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Craniofacial osteoclasts are essential for site-specific processes such as alveolar bone resorption, tooth eruption, and orthodontic tooth movement. Much of the current understanding of osteoclast development and function comes from studies using long bone-derived cells. Minimal investigation has been done to explore skeletal site differences. The overall goal of this study was to determine if mandibular- and femoral-derived osteoclasts represent distinct populations. To test this hypothesis, bone marrow cells were initially analyzed from the mandible and femur of 2-month-old mice. It was shown that mandibular-derived osteoclasts have enhanced size (mm 2 ) compared with femoral-derived osteoclasts. Since bone marrow macrophages are a heterogenous population, we additionally selected for monocytes and demonstrated that mandibular-derived monocytes also form osteoclasts with increased size compared with femoral-derived monocytes. Osteoclast precursor populations from both skeletal sites were analyzed by flow cytometry. A newly described Ly6C High+ population as well as the Ly6C int population was increased in the mandibular-derived cells. The difference in differentiation potential between monocyte cultures suggests that the increase in the Ly6C High+ population may explain the enhanced differentiation potential in mandibular-derived cells. Monocyte genes such as Pu.1 , C/ebp-a, and Prdm1 are increased in expression in mandibular-derived monocytes compared with femoral-derived monocytes. As expected with enhanced differentiation, osteoclast genes including Nfatc1, Dc-stamp, Ctsk , and Rank are upregulated in mandibular-derived osteoclast precursors. Future studies will determine how changes in the environment of the mandible lead to changes in percentages of osteoclast progenitors and their differentiation potential., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Myocyte enhancement factor 2C (MEF2C) is a transcription factor studied in the development of skeletal and smooth muscles. Bone resorption studies have exhibited that the reduced expression of MEF2C contributes to osteopetrosis and the dysregulation of pathological bone remodeling. Our current study aims to determine how MEF2C contributes to osteoclast differentiation and to analyze the skeletal phenotype of Mef2c- cKO mice ( Cfms-cre ; Mef2c fl/fl ). qRT-PCR and Western blot demonstrated that Mef2c expression is highest during the early days of osteoclast differentiation. Osteoclast genes, including c -Fos, c-Jun , Dc-stamp , Cathepsin K , and Nfatc1 , had a significant reduction in expression, along with a reduction in osteoclast size. Despite reduced CTX activity, female Mef2c cKO mice were osteopenic, with decreased bone formation as determined via a P1NP ELISA, and a reduced number of osteoblasts. There was no difference between male WT and Mef2c- cKO mice. Our results suggest that Mef2c is critical for osteoclastogenesis, and that its dysregulation leads to a sex-specific osteopenic phenotype.

Developing multifunctional nanostructures that promote bone repair while fighting infection is highly desirable in bone regenerative therapies. Previous efforts have focused on achieving one property or another by altering the chemical makeup of nanostructures or using growth factors or antibiotics. We present nanostructures with several simultaneous functional attributes including positive effects of strontium on bone formation and prevention of osteoclast differentiation along with incorporation of antimicrobial peptides (AMP) to prevent infection. To form these multifunctional nanostructures, mesoporous calcium silicate (CaMSN) was modified with high levels of strontium. For this, CaMSNs were either partially substituted (20 wt% Ca) or completely replaced with strontium (Sr) to form Sr-CaMSN or SrMSN. The mesoporous nature of these bioactive silicate nanostructures rendered a configuration for substantial AMP loading as well as their effective delivery. The physico-chemical and structural characterization of synthesized MSNs confirmed the mesoporous nature of the synthesized MSNs and their total surface area, pore size, pore volume and SBF-mediated bioactivity remained unaltered with the incorporation of Sr. However, biological evaluation confirmed that synthesized SrMSN upregulated osteogenic differentiation of mesenchymal stromal cells and significantly downregulated osteoclast differentiation. Also, the AMP-loaded MSNs prevented formation and growth of methicillin resistant Staphylococcus aureus (MRSA) biofilms. Thus, high Sr-containing AMP-loaded SrMSNs may combat MRSA-associated infection while promoting bone regeneration. The controlled availability of therapeutic Sr and AMP release as SrMSN degrade enables its potential application in bone tissue regeneration., (Copyright © 2022 Elsevier B.V. All rights reserved.)

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII(fl/fl);lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.

Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells., ((c) 2009 Wiley-Liss, Inc.)