Developmental origin, functional maintenance and genetic rescue of osteoclasts.

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss.

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2-/- ) and wild-type (Tfr2+/+ ) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2+/+ and Tfr2-/- mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Association between bone turnover markers and periodontitis: A population-based cross-sectional study.

AIM: To examine the associations between bone turnover markers and periodontitis in two cross-sectional population-based studies. MATERIALS AND METHODS: We used data from two independent adult samples (N = 4993), collected within the Study of Health in Pomerania project, to analyse cross-sectional associations of N-procollagen type 1 amino-terminal propeptide (P1NP), C-terminal cross-linking telopeptide, osteocalcin, bone-specific alkaline phosphatase (BAP), fibroblast growth factor 23, wingless-type mouse mammary tumour virus integration site family member 5a (WNT5A), and sclerostin values with periodontitis. Confounder-adjusted gamma and fractional response regression models were applied. RESULTS: Positive associations were found for P1NP with mean pocket probing depth (PPD; eß=1.008 ; 95% confidence interval [CI]: 1.001-1.015), mean clinical attachment loss (mean CAL; eß=1.027 ; 95% CI: 1.011-1.044), and proportion of sites with bleeding on probing (%BOP; eß=1.055 ; 95% CI: 1.005-1.109). Similar associations were seen for BAP with %BOP ( eß=1.121 ; 95% CI: 1.042-1.205), proportion of sites with PPD ≥4 mm (%PPD4) ( eß=1.080 ; 95% CI: 1.005-1.161), and sclerostin with %BOP ( eß=1.308 ; 95% CI: 1.005-1.704). WNT5A was inversely associated with mean PPD ( eß=0.956 ; 95% CI: 0.920-0.993) and %PPD4 ( eß=0.794 ; 95% CI: 0.642-0.982). CONCLUSIONS: This study revealed scattered associations of P1NP, BAP, WNT5A, and sclerostin with periodontitis, but the results are contradictory in the overall context. Associations reported in previous studies could not be confirmed.

Toward Biofabrication of Resorbable Implants Consisting of a Calcium Phosphate Cement and Fibrin-A Characterization In Vitro and In Vivo.

Cleft alveolar bone defects can be treated potentially with tissue engineered bone grafts. Herein, we developed novel biphasic bone constructs consisting of two clinically certified materials, a calcium phosphate cement (CPC) and a fibrin gel that were biofabricated using 3D plotting. The fibrin gel was loaded with mesenchymal stromal cells (MSC) derived from bone marrow. Firstly, the degradation of fibrin as well as the behavior of cells in the biphasic system were evaluated in vitro. Fibrin degraded quickly in presence of MSC. Our results showed that the plotted CPC structure acted slightly stabilizing for the fibrin gel. However, with passing time and fibrin degradation, MSC migrated to the CPC surface. Thus, the fibrin gel could be identified as cell delivery system. A pilot study in vivo was conducted in artificial craniofacial defects in Lewis rats. Ongoing bone formation could be evidenced over 12 weeks but the biphasic constructs were not completely osseous integrated. Nevertheless, our results show that the combination of 3D plotted CPC constructs and fibrin as suitable cell delivery system enables the fabrication of novel regenerative implants for the treatment of alveolar bone defects.

Loss of Dkk-1 in Osteocytes Mitigates Alveolar Bone Loss in Mice With Periodontitis.

Background: Periodontitis is a highly prevalent infection-triggered inflammatory disease that results in bone loss. Inflammation causes bone resorption by osteoclasts, and also by suppression of bone formation via increase of Dickkopf-1 (Dkk-1), an inhibitor of Wnt signaling. Here, we tested the hypothesis that osteocytic Dkk-1 is a key factor in the pathogenesis of periodontitis-induced alveolar bone loss (ABL). Methods: Twelve-week-old female mice with a constitutive deletion of Dkk-1 specifically in osteocytes (Dkk-1fl/fl;Dmp1:Cre) were subjected to experimental periodontitis (EP). Cre-negative littermates served as controls. EP was induced by placing a ligature around the upper 2nd left molar, the contralateral side was used as control. Mice were killed after 11 days and maxillae removed for micro-CT and histological analyses. The mRNA expression of Dkk-1, Runx2, Osteocalcin, OPG, RANKL, RANKL/OPG ratio, LEF-1, and TCF-7 were assessed in maxillae, while mRNA expressions of TNF and IL-1 were evaluated on gingiva using real-time PCR. Blood samples were collected for Dkk-1, CTX, and P1NP measurement by ELISA. Results: The deletion of Dkk-1 in osteocytes prevented ABL in mice with EP, compared to Cre-negative control mice with EP. Micro-CT analysis showed a significant reduction of bone loss (-28.5%) in EP Dkk-1fl/fl;Dmp1:Cre-positive mice compared to their littermate controls. These mice showed a greater alveolar bone volume, bone mineral density, trabecular number, and trabecular thickness after EP when compared to the Cre-negative controls. The local expression in maxillae as well as the serum levels of Dkk-1 were reduced in Dkk-1fl/fl;Dmp1:Cre-positive mice with EP. The transgenic mice submitted to EP showed increase of P1NP and reduction of CTX-I serum levels, and increase of TCF-7 expression. Histological analysis displayed less inflammatory infiltrates, a reduction of TNF and IL-1 expressions in the gingiva and fewer osteoclasts in Cre-positive animals with EP. Moreover, in mice with EP, the osteocytic deletion of Dkk-1 enhanced bone formation due to increased expressions of Runx2 and Osteocalcin and decreased expression of RANKL in maxillae. Conclusion: In summary, Dkk-1 derived from osteocytes plays a crucial role in ABL in periodontitis.

Stimulation of bone formation by monocyte-activator functionalized graphene oxide in vivo.

Nanosystems are able to enhance bone regeneration, a complex process requiring the mutual interplay between immune and skeletal cells. Activated monocytes can communicate pro-osteogenic signals to mesenchymal stem cells and promote osteogenesis. Thus, the activation of monocytes is a promising strategy to improve bone regeneration. Nanomaterials specifically selected to provoke immune-mediated bone formation are still missing. As a proof of concept, we apply here the intrinsic immune-characteristics of graphene oxide (GO) with the well-recognized osteoinductive capacity of calcium phosphate (CaP) in a biocompatible nanomaterial called maGO-CaP (monocytes activator GO complexed with CaP). In the presence of monocytes, the alkaline phosphatase activity and the expression of osteogenic markers increased. Studying the mechanisms of action, we detected an up-regulation of Wnt and BMP signaling, two key osteogenic pathways. The role of the immune activation was evidenced by the over-production of oncostatin M, a pro-osteogenic factor produced by monocytes. Finally, we tested the pro-osteogenic effects of maGO-CaP in vivo. maGO-CaP injected into the tibia of mice enhanced local bone mass and the bone formation rate. Our study suggests that maGO-CaP can activate monocytes to enhance osteogenesis ex vivo and in vivo.

Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function.