Fasciola gigantica: Ultrastructural localisation of neoblast recruitment in somatic tissues during growth and development in the hepatic parenchyma of experimentally infected mice.

Application of 'omics' technology, and advances in in vitro methods for studying the growth of Fasciola hepatica, have highlighted the central role of migrating neoblasts in driving forward development and differentiation towards the adult-like form. Neoblast populations present molecular heterogeneity, morphological variation and changes associated with recruitment of these stem cells into their final tissue locations. However, terminal differentiation towards function, has received much less attention than has been the case for the free-living Platyhelminths. An actively replicating neoblast population, comprising cells with heterochromatic nuclei consistent with regulation of gene expression, has been identified in the parenchyma of juvenile Fasciola gigantica migrating in the liver of experimentally infected mice. In some of these cells, early cytoplasmic differentiation towards myocyte function was noted. Neoblasts have also been identified close to, and incorporated in, the subtegumental zone, the gastrodermis and the excretory ducts. In these locations, progressive morphological differentiation towards terminal function has been described. This includes the appearance of specific progenitors of type-1, type-2 and type-3 tegumental cells, the latter possibly contributing to tegumental spine development. 'Cryptic' surface molecular differentiation is postulated to account for recognition and 'docking' of migrating neoblasts with their final site for terminal differentiation.

Communication between ephrinB2 and EphB4 within the osteoblast lineage.

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Mechanisms involved in skeletal anabolic therapies.

Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.

Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.

Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions.

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Rodent osteoblast-like cells support osteoclastic differentiation of human cord blood monocytes in the presence of M-CSF and 1,25 dihydroxyvitamin D3.

Fracture repair requires the involvement of osteoclasts (OC), multinucleated cells which are responsible for bone resorption and form by fusion of circulating mononuclear haemopoietic precursors. The nature of these circulating precursor cells, in particular their relationship to blood monocytes, is uncertain. To define further the nature of the circulating human OC precursor, and to determine the role bone stromal cells and humoral factors play in the differentiation of OCs, we co-cultured human umbilical cord blood monocytes with UMR106.01 osteoblast-like cells in the presence and absence of 1,25 dihydroxyvitamin D3 [1,25 (OH)2D3], macrophage-colony stimulating factor (M-CSF) and dexamethasone on both bone slices and coverslips. Isolated cells were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14 and HLA-DR) and negative for OC markers [tartrate resistant acid phosphatase (TRAP), vitronectin receptors (VNR) and calcitonin receptors (CT receptors)] and did not form resorption pits on bone slices after 24 hr in culture. However, after 14 days in co-culture with UMR106.01 cells, in the presence of 1,25 (OH)2D3 and M-CSF, numerous TRAP, CT receptor and VNR positive multinucleated cells capable of extensive lacunar bone resorption were formed in these co-cultures. The presence of 1,25 (OH)2D3, M-CSF and a bone-derived stromal cell population were absolute requirements for OC differentiation. It is concluded that mononuclear phagocytes are capable of differentiating into mature functional OCs when cultured under specific cellular and hormonal conditions. This is vitro model of human OC differentiation should prove useful in further analysing factors controlling OC generation in bone remodelling and repair.

Cellular and hormonal factors influencing monocyte differentiation to osteoclastic bone-resorbing cells.

Osteoclasts are multinucleated cells which form by fusion of circulating mononuclear hemopoietic precursors. The nature of these precursor cells and the roles bone stromal cells and hormonal factors play in their differentiation to osteoclasts are unknown. We cocultured adherent murine blood monocytes (nonspecific esterase and F4/80 positive; tartrate-resistant acid phosphatase negative) with osteoblastic and fibroblastic stromal cell lines in the presence of 2 x 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Tartrate-resistant acid phosphatase and calcitonin (CT) receptor-positive osteoclastic cells, which formed numerous resorption pits in vitro, were noted after only 4 days in coculture with UMR106 osteoblast-like cells. Resorption was seen in cocultures to which as few as 100 peripheral blood mononuclear cells had been added. 1,25-(OH)2D3 and contact with live bone stromal cells were absolute requirements for monocyte differentiation into bone-resorbing cells. Both salmon CT (5 IU/ml) and prostaglandin E2 (10(-6) M) significantly inhibited bone resorption. Thus, a significant proportion of the peripheral blood mononuclear cells in the monocyte fraction are capable of differentiating into cells showing the cytochemical and functional characteristics of osteoclasts. The presence of specific hormonal [1,25-(OH)2D3] and bone stromal cell elements is necessary for this process to occur; the resultant resorption can be modulated by known inhibitors of bone resorption, CT and prostaglandin E2.

A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro.

Both human and murine osteoclasts can be derived in vitro from hematopoietic cells or monocytes that are co-cultured with osteoblasts or marrow-derived stromal cells. The osteoclastogenic stimulus provided by murine osteoblasts and marrow-derived stromal cells is now known to be mediated by osteoclast differentiation factor (ODF), a membrane-bound tumor necrosis factor-related ligand. This study demonstrates that mouse spleen cells and monocytes form osteoclasts when cultured in the presence of macrophage-colony stimulating factor (M-CSF) and a soluble form of murine ODF (sODF). Numerous multinucleated osteoclasts expressing tartrate resistant acid phosphatase (TRAP) and calcitonin receptor (CTR) formed within 7 days of culture and engaged in extensive lacunar bone resorption. Osteoclast number and bone resorption area was dependent on sODF concentration. Long-term cultured human monocytes also formed bone resorbing osteoclasts in response to co-stimulation by sODF and M-CSF, although this required more than 11 days in culture. This human osteoclast differentiation was strongly inhibited by granulocyte-macrophage colony stimulating factor. This study further characterises murine osteoclast differentiation caused by sODF and M-CSF co-stimulation in vitro, and shows that the same co-stimulation causes human osteoclast differentiation to occur. We propose that this methodology can be employed to investigate the direct effects of cytokines and other factors on human osteoclast differentiation.

The human osteoclast precursor circulates in the monocyte fraction.

The osteoclast is known to be formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. The precise nature of these circulating cells and, in particular, their relation to monocytes is unknown. We have developed an in vitro system of human osteoclast formation whereby human monocytes [CD14, CD11a, CD11b and HLA-DR positive, and tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), vitronectin receptor (VNR) negative] were isolated and cocultured for up to 21 days with UMR106 rat osteoblast-like cells or ST2 mouse preadipocytic bone marrow stromal cells in the presence of 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) and macrophage colony stimulating factor (M-CSF). Numerous TRAP, VNR and CTR positive multinucleated cells, capable of extensive lacunar bone resorption, formed in these cocultures; the absolute requirements for this to occur were contact with the above bone stromal cells, 1,25(OH)2D3, and M-CSF. These results show that the human mononuclear osteoclast precursor circulates in the monocyte fraction and exhibits a monocyte phenotype, acquiring osteoclast phenotypic features in the process of differentiation into mature functional osteoclasts.

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

The generation of highly enriched osteoclast-lineage cell populations.

Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.

Insulin receptor expression in primary and cultured osteoclast-like cells.

Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.

Calcitonin receptor antibodies in the identification of osteoclasts.

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.

Giant cells in pigmented villo nodular synovitis express an osteoclast phenotype.

AIM: To determine the cytochemical and functional phenotype of multinucleated giant cells in pigmented villo nodular synovitis (PVNS). METHODS: Giant cells isolated from a patient with PVNS of the knee were assessed for a number of markers used to distinguish osteoclasts from macrophages/ macrophage polykaryons: evidence of tartrate resistant acid phosphatase (TRAP) activity; expression of CD11b, CD14, CD51, and calcitonin receptors; and the ability of the giant cells to carry out lacunar resorption. RESULTS: Isolated giant cells expressed an osteoclast antigenic phenotype (positive for CD51, negative for CD11b and CD14) and were TRAP and calcitonin receptor positive. They also showed functional evidence of osteoclast differentiation, producing numerous lacunar bone resorption pits on bone slices in short term culture. CONCLUSIONS: The giant cells in this case of PVNS express all the phenotypical features of osteoclasts including the ability to carry out lacunar resorption. This may account for the bone destruction associated with this aggressive synovial lesion.

Uranyl nitrate: 91-day toxicity studies in the New Zealand white rabbit.

These studies were undertaken to derive a lowest-observed-adverse-effect level (LOAEL) in the New Zealand White rabbit following a 91-day exposure to uranium (U, as uranyl nitrate hexahydrate, UN) in drinking water. Males were exposed for 91 days to UN in their drinking water (0.96, 4.8, 24, 120, or 600 mg UN/L). Subsequently, females were similarly exposed for 91 days (4.8, 24, or 600 mg UN/L). Control groups were given tap water (< 0.001 mg U/L). Regular observations were recorded, and urine was collected periodically. Four males showed evidence of Pasteurella multocida infection and were excluded from the study. Following the study, all animals were euthanized, and multiple hematological and biochemical parameters were determined. Necropsies were conducted, and histopathological examination was performed. The hematological and biochemical parameters were not affected in a significant exposure-related manner. Dose-dependent differences consisted of histopathological changes limited primarily to kidney. Changes in renal tubules were characteristic of uranium toxicity. Based on changes in the tubular nuclei, the 91-day LOAEL for males in this study is 0.96 mg UN/L drinking water. The females drank 65% more water than the males, yet appeared to be less affected by the exposure regimen, although they also developed significant tubular nuclear changes in their lowest exposure group, deriving a LOAEL of 4.8 mg UN/L. Tissue uranium residue studies suggested that pharmacokinetic parameters for the males and females differ, possibly accounting for the difference in observed sensitivity to UN. An adverse effect of P. multocida infection cannot be excluded.

Uranyl nitrate: 28-day and 91-day toxicity studies in the Sprague-Dawley rat.

Although uranium (U) is a classic experimental nephrotoxin, there are few data on its potential long-term chemical toxicity. These studies were undertaken to derive a no-observed-adverse-effect level (NOAEL) in male and female Sprague-Dawley rats following 91-day exposure to uranium (as uranyl nitrate hexahydrate, UN) in drinking water. Following a 28-day range-finding study, five groups of 15 male and 15 female weanling rats were exposed for 91 days to UN in drinking water (0.96, 4.8, 24, 120, or 600 mg UN/L). A control group was given tap water (< 0.001 mg U/L). Daily clinical observations were recorded. Following the study, animals were euthanized and exsanguinated, and multiple hematological and biochemical parameters were determined. Necropsies were conducted, and multiple tissues were sampled for histopathological examination. The hematological and biochemical parameters were not affected in a significant exposure-related manner. Although there were qualitative and slight quantitative differences between males and females, histopathological lesions were observed in the kidney and liver, in both males and females, in all groups including the lowest exposure groups. Renal lesions of tubules (apical nuclear displacement and vesiculation, cytoplasmic vacuolation, and dilation), glomeruli (capsular sclerosis), and interstitium (reticulin sclerosis and lymphoid cuffing) were observed in the lowest exposure groups. A NOAEL was not achieved in this study, since adverse renal lesions were seen in the lowest exposed groups. A lowest-observed-adverse-effect level of 0.96 mg UN/L drinking water can be reported for both the male and the female rats (average dose equivalent 0.06 and 0.09 mg U/kg body wt/day, respectively).

Uranyl nitrate: 91-day exposure and recovery studies in the male New Zealand white rabbit.

This study was undertaken to examine the reversibility of renal injury in the male New Zealand White rabbit subsequent to a 91-day exposure to uranyl nitrate (UN) in drinking water, followed by various recovery periods. Specific pathogen-free (SPF) animals were exposed for 91 days to UN in their drinking water (24 or 600 mg UN/L). Control groups were given municipal tap water (< 0.001 mg U/L). Regular clinical observations were recorded, and urine was collected periodically. Recovery periods between the last UN exposure and termination were 0, 8, 14, 45, or 91 days. Following the study, all animals were anesthetized and terminated by exsanguination, and multiple hematological and biochemical parameters were determined. Necropsies were conducted, and histopathological examination was performed. Exposure-related histopathological changes were observed only at much higher doses than in our previous male rabbit study where non-SPF-free animals had been used. Minor increases in kidney to body weight ratios were observed in the high-dose groups following exposure and early recovery. Renal tubular injury with degenerative nuclear changes, cytoplasmic vacuolation, and tubular dilation was seen in the high-dose group, without consistent resolution even after 91 days recovery. Animals ingested approximately 33% more uranium per day in this study than did males in a comparable dose group in the previous study, yet their kidney tissue uranium residues were 30% lower. These results suggest that SPF rabbits are less sensitive to uranyl injury than the non-SPF animals. The lowest-observed-adverse-effect level is estimated to lie at or below 24 mg UN/L.

In Vitro Cellular Systems for Studying OC Function and Differentiation : Primary OC Cultures and the FLG 29.1 Model.

Several pieces of evidence have shown that osteoclasts (OCs) are derived from progenitors originating from hemopoietic stem cells (1-3). More specifically, early OC precursors seem to be closely related to the colony-forming unit for granulocytes and macrophages (CFU-GM) (3-5. However, compared with other bone or marrow cells, OCs are found in extremely low numbers in normal adult bone. In addition, active OCs are strongly adherent to the bone surface. For these reasons, it is impossible to obtain pure or highly enriched cultures of intact OCs, although it is possible to obtain large numbers of OCs if good source tissue is available. OCs are found in large numbers only in bone undergoing extensive physiological remodeling (e.g., fetal bone and growing bone metaphyses) or pathological osteolysis (e g., fracture callus) Since human tissue is often difficult to obtain, most OC research has employed animal models, notably rabbit, rat, and chick.

Biomaterial particle phagocytosis by bone-resorbing osteoclasts.

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic biomaterials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells.