestations of ADO2 included histories of fractures (100%), osteomyelitis (16.7%), vision loss (50%), and anemia (58.3%). Baseline CTX and NTX/Cr were generally low-normal. Procollagen type I N-terminal propeptide was elevated or in the upper-normal range in 11 of 12 (91.6%) subjects. Elevations of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were common. One subject withdrew due to rash. Five subjects achieved doses of 50 µg/m 2 3 days a week, while six reached the full dose of 100 µg/m 2 3 days a week. Only 3 of 11 (27.3%) completing subjects achieved the primary outcome of increasing CTX ≥25% above baseline at week 14. The mean ± SD change from baseline in CTX at week 14 was +2.2% ± 43.2%, p = 0.86). Likewise, there was no significant change in NTX/Cr (mean change -2.1%, p = 0.81). Interferon gamma-1b was poorly tolerated. Most subjects had adverse events, and the Mental Health and Mental Component Scales of the SF-36v2 health survey declined slightly (p < 0.05). Over 14 weeks, interferon gamma-1b failed to significantly increase bone turnover markers in ADO2 and was poorly tolerated. Consequently, interferon gamma-1b is unlikely to be effective for decreasing bone mass in ADO2. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Background: Osteopetrosis is a heterogeneous group of uncommon congenital disorders that causes bony sclerosis and remodeling. Patients who are symptomatic can show significant neurologic consequences with the involvement of cranial nerves and symptoms of increased intracranial pressure (ICP)., Case Description: We report an unusual case of a 26-year-old woman with an autosomal-dominant type of osteopetrosis who presented with headache and severe visual deterioration, both attributed to increased ICP. A hemicranioplasty was preformed, resulting in the resolution of her symptoms of ICP and stabilization of her vision. Postoperative imaging showed expansion of the ventricles and the subarachnoid spaces with an improvement of the associated cerebellar herniation., Conclusions: In conclusion, in patients with symptomatic osteopetrosis, cranioplasty can be considered as an option to treat high ICP-related symptoms., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Osteopetrosis is a generic term for generalized sclerotic conditions caused by rare genetic disorders. Decreased osteoclastic activities disturb bone remodeling, resulting in greater mineral density and greater compressive strength; therefore, bone fracture is a major physical symptom of osteopetrosis. Osteomyelitis of the maxilla or mandible is a common and well-documented complication of osteopetrosis. Local infection, such as odontogenic infection, is more likely to lead to osteomyelitis, and treatment strategies can be challenging. However, detailed ultrastructural analyses of bone from patients with osteopetrosis and odontogenic infection are limited. This report describes a case of osteomyelitis of the maxilla and mandible secondary to osteopetrosis in an adult patient and presents ultrastructural data of alveolar bone tissue analyzed by contact microradiography, electron probe microanalysis, and x-ray diffraction. Cases of osteomyelitis of the jaw secondary to osteopetrosis also are reviewed., (Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Osteopetrosis is a rare genetic condition of reduced osteoclastic bone resorption which causes defective bone remodeling and skeletal sclerosis during growth, having effects on many organs and tissues. Mutation of T-cell immune regulator 1 (TCRG1) gene is the most common genetic defect leading to osteopetrosis, with poor prognosis. The autosomal recessive form presents in the infantile period (also known as malignant infantile osteopetrosis--MIOP), and is characterized by fractures, short stature, hepatosplenomegaly, compressive neuropathies, hypocalcemia and pancytopenia. Being a rare disease with non-specific clinical manifestations, the diagnosis is difficult and usually delayed. Rickets is a characteristic feature of MIOP which results from the defect in osteoclasts to provide a normal Ca/P balance resulting in the poor mineralization of the osteoid. Various treatment options have been suggested for osteopetrosis, but hematopoietic stem cell transplantation still remains the only curative treatment option presently. The authors report the case of a 46-day-old girl with late-onset neonatal hypocalcemia and rickets that was later diagnosed as osteopetrosis. This case report emphasizes that infantile osteopetrosis is an important cause of neonatal hypocalcemia. As irreversible complications develop within the first months of life, immediate diagnosis and early intervention are crucial and may be life-saving., (© The Author [2015]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study aimed to generate a monoclonal antibody (mAb) targeting both tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL) and to evaluate the therapeutic effects of this antibody on acute inflammation and osteoporosis. We used hybridoma techniques to generate potential mAbs and enzyme-linked immunosorbent assay (ELISA) to determine their specificity. Crystal violet staining was performed to measure the effective dose of the candidate mAbs. The neutralizing effect of the mAbs was evaluated by TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays. We further assessed the therapeutic effect of the mAbs in BALB/c mice with carrageenan-induced acute inflammation and ovariectomy-induced osteoporosis. We successfully generated an IgG1 isotype mAb that recognizes human TNF-α and RANKL, which we named 8G12. The 50% effective dose of 8G12 was approximately 1μg/mL. L929 cells treated with 8G12 exhibited decreased levels of apoptosis (20.04% compared to 63.28% in the positive controls). In addition, treatment with 8G12 inhibited osteoclastogenesis in a dose-dependent manner in vitro. Carrageenan-induced paw edema was significantly reduced in the 8G12-treated mice compared to the positive controls. Treatment with 8G12 also reduced the number of infiltrating leukocytes by more than 50%. The 8G12 treatment not only prevented bone loss but also increased the number, thickness and volume of trabeculae and reduced trabecular separation in ovariectomized mice. Our data suggest that the 8G12 effectively neutralizes the bioactivity of TNF-α and RANKL, ameliorating osteoporosis and inflammation. We therefore propose that 8G12 could be a candidate for generating therapeutic antibodies for treating inflammatory bone diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.

In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial., (Copyright © 2012 American Society for Bone and Mineral Research.)

The unique ability of the osteoclasts to resorb the calcified bone matrix is dependent on secretion of hydrochloric acid. This process is mediated by a vacuolar H+ ATPase (V-ATPase) and a chloride-proton antiporter. The structural subunit of the V-ATPase, a3, is highly specific for osteoclasts, and mutations in a3 lead to infantile malignant osteopetrosis, a phenomenon characterized by increased bone mass, an increased number of non-resorbing osteoclasts, and a complete lack of bone resorption. Importantly, these individuals have normal or even increased osteoblast numbers and bone formation suggesting that the osteoclasts, but not their resorptive capability, relay an anabolic signal, and, hence, that bone formation can be uncoupled from bone resorption when the a3 subunit is eliminated by mutations, or possibly by pharmacological intervention. The pharmacological profile of the a3 subunit as a highly specific target with a mode of action profile augmenting uncoupling and sustained bone formation, as derived from osteopetrotic patients and mice, highlights the relevance of the V-ATPase in future osteoporosis drug development. However, as illustrated by numerous attempts at developing specific inhibitors of the osteoclastic V-ATPase it is a very difficult target to work with, and an inhibitor possessing the desired profile remains elusive, although highly promising approaches recently have been launched.

The biological properties of the lanthanides, primarily based on their similarity to calcium, have been the basis for research into potential therapeutic applications of lanthanides since the early part of the twentieth century. Up to date, cerium nitrate has been used as a topical cream with silver sulfadiazene for the treatment of burn wounds. A lanthanide texaphyrin complex (motexafin gadolinium) has been evaluated through Phase III clinical trials for the treatment of brain metastases in non-small cell lung cancer. Lanthanum carbonate (Fosrenol) as a phosphate binder has been approved for the treatment of hyperphosphatemia in renal dialysis patients in both the USA and Europe. This review will highlight therapeutic applications of the lanthanides for burn wounds, cancer, hyperphosphatemia, immune function, magnetic resonance imaging (MRI) contrast agents and osteoporosis, and discuss their future potential in the medical fields.

Estrogen replacement is a potent therapy for postmenopausal osteoporosis. However, its carcinogenic effects on breasts and the uterus limit its utilization. Raloxifene has estrogen-like effects on bones without the carcinogenic symptoms on breast or uterine tissue. Their individual effects are well characterized, but the results of their interaction remains elusive. In this work, we investigate the consequences of a combined raloxifene/estrogen therapy on bone and uterus with experimental osteoporosis. 40 Wistar rats began treatment 3 months post-ovariectomy. Estrogen and raloxifene were administered 0.03 mg/kg/day and 1.5mg/kg/day separately and together for 5 times per week for 12 weeks. Biomechanical tests and bone mineral density measurements, histology of uterus, and blood markers were analyzed. The co-administration group had higher toughness and ultimate strength than the ovariectomized controls (P<0.01). E+R had better biomechanical properties than the single treatments; yet the differences were not significant. Uterus histology signified high degeneration in the estrogen group. The raloxifene group had less degeneration but higher vascularization. Less immune reaction and vascularization were observed in the group with combined dosage than in those with individual treatments. Hence, the uterus of the combined treatment had fewer side effects than the ones that were individually treated. Mutual antagonization might be possible between raloxifene and estrogen, and that might have caused a decrease in the adverse effects. Overall, combined therapy might be useful to minimize the individual side effects of raloxifene and estrogen on the uterus and still provide bone strength and toughness., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Osteopetrorickets is a rare autosomal recessive disorder of osteoclast function characterized by abnormally dense bone and failure of resorption of calcified cartilage. Rickets is a paradoxical complication of osteopetrosis, resulting from the inability of the osteoclasts to maintain a normal calcium-phosphorus balance in the extracellular fluid. We report a patient with an unusual case of infantile osteopetro-rickets who was admitted with anterior fontanel bulging and was treated with haploidentical bone marrow transplantation.

CLC-7 is a member of the voltage-gated chloride channels family. It resides mainly in the late endosomes, lysosomes and the ruffled membrane of osteoclasts. Mice deficient in the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. In the present review, some of the known features of CLC-7 such as structure, function and its roles in physiological or pathophysiological processes are highlighted.

Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCgamma-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.

Numerous experimental and clinical observations suggest that overall changes in bone resorption during menopause or treatment with hormone replacement therapy (HRT) are combined effects of changes in osteoclast number and function. Moreover, due to a coupling between osteoclastic bone resorption and osteoblastic bone formation, pronounced alteration of osteoclast number will eventually lead to alteration of osteoblastic bone formation. Fragments of type I collagen, such as the C- and N-terminal telopeptides of collagen type I (CTX and NTX, respectively), are generated during bone resorption and hence can be used as surrogate markers of osteoclast function. Circulating levels of different enzymes in the serum, such as TRAP 5b and cathepsin K are proportional to the number of osteoclasts, and hence can be used as surrogate markers of osteoclast number. Since antiresorptive effects can be obtained in different ways, we felt it was timely to discuss the different scenarios, highlight differences specific to different pharmacological interventions with different mechanisms of action, and discuss how these bone markers can assist us in a deeper analysis of the pharmacodynamics and safety profile of existing and upcoming drug candidates.

Malignant infantile osteopetrosis is a rare autosomal recessive disorder characterized by presentation in the first few months of life with manifestations relating to an underlying defect in osteoclastic bone resorption. This report describes a 10 day-old boy in whom neonatal hypocalcemia was present and whose brother had died with the diagnosis of osteopetrosis.

(1) Severe malignant osteopetrosis is a very rare disease. The principal manifestations are anaemia, infections, sensory disorders and fractures, due to generalised bone condensation. The disease is generally fatal in childhood. The only treatment capable of modifying the natural outcome is bone marrow transplantation. The benefits of high-dose steroids and calcitriol are usually modest and transient. (2) Severe malignant osteopetrosis is a new licensed indication for interferon gamma-1b, a drug known to reduce the incidence of severe infections in children with chronic septic granulomatosis. (3) An unblinded trial involving 15 children with a mean age of about one year compared calcitriol plus interferon gamma-1b with calcitriol alone. The time to treatment failure was longer with the combination, based on a combined endpoint chosen to make the statistical analysis more sensitive. (4) A clinical trial involving 15 patients, who were compared with a historical series of 94 untreated patients, provided ambiguous results. (5) In these trials the main adverse effect of interferon gamma-1b was a flu-like syndrome. (6) Given the gravity of severe malignant osteopetrosis, the limited available treatment options, and the rarity of serious adverse events with interferon gamma-1b, evaluation of this therapy should continue.

Vitamin D-binding protein (DBP) is a multi-functional plasma protein with many important functions. These include transport of vitamin D metabolites, control of bone development, binding of fatty acids, sequestration of actin and a range of less-defined roles in modulating immune and inflammatory responses. Exploitation of the unique properties of DBP could enable the development of important therapeutic agents for the treatment of a variety of diseases.

Osteopetrosis is a heterogeneous family of rare human genetic disorders due to markedly decreased bone resorption. It is one among disorders causing osteosclerosis of the trabecular bone and/or hyperostosis of the cortical bone. Four types of human osteopetrosis have been clearly defined, but patients with atypical symptoms are frequent, suggesting that there are additional forms. The most severe expression of this condition in its malignant form is inherited as an autosomal recessive disorder and it is usually fatal before school age. It presents with failure to thrive, severe hepatosplenomegaly, pancytopenia and nerve compression leading to blindness and deafness during infancy. The case of a 2-month-old female child with severe hepatosplenomegaly, failure to thrive, nystagmus, pancytopenia, gengival hyperplasia, optic atrophy, absent evoked visual potential and increased bone density within the total skeleton, is reported. Diagnosis of autosomal recessive malignant osteopetrosis was established by transiliac bone biopsy. She underwent bone marrow transplantation, but died soon afterwards. This rare and mortal disorder of bone formation requires early diagnosis and immediate pharmacological treatment, consisting in administration of vitamin D, in order to enhance bone resorption and of prednisone to improve hematological indexes and, if possible, bone marrow transplantation in order to ameliorate quality of life and survival.

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone-in-bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X-ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow-up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.

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.

We have used dual-energy x-ray absorptiometry (DXA) in evaluation and follow-up of a patient with osteopetrosis, before and after cord blood transplantation. Other methods of follow-up in such cases have been described, but the use of DXA has not previously been reported. We have shown that DXA offers a safe means of assessing disease progression, the timing of treatment, and response after therapy for osteopetrosis.

The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in this mutation is unaffected by either molecule. Thus, the tl mutation intercepts the function of a gene required for both normal endochondral ossification and bone resorption, thereby uncoupling the coordination of skeletal metabolism required for normal long bone growth.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.

Although young mice homozygous for the osteopetrosis (op) mutation usually developed prominent osteopetrosis, its severity was markedly reduced in aged op/op mice. This age-associated reversal of osteopetrosis was accompanied by the expansion of bone marrow cavities and increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive cells and of macrophages in the bone marrow. The TRAP-positive cells were mononuclear and developed ruffled borders and numerous vesicles, vacuoles, and granules. Enzyme-linked immunosorbent assay demonstrated a significant elevation of serum granulocyte/ macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 levels in the aged op/op mice. To examine whether GM-CSF and/or IL-3 could correct osteopetrosis in young op/op mice, 5 ng of recombinant murine (rm)GM-CSF and/or 100 ng of rmIL-3 were injected daily into young op/op mice. In these treated young op/op mice, the bone marrow cavities were expanded significantly at 2 weeks after administration, associated with significantly increased numbers of TRAP-positive cells and bone marrow macrophages. TRAP-positive cells increased in number with days after injection. These results suggest that GM-CSF and IL-3 induce the development of osteoclasts to correct osteopetrosis in the op/op mice with aging.

The osteopetrotic mib mutation in rats causes defects in the skeletal bone tissue in young animals. These defects, i.e. slow bone remodelling, changes in both crystallinity and mineral content, are transient and undergo normalization, even without any treatment in 6-wk-old animals. Treatment with CSF-1 (colony stimulating factor-1) accelerates the normalization process in skeletal bones. The periodontal tissues around the apices of incisors show abnormalities caused by the slow remodelling process of the mandible bone tissue, the deficiency of osteoclasts and their abnormal morphology, as well as the disorganization of periodontal ligament fibres. In contrast to the skeletal tissues, these abnormalities would not undergo spontaneous normalization. Under treatment with colony stimulating factor 1 (CSF-1), the primitive bone trabeculae of mandible are resorbed and the normalization of the number of osteoclasts and their cytology occurs. The organization of the periodontal ligament fibres is partially restored, resembling the histological structure of the normal one.

Two physiological regulators of osteoclast maturation have recently been identified: the secreted protein osteoprotegerin and the cell-surface ligand to which it binds. These proteins are likely to play an important part in the control of bone resorption, but are also likely to have important roles in other tissues.

Native bone morphogenetic protein and associated noncollagenous proteins induced the formation of heterotopic bone in the hindquarter muscles of osteopetrotic (op/op) mice and those of their phenotypically normal littermates (+/?). In op/op mice, the heterotopic bone consisted of a disorganized, densely packed mixture of irregular calcified cartilage, osteoid, chondro-osteoid, and fibrous tissue. Injections of recombinant human macrophage colony-stimulating factor initiated bone resorption that began in the peripheral vascularized regions of the metaphyses and continued in central areas of uncalficified avascular chondro-osteoid. On vascularized surfaces, osteoclasts were stained with tartrate-resistant acid phosphatase. In op/op mice treated with macrophage colony-stimulating factor, the osteoclasts were small, with only two or three nuclei, and they did not exhibit tartrate-resistant acid phosphatase staining. In untreated op/op mice, surgical blood clots persisted in the heterotopic sites as late as 3 weeks after the operation, whereas in treated op/op mice, the blood clots were absorbed almost as rapidly as in normal mice. Histologically, recombinant human macrophage colony-stimulating factor restored normal macrophage functions: absorption and organization of blood clot, osteoclastogenesis, synthesis of tartrate-resistant acid phosphatase, bone remodeling, islands of myelopoiesis, and construction of an ossicle complete with a cortex and a medulla filled with functioning hematopoietic bone marrow.

Osteopetrotic (op/op) mice show severe osteosclerosis caused by an inherited deficiency of osteoclast and resultant failure of tooth eruption, which can be cured by the injection of macrophage colony-stimulating factor (M-CSF). The present study revealed that consecutive injections of M-CSF in these mutant mice brought about a recovery of bone resorption resulting in the resumption of growth of tooth root and periodontal ligament. Bone resorption at the inner surface of bony crypts was noted on the 5th day after the start of M-CSF injections. This activity was reduced with the progress of root and periodontal ligament formation, being confined to the basal and crestal portion of bony crypts by the 15th day of the experiment. Second molars emerged into the oral cavity on the 15th day, but no eruption of first molars was observed until the 20th day. Throughout the experiment, first molars exhibited appreciable root deformity, which was less severe in second molars. Delayed eruption of first molars was thought to be related to the severity of the disturbance of root formation.

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

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.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

In mice homozygous for the osteopetrosis (op) mutation, loss of osteoclasts in the postnatal period and their development, differentiation, and maturation following daily M-CSF administration in adult life were investigated. Histochemical, immunohistochemical, and ultrastructural approaches, as well as [3H]thymidine autoradiography, clarified the role of M-CSF on osteoclast development and differentiation. In op/op mice osteoclasts appeared normal at birth. However, osteoclast numbers were reduced within a few days after birth, and osteoclasts were undetectable by 3-4 days of age. In adult op/op mice there were no multinuclear osteoclasts; however, small numbers of mononuclear cells (so-called 'preosteoclasts') were observed on the endosteal surface of bone. These preosteoclasts expressed tartrate-resistant acid phosphatase and showed ultrastructural features of immature osteoclasts. After daily M-CSF administration in op/op mice, osteoclasts developed from the fusion of preosteoclasts and osteoclasts numbers increased to the levels of normal littermates at 3 days. Autoradiographic analysis with [3H]thymidine revealed no labeling in osteoclasts and preosteoclasts. In the mutant mice, M-CSF administration induced numerical increases of monocytes, promonocytes, and earlier precursor cells in bone marrow, ER-MP12- or, ER-MP58-positive granulocyte/macrophage colony-forming cells (GM-CFCs). Among these macrophage precursors, ER-MP58-positive cells were considered preosteoclast precursors, and possessed marked proliferative potential. These data suggest that an ER-MP58-positive cell subpopulation of GM-CFCs proliferates in response to M-CSF, differentiates into preosteoclasts which fuse with each other to develop into mature osteoclasts.

Osteopetrosis is a heterogeneous group of bone diseases characterized by an excess accumulation of bone and a variety of immune defects. Osteopetrosis (op) and incisors absent (ia) are two nonallelic mutations in the rat which demonstrated these skeletal defects as a result of reduced bone resorption. Osteopetrotic (op) rats have severe sclerosis as a result of reduced numbers of osteoclasts which are structurally abnormal. The sclerosis in ia rats is not as severe as in op mutants; they have elevated numbers of osteoclasts, but they are also morphologically abnormal, lacking a ruffled border. Both of these mutations have defects in the inflammation-primed activation of macrophages. They demonstrate independent defects in the cascade involved in the conversion of vitamin D binding protein (DBP) to a potent macrophage activating factor (DBP-MAF). Because this factor may also play a role in the pathogenesis of osteoclastic dysfunction, the effects of ex vivo-generated DBP-MAF were evaluated on the skeletal system of these two mutations. Newborn ia and op rats and normal littermate controls were injected with DBP-MAF or vehicle once every 4 days from birth until 2 weeks of age, at which time bone samples were collected to evaluate a number of skeletal parameters. DBP-MAF treated op rats had an increased number of osteoclasts and the majority of them exhibited normal structure. There was also reduced bone volume in the treated op animals and an associated increased cellularity of the marrow spaces. The skeletal sclerosis was also corrected in the ia rats; the bone marrow cavity size was significantly enlarged and the majority of the osteoclasts appeared normal with extensive ruffled borders.

It has recently been shown that following treatment with colony-stimulating factor-1 (CSF-1) the osteopetrotic condition in toothless (tl) rats greatly improves and growth is accelerated. We have examined the effects of such treatment on the microvasculature of the distal femoral chondro-osseous junction, a site where bone growth in length is coordinated with angiogenesis. Vascular casts and ultrastructural analyses of this region showed that, compared to untreated normal rats, untreated mutants showed little bone growth or angiogenesis. When mutants were treated with CSF-1 angiogenesis was markedly accelerated. These data show a remarkable effect of this growth factor on angiogenesis in this osteopetrotic mutation. Whether this effect of CSF-1 on angiogenesis is direct or indirect is not known and indicates that its effects on the normal microvasculature deserve further study.

The osteopetrotic (op) mutation in mice is characterized by general 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. Contrary to previous reports, we have noted persistent metaphyseal sclerosis in op mice treated with CSF-1 at doses above physiological concentrations of circulating CSF-1. We pursued this observation by quantitating osteoclasts and macrophages in the first 500 microns (area A) and the subsequent 1000 microns (area B) in the proximal tibial metaphysis using tartrate-resistant acid phosphatase and F4/80 as cell markers. In untreated normal mice, osteoclasts and macrophages were found in areas A (9.1 and 13.8 cells/1000 microns2) and B (4.1 and 9.4 cells/1000 microns2), respectively. In untreated mutants, osteoclasts and macrophages as percentages of normal were, respectively, 0% and 2% (area A) and 30% and 13% (area B). After CSF-1 treatment (0.15, 0.3, 0.5, and 1.0 x 10(6) U/day) for 28 days, marrow cavity size and numbers of osteoclasts and macrophages increased significantly in area B. However, area A remained sclerotic, with few macrophages (3% to 20%), and although osteoclast numbers were normal, their distribution was not, being absent in subepiphyseal sites. High CSF-1 gene expression occurs at bone modeling sites, co-localizes with osteoblasts, and temporally correlates with their differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteopetrois is an inherited bone disease characterized by an excessive accumulation of bone throughout the skeleton. The disease in the ia (incisors absent) rat is the result of reduced bone resorption caused by defective, although numerous osteoclasts. In addition to the bone defects, ia rats have suppressed natural immunity, even though these animals have excessive numbers of natural killer (NK) cells. The osteopetrotic condition also appears to have an associated abnormality in vitamin D metabolism. Because 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] stimulates bone resorption and has a role in the immunoregulation of NK cells, mutant and normal rats were infused with 1,25-(OH)2D3 for 14 days in an attempt to correct the defects in this mutant. Serum levels of osteocalcin, 25-OHD3, and 1,25-(OH)2D3, as well as NK function and parameters of bone resorption, were evaluated after the infusion period. Serum levels of osteocalcin and 1,25-(OH)2D3 were elevated in both ia and normal rats treated with 1,25-(OH)2D3. Serum 25-OHD3 levels were significantly reduced in the treated animals. The elevated percentage of NK cells normally found in ia rats was reduced to normal in the treated mutants, and NK cell function was elevated to normal levels of lytic activity. The percentage of NK cells and NK function remained unchanged in the treated normal rats. The bone marrow cavity size was significantly increased in the 1,25-(OH)2D3-treated mutants, as was the percentage of osteoclasts exhibiting normal morphology. Radiographically, the mutant bones were less dense.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructural features of macrophages and dendritic cells of mice homozygous for osteopetrosis (op/op) mutation were studied. The mutant mice are characterized by defective differentiation of osteoclasts, monocytes, and tissue macrophages due to the lack of functional macrophage colony stimulating factor (M-CSF/CSF-1) activity. In op/op mice, tissue macrophages were reduced in number and smaller than in normal littermates. Macrophages in op/op mice showed various degrees of phagocytosis but the development of intracytoplasmic organelles and microvillous projections was poor. After administration of CSF-1 daily for 2 weeks, macrophages in op/op mice developed lysosomes and microvillous projections. In the thymic medulla, T-cell zone of lymph nodes, splenic white pulp and epidermis of the op/op mice, the number of dendritic cells was similar to that in normal littermates and the dendritic cells developed a tubulovesicular system typical of interdigitating cells. Birbeck granules in epidermal Langerhans cells were detected in unmanipulated op/op mice, op/op mice injected with CSF-1, and normal littermates or control mice. However, in untreated op/op mice, dendritic cells projected shorter cytoplasmic processes than in normal littermates, normal control mice and CSF-1 injected op/op mice. These results indicate that the differentiation and maturation of tissue macrophages are mediated by CSF-1, but the dendritic cell differentiation is controlled by other factor(s) than CSF-1, most probably by GM-CSF.