Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.

Introduction: Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes.Methods: Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy.Results: Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation.Conclusions: This method should prove valuable as a means of studying cbondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies. (c) 2005 Elsevier Inc. All rights reserved.

Introduction: Many of the insights into the factors that regulate the differentiation and activation of osteoclasts are gained from different spontaneous and genetically induced osteopetrotic animal models. The osteopetrotic incisors absent (ia) rat exhibits a generalized skeletal sclerosis and a delay of tooth eruption. Although the is rat has well been studied phenotypically, the genetic defect still remains unknown.Material and Methods: To map the is locus, we outcrossed the inbred is strain with the inbred strain Brown Norway. Intercrossing F1 animals produced the F2 generation. Thirty-one mutant F2 animals and six mutant F4 animals were available for segregation analysis.Results: Segregation analysis enabled us to assign the disease causing gene to rat chromosome 10q32.1. Homozygosity for the is allele was obtained for two of the markers analyzed (D10Rat18 and D10Rat84). Key recombinations delineate a candidate region of 4.7 cM flanked by the markers D10Rat99 and D10Rat17Conclusion: We have delineated a 4.7-cM region on rat chromosome 10q32.1 in which the gene responsible for the osteopetrotic phenotype of the is rat is located. Although the sequence of this chromosomal region is not complete, over 140 known or putative genes have already been assigned to this region. Among these, several candidate genes with a putative role in osteoclast functioning can be identified. However, at this point, it cannot be excluded that one of the genes with a currently unknown function is involved in the pathogenesis of the is rat. Further analysis of the genes mapped in this region will provide us more insight into the pathogenesis of this osteopetrotic animal model.

The proliferation and hypertrophy of growth plate chondrocytes set the pace and pattern for growth of endochondral bones. Complex signaling pathways regulating chondrocyte differentiation during development and growth have been discovered in recent years, but as yet little is known about how chondrocytes are able to orient themselves to align properly with respect to the direction of bone growth. Histomorphometric methods developed for analysis of growth plates rely to a significant extent on assessments of the relative heights of the zones of proliferating and hypertrophic chondrocytes. In a growing number of osteopetrotic mutations, however, it is becoming apparent that growth plates lack clearly demarcated zones of chondrocyte differentiation, and they show other notable histological abnormalities that cannot be measured with standard approaches. This is particularly true of mutations in which osteoclasts are altogether absent. We therefore developed a new approach that measures the salient features of this type of chondrodysplasia and have applied it to the toothless (tl) rat. The tl rat has a frameshift mutation in the Csf-1 gene that renders it null, resulting in severe osteopetrosis. An accompanying pathology is a severe, progressive growth plate chondrodysplasia. We measured cell orientation, cell area, and local columnar organization as functions of distance from the upper margin of the growth plate, in addition to growth plate thickness and cell density. All these parameters were markedly abnormal in the tl rats, thus implicating Csf-1 not only in its well-established role in regulating osteoclastic bone resorption, but also in endochondral ossification. This approach should prove useful in distinguishing among growth plate chondrodysplasias, most especially in the growing number of osteopetrotic mutations having growth plates that lack the normal zonal organization and in which the chondrocytes are mis-oriented. In turn, detailed assessments of chondrocyte misorientation may give insights into how they normally are able to arrange themselves with such precision.

We report the skeletal manifestations of restoring RANKL (TNFSF11/TRANCE; see foot note* on nomenclature) expression in null mice using a lymphocyte-specific promoter. RANKL was discovered independently by immunologists and bone researchers by virtue of its essential roles in lymph node organogenesis, normal cellular immunity, and osteoclastogenesis. "Rescue" of RANKL knockout mice by a T- and B-cell expressed transgene reversed many immunological manifestations of the knockout, while it had highly selective effects on the skeletal pathology. RANKL-null mice exhibit severe osteopetrosis, no tooth eruption, markedly reduced skeletal growth, and growth plate chondrodystrophy. The transgene induced tartrate-resistant acid phosphatase (TRAP) positive cells in long bones as early as 3 days postpartum, restored marrow spaces in long bones, produced lamellar bone in the diaphyses, and restored osteoclasts at many endosteal sites, but not in periosteum nor the jaws. It did not improve the chondrodystrophy, chondroosseous junction defects, or tooth eruption. The ends of limb and axial skeletal elements remained highly sclerotic while diaphyses became osteopenic, and growth retardation persisted. Together, these results demonstrate the importance of local delivery of RANKL for many skeletal processes.

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

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1(null). All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.

The toothless (tl) osteopetrotic mutation in the rat affects an osteoblast-derived factor that is required for normal osteoclast differentiation. Although the genetic locus remains unknown, the phenotypic impact of the tl mutation on multiple systems has been well characterized. Some of its actions are similar to tumornecrosis factor superfamily member 11(TNFSF11; also called TRANCE, RANKL, ODF and OPGL) null mice. TNFSF11 is a recently described member of the tumor necrosis factor superfamily which, when expressed by activated T cells, enhances the survival of antigen-presenting dendritic cells, and when expressed by osteoblasts, promotes the differentiation and activation of osteoclasts. The skeletal similarities between tl rats and TNFSF11(-/-) mice include 1) profound osteoclastopenia (TNFSF11-null mice, 0% and tl rats 0-1% of normal); 2) persistent, non-resolving osteopetrosis that results from 3) a defect not in the osteoclast lineage itself, but in an osteoblast-derived, osteoclastogenic signal; and 4) a severe chondrodysplasia of the growth plates of long bones not seen in other osteopetrotic mutations. The latter includes thickening of the growth plate with age, disorganization of chondrocyte columns, and disturbances of chondrocyte maturation. These striking similarities prompted us to undertake studies to rule in or out a TNFSF11 mutation in the tl rat. We looked for expression of TNFSF11 mRNA in tl long bones and found it to be over-expressed and of the correct size. We also tested TNFSF11 protein function in the tl rat. This was shown to be normal by flow cytometry experiments in which activated, spleen-derived T-cells from tl rats exhibited normal receptor binding competence, as measured by a recombinant receptor assay. We also found that tl rats develop histologically normal mesenteric and peripheral lymph nodes, which are absent from TNFSF11-null mice. Next, we found that injections of recombinant TNFSF11, which restores bone resorption in null mice, had no therapeutic effect in tl rats. Finally, gene mapping studies using co-segregation of polymorphic markers excluded the chromosomal region containing the TNFSF11 gene as harboring the mutation responsible for the tl phenotype. We conclude that, despite substantial phenotypic similarities to TNFSF11(-/-) mice, the tl rat mutation is not in the TNFSF11 locus, and that its identification must await the results of further studies.