We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen ␣1 type I promoter (Col2.3⌬TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3⌬TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow. (Blood. 2004; 103:3258-3264)

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (

Apoptosis through Fas/Fas ligand (FasL) is an important regulator of immune system homeostasis but its role in bone homeostasis is elusive. We systematically analyzed: a) the expression of Fas/FasL during osteoblastogenesis and osteoclastogenesis in vitro, b) the effect of FasL on apoptosis and osteoblastic/osteoclastic differentiation, and c) osteoblastogenesis and osteoclastogenesis in mice deficient in Fas or FasL. The expression of Fas increased with osteoblastic differentiation. Addition of FasL weakly increased the proportion of apoptotic cells in both osteoclastogenic and osteoblastogenic cultures. In a colony forming unit assay, FasL decreased the proportion of osteoblast colonies but did not affect the total number of colonies, indicating specific inhibitory effect of Fas/FasL on osteoblastic differentiation. The effect depended on the activation of caspase 8 and was specific, as addition of FasL to osteoblastogenic cultures significantly decreased gene expression for runt-related transcription factor 2 (Runx2) required for osteoblastic differentiation. Bone marrow from mice without functional Fas or FasL had similar osteoclastogenic potential as bone marrow from wild-type mice, but generated more osteoblast colonies ex vivo. These colonies had increased expression of the osteoblast genes Runx2, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin, and osteoprotegerin. Our results indicate that Fas/FasL system primarily controls osteoblastic differentiation by inhibiting progenitor differentiation and not by inducing apoptosis. During osteoclastogenesis, Fas/FasL system may have a limited effect on osteoclast progenitor apoptosis. The study suggests that Fas/FasL system plays a key role in osteoblastic differentiation and provides novel insight into the interactions between the immune system and bone.

Citation and quotation errors are common in medical journals. We assessed the prevalence of those errors in gross anatomy journals, where articles often cite old anatomical studies. The study included 199 randomly selected references from articles published in the first 2001 issue of three major gross anatomy journals: Annals of Anatomy, Clinical Anatomy, and Surgical and Radiologic Anatomy. The selected references were checked for accuracy against the original articles. Citation errors were classified as major, intermediate, and minor. Quotation errors were classified as major and minor. Citations errors were found in 27% (54/199) of the references and 38% of them were major errors. Errors occurred in 19% (52/272) of quotations and nearly all (94%) were major. Furthermore, 24% of the quotations were indirect references to a secondary, instead of original, source. There was no statistically significant difference in the rates of citation or quotation errors between the references published before or after the introduction of MEDLINE (chi2 test, P > 0.05) in 1963, and the prevalence of these errors in gross anatomy journals was similar to that found in other medical fields. A high proportion of major citation errors, a very high proportion of major quotation errors, and the substantial number of indirect quotations call for serious editorial action in anatomy journals.

Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (μCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (ptx3−/−) had lower trabecular bone volume than their wild-type (ptx3+/+) littermates (BV/TV by μCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females, p < 0.0001; BV/TV 9.06 ± 1.89 vs 10.47 ± 1.97 for males, p = 0.0435). In addition, μCT revealed lower trabecular bone volume in second lumbar vertebra of ptx3−/− mice. PTX3 was increasingly expressed during osteoblast maturation in vitro and was able to reverse the negative effect of fibroblast growth factor 2 (FGF2) on osteoblast differentiation. This effect was specific for the N-terminal domain of PTX3 that contains the FGF2-binding site. By using the closed transversal tibial fracture model, we found that ptx3−/− female mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to ptx3+/+ mice (BV/TV 17.05 ± 4.59 vs 20.47 ± 3.32, p = 0.0195). Non-hematopoietic periosteal cells highly upregulated PTX3 expression during the initial phase of fracture healing, particularly CD51+ and αSma+ osteoprogenitor subsets, and callus tissue exhibited concomitant expression of PTX3 and FGF2 around the fracture site. Thus, PTX3 supports maintenance of the bone mass possibly by inhibiting FGF2 and its negative impact on bone formation. Moreover, PTX3 enables timely occurring sequence of callus mineralization after bone fracture injury. These results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair, a reflection of the function of this molecule in tissue homeostasis and repair.

BackgroundThe peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).MethodsPB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB [RANK]) and selected chemokines (CC chemokine receptor 1 [CCR1], CCR2, CCR4, CXC chemokine receptor 3 [CXCR3], CXCR4) were determined in an OCP-rich subpopulation (CD3−CD19−CD56−CD11b+CD14+) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro.ResultsThe OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK+ subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4+ subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs.ConclusionsIn our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-017-1337-6) contains supplementary material, which is available to authorized users.

The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes.The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220 -CD3 -CD11b -/lo CD115 + CD117 + CX3CR1 + and possibly also Ter119 -CD11c -CD135 lo Ly6C + RANK -. In the peripheral blood OCP population bears the monocytoid phenotype B220 -CD3 -NK1.1 -CD11b + Ly-6C hi CD115 + CX3CR1 + , presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3 -CD19 -CD56 -), within immature monocyte subset (CD11b + CD14 + CD16 -), expressing receptors for M-CSF and RANKL (CD115 + RANK + ). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3 -CD19 -CD56 -CD11b + CD14 + , and the disease activity score (DAS28) in the follow-up samples from patient with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption.Control of the activity and migratory behavior of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.Keyword: osteoclast progenitors, arthritis, inflammation, cytokines, chemokines, osteoresorption 3 Inflammation induced bone lossBone is a highly dynamic tissue important for its mechanical and metabolic functions, and characterized by rapid response to numerous physical, endocrine and paracrine stimuli in physiological and pathological conditions. Among others signals, osteoresorptive mediators (such as interleukin (IL)-1, IL-6, IL-15, IL-17, IL, 21, IL-33, tumor necrosis factor (TNF)-α and receptor activator of nuclear factor-κB ligand (RANKL), CCL2, CXCL12) produced by inflammatory/immune cells create conditions that promote maturation and function of osteoclasts [1][2][3][4]. Bone resorption and osteolysis are the prominent features and causes of substantial morbidity in inflammatory processes associated with arthritis as well as with localized bacterial infections of bone and adjacent tissues, peri-prosthetic loosening, vasculitis, connective tissue diseases, chronic viral infections and inflammatory bowel diseases [5][6][7][8]. Bone resorption in arthritisInflammatory arthritides comprise a heterogeneous group of joint disorders that are characterized by chronic inflammatory response as we...

We investigated the bone phenotype of mice with generalized lymphoproliferative disorder (gld) due to a defect in the Fas ligand-mediated apoptotic pathway. C57BL/6-gld mice had greater whole body bone mineral density and greater trabecular bone volume than their wild-type controls. gld mice lost 5-fold less trabecular bone and had less osteoclasts on bone surfaces after ovariectomy-induced bone resorption. They also formed more bone in a model of osteogenic regeneration after bone marrow ablation, had less osteoclasts on bone surfaces and less apoptotic osteoblasts. gld and wild-type mice had similar numbers of osteoclasts in bone marrow cultures, but marrow stromal fibroblasts from gld mice formed more alkaline phosphatase-positive colonies. Bone diaphyseal shafts and bone marrow stromal fibroblasts produced more osteoprotegerin mRNA and protein than wild-type mice. These findings provide evidence that the disturbance of the bone system is a part of generalized lymphoproliferative syndrome and indicates the possible role of osteoprotegerin as a regulatory link between the bone and immune system.