RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border.However,the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown.Here,we show with GeneChip analysis that RUN and FYVE domain-containing protein 4(RUFY4)is strongly upregulated during osteoclastogenesis.Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo.Furthermore,deleting Rufy4 did not affect osteoclast differentiation,but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes,their trafficking to the membrane,and their secretion of cathepsin K into the extracellular space.Mechanistically,RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes.Consequently,Rufy4-deficient mice were highly protected from lipopolysaccharide-and ovariectomy-induced bone loss.Thus,RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor

DNAX-associated protein 12 kD size(DAP12)is a dominant immunoreceptor tyrosine-based activation motif(ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis.Although several DAP12-associated receptors(DARs)have been identified in osteoclasts,including triggering receptor expressed on myeloid cells 2(TREM-2),C-type lectin member 5 A(CLEC5A),and sialic acid-binding Ig-like lectin(Siglec)-15,their precise role in the development of osteoclasts and bone remodeling remain poorly understood.In this study,mice deficient in Trem-2,Clec5a,Siglec-15 were generated.

Effects of acupotomy on the activity of osteoclasts and osteoblasts in the subchondral bone of rabbits with early and mid-stage knee osteoarthritis models

Objective:To investigate whether acupotomy could inhibit subchondral bone remodeling in knee osteoarthritis(KOA)rabbits by regulating the activity of osteoblasts and osteoclasts.Methods:KOA rabbits were prepared by immobilization for 6 and 9 weeks by Videman method.Nine groups of rabbits(control,6 weeks and 9 weeks model,6 weeks and 9 weeks acupotomy,6 weeks and 9 weeks electroacupuncture,and 6 weeks and 9 weeks drug groups)received acupotomy,electroacupuncture and risedronate sodium intervention,respectively,for 3 weeks.Results:Acupotomy can inhibit the activity of osteoclasts and osteoblasts in subchondral bone by reducing the proteins expression of cathepsin K(CK)and tartrate-resistant acid phosphatase(TRAP)and decreasing the proteins expression of osteocalcin(OCN)and alkaline phosphatase(ALP),to intercept the abnormal bone resorption and bone formation of subchondral bone in 6-week and 9-week immobilization-induced KOA rabbits.Conclusion:These findings indicated that acupotomy may be more advantageous than risedronate sodium intervention in modulating subchondral bone remodeling in KOA rabbits,especially in 9-week immobilization-induced KOA rabbits.

基于优化极限学习机模型的反应堆中子通量与k_(eff)预测方法研究

通过模拟和扩展人类智能,人工智能能够解决预测反应堆k_(eff)和中子通量等问题。本研究选用国际原子能机构(International Atomic Energy Agency,IAEA)反应堆作为研究对象,以稳态时的堆芯中子通量和k_(eff)为预测量,通过堆芯物理分析程序ADPRES生成数据样本后,利用极限学习机(Extreme Learning Machine,ELM)构建中子通量和k_(eff)的基础神经网络模型,随后通过随机森林(Random Forest,RF)评估特征值重要程度以建立各模型最佳输入特征子集,采用遍历方法确定隐藏层最佳神经元数目,最后使用鲸鱼优化算法(Whale Optimization Algorithm,WOA)对其初始权值与阈值进行优化,进一步提高了模型的精度。研究结果显示:经降维优化处理后,神经网络的预测能力有较大提升,其中k_(eff)的预测精度提高了两个量级,中子通量的预测误差降低了87.24%,并且减少了模型训练时间。本文构建方法对快速评估堆芯物理特性有一定参考意义。

Effect of Lanthanum(Ⅲ) on Cytosolic Free Calcium in Isolated Rabbit Mature Osteoclasts

The effect of lanthanum （ Ⅲ ） （La^3 ＋ ） on cytosolic free calcium （ [ Ca^2 ＋ ] i ） in isolated rabbit mature osteoclasts was studied with the employment of fluo-3/AM as an intracellular calcium-sensitive fluorescent probe by using a confocal laser scanning microscope. La^3＋ does not alter basal [Ca^2＋ ]i levels and cell spread area at the concentration of 1.00 × 10^- 8 mol· L ^- 1. However, La^3 ＋ at higher concentrations （ 1. 00 × 10^ - 5 and 1.00 × 10^- 7 mol· L^- 1 ） decreases [ Ca^2 ＋ ] i levels and cell spread area, and greater decreases are observed for the higher concentrations of La^3 ＋ . Since [Ca^2 ＋ ]i affects cytoskeleton and the adhesion properties of osteoclasts, our results seem to suggest that La^3 ＋ inhibit bone resorption by decreasing [Ca^2＋]i in rabbit mature osteoclasts.

Effects of Lanthanum on Bone Resorbing Activity of Rabbit Mature Osteoclasts Co-Cultured with Osteoblasts

The effects of lanthanum （Ⅲ） on the bone resorbing activity of rabbit mature osteoclasts （OCs） in the presence of osteoblasts （OBs） were studied in vitro by measuring the number and area of absorption pits. La（ Ⅲ ） at concentrations ranging from 1.00 × 10^-5 to 1.00 × 10^-8 mol·L^-1 show no effect on mature OC number （P 〉 0.05）. In the OC-OB coculture systems without La（Ⅲ ）, osteoblasts alone did not influence the pit number and area whether the two kinds of cells were in contact or not （ P 〉 0.05）. Under the OC-OB not-in-contact condition, the effect of La（ Ⅲ ） on the bone-resorbing activity of OCs was similar to that of La（Ⅲ） in the absence of OBs （P 〉 0.05）. However, while OCs were in direct contact with OBs, the inhibitory effects of La（ Ⅲ ） on OCs＇ bone-resorbing activity decreased at the concentrations of 1.00 × 10^-5, 1.00×10^-6 and 1.00×10^-7mol·L^-1, and the promotion effects increased at 1.00×10^-8mol·L^-1 （P 〈0.05）. The results suggest that direct cell-cell contact between OC and OB be essential for OBs to play their role in regulating the response of OCs to La（ Ⅲ ）.

Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis,which is characterized by increased bone resorption and inadequate bone formation.As novel antiosteoporotic therapeutics are needed,understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets.This study aimed to provide an overview of transcriptional reprogramming during human osteoclast differentiation.Osteoclasts were differentiated from CD14+monocytes from eight female donors.RNA sequencing during differentiation revealed 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors.These patterns revealed distinct molecular functions associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies and bone mineral density SNPs.Network analyses revealed mutual dependencies between temporal expression patterns and provided insight into subtype-specific transcriptional networks.The donor-specific expression patterns revealed genes at the monocyte stage,such as filamin B(FLNB)and oxidized low-density lipoprotein receptor 1(OLR1,encoding LOX-1),that are predictive of the resorptive activity of mature osteoclasts.The expression of differentially expressed G-protein coupled receptors was strong during osteoclast differentiation,and these receptors are associated with bone mineral density SNPs,suggesting that they play a pivotal role in osteoclast differentiation and activity.The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5 A receptor 1(C5AR1),somatostatin receptor 2(SSTR2),and free fatty acid receptor 4(FFAR4/GPR120).Activating C5AR1 enhanced osteoclast formation,while activating SSTR2 decreased the resorptive activity of mature osteoclasts,and activating FFAR4 decreased both the number and resorptive activity of mature osteoclasts.In conclusion,we report the occurrence of transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as antiresorptive G-protein coupled receptors and FLNB and LOX-1 as potential molecular markers of osteoclast activity.These data can help future investigations identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel antiosteoporotic targets.

CD97 inhibits osteoclast differentiation via Rap1a/ERK pathway under compression

Acceleration of tooth movement during orthodontic treatment is challenging, with osteoclast-mediated bone resorption on the compressive side being the rate-limiting step. Recent studies have demonstrated that mechanoreceptors on the surface of monocytes/macrophages, especially adhesion G protein-coupled receptors (aGPCRs), play important roles in force sensing.However, its role in the regulation of osteoclast differentiation remains unclear. Herein, through single-cell analysis, we revealed that CD97, a novel mechanosensitive aGPCR, was expressed in macrophages. Compression upregulated CD97 expression and inhibited osteoclast differentiation;while knockdown of CD97 partially rescued osteoclast differentiation. It suggests that CD97 may be an important mechanosensitive receptor during osteoclast differentiation. RNA sequencing analysis showed that the Rap1a/ERK signalling pathway mediates the effects of CD97 on osteoclast differentiation under compression. Consistently, we clarified that administration of the Rap1a inhibitor GGTI298 increased osteoclast activity, thereby accelerating tooth movement. In conclusion,our results indicate that CD97 suppresses osteoclast differentiation through the Rap1a/ERK signalling pathway under orthodontic compressive force.

Method of fabricating artificial rock specimens based on extrusion free forming(EFF)3D printing

Three-dimensional(3D)printing technology has been widely used to create artificial rock samples in rock mechanics.While 3D printing can create complex fractures,the material still lacks sufficient similarity to natural rock.Extrusion free forming(EFF)is a 3D printing technique that uses clay as the printing material and cures the specimens through high-temperature sintering.In this study,we attempted to use the EFF technology to fabricate artificial rock specimens.The results show the physico-mechanical properties of the specimens are significantly affected by the sintering temperature,while the nozzle diameter and layer thickness also have a certain impact.The specimens are primarily composed of SiO_(2),with mineral compositions similar to that of natural rocks.The density,uniaxial compressive strength(UCS),elastic modulus,and tensile strength of the printed specimens fall in the range of 1.65–2.54 g/cm3,16.46–50.49 MPa,2.17–13.35 GPa,and 0.82–17.18 MPa,respectively.It is capable of simulating different types of rocks,especially mudstone,sandstone,limestone,and gneiss.However,the simulation of hard rocks with UCS exceeding 50 MPa still requires validation.

Effect of Zhuang Medicine Feilongzhangxue on the Expression of HMGB1-TLR4 / RANKL-NF-κB Signaling Pathway Related Factors in Osteoclasts

Objective:To study the regulatory effect of feilongzhangxue on the levels of HMGB1-TLR4/RANKL-NF-κB signaling pathway related factors HMGB1,RANKL,rank,TRAF-6 and NF-κBp65 in osteoclasts,so as to explore the mechanism of feilongzhangxue intervention in RA;Methods:The osteoclasts with good activity were randomly divided into blank group,methotrexate control group and Zhuang medicine feilongzhang blood containing serum treatment group,which were divided into OC+blank group,OC+methotrexate control group,OC+Zhuang medicine feilongzhang blood containing serum group;The expression of HMGB1,RANKL,rank,TRAF-6 and NF-κBp65 mRNA was detected by RT-PCR;The protein expressions of HMGB1,RANKL,RANK,TRAF-6 and NF-κBp65 were detected by immunofluorescence.Results:PCR results showed that:Compared with the blank group,feilongzhangxue could effectively inhibit the expression levels of HMGB1,RANKL,rank,TRAF-6 and NF-κB p65 mRNA in OC cells,and the inhibitory effect was stronger than methotrexate.Immunofluorescence test results showed that:Compared with the blank group and methotrexate group,feilongzhangxue could effectively inhibit the protein expression of HMGB1,RANKL,rank,TRAF-6 and NF-κB p65 in OC cells.Conclusion:The effect of Zhuang medicine feilongzhangxue on hmgbl-tlr4/rankl-nf-κB signaling pathway of osteoclasts is through the regulation of related factors HMGB1,RANKL,rank,TRAF-6 and NF-κB p65,which may be the key mechanism of Zhuang medicine feilongzhangxue on rheumatoid arthritis.

A Simplified Method for Purifying Osteoclasts from Human Giant Cell Tumor of Bone

Objective: To purify and identify the osteoclasts from the tissue of humangiant cell tumor of bone. Methods: We have developed a new method that allows the purification oflarge numbers of authentic osteoclasts (OCs). The OCs were isolated from tissue of human giant celltumor of bone by 0.25% trypsin and collagenase. We characterized OCs in terms of the expression ofdifferent phenotypic markers of OCs. The phenotypic markers of OC included Tartrate-resistant acidphosphatase staining (TRAP). The expression of calcitonin receptor (CTR), cathepsin K and receptoractivator of necrosis factor κB (RANK) mRNA were examined by RT-PCR. Results: The OC cell purifiedby above method functioned normally in vitro. The purity was about 79.7%. They showed the normalosteoclast phenotypes markers of OC. Conclusion: The method provides a system for performingbiochemical and molecular studies of OCs. The study indicates that the method of purifying theosteoclasts from human GCT cell can be used for research of bone metabolism.

Paracrine and endocrine actions of bone——the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin（SOST） that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or ＂osteokines＂from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin（OCN） secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2（LCN2） is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

Osteoclasts:New Insights

Osteoclasts, the bone-resorbing cells, play a pivotal role in skeletal development and adult bone remodeling. They also participate in the pathogenesis of various bone disorders. Osteoclasts differentiate from cells of the monocyte/macrophage lineage upon stimulation of two essential factors, the monocyte/ macrophage colony stimulating factor （M-CSF） and receptor activation of NF-κB ligand （RANKL）. M-CSF binds to its receptor c-Fms to activate distinct signaling pathways to stimulate the proliferation and survival of osteoclast precursors and the mature cell. RANKL, however, is the primary osteoclast differentiation factor, and promotes osteoclast differentiation mainly through controlling gene expression by activating its receptor, RANK. Osteoclast function depends on polarization of the cell, induced by integrin avβ3, to form the resorptive machinery characterized by the attachment to the bone matrix and the formation of the bone-apposed ruffled border. Recent studies have provided new insights into the mechanism of osteoclast differentiation and bone resorption. In particular, c-Fms and RANK signaling have been shown to regulate bone resorption by cross-talking with those activated by integrin avβ3. This review discusses new advances in the understanding of the mechanisms of osteoclast differentiation and function.

The effects of interleukin-1β in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase （MAPK） signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β （IL-1β）-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-11~ restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

Prostaglandin E2(PGE2), a major cyclooxygenase-2(COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis(OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts(EP4 Lys M) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4 Lys M mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of plateletderived growth factor-BB(PDGF-BB) was also lower in the EP4 Lys Mmice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4 Lys Mmice. Finally, we showed that the Gαs/PI3 K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

Two macrophages, osteoclasts and microglia: from development to pleiotropy

Tissue-resident macrophages are highly specialized to their tissue-specific microenvironments,activated by various inflammatory signals and modulated by genetic and environmental factors.Osteoclasts and microglia are distinct tissue-resident cells of the macrophage lineage in bone and brain that are responsible for pathological changes in osteoporosis and Alzheimer’s disease(AD),respectively.Osteoporosis is more frequently observed in individuals with AD compared to the prevalence in general population.Diagnosis of AD is often delayed until underlying pathophysiological changes progress and cause irreversible damages in structure and function of brain.As such earlier diagnosis and intervention of individuals at higher risk would be indispensable to modify clinical courses.Pleiotropy is the phenomenon that a genetic variant affects multiple traits and the genetic correlation between two traits could suggest a shared molecular mechanism.In this review,we discuss that the Pyk2-mediated actin polymerization pathway in osteoclasts and microglia in bone and brain,respectively,is the horizontal pleiotropic mediator of shared risk factors for osteoporosis and AD.

A 3D-printed molybdenum-containing scaffold exerts dual pro-osteogenic and anti-osteoclastogenic effects to facilitate alveolar bone repair

The positive regulation of bone-forming osteoblast activity and the negative feedback regulation of osteoclastic activity are equally important in strategies to achieve successful alveolar bone regeneration. Here, a molybdenum(Mo)-containing bioactive glass ceramic scaffold with solid-strut-packed structures(Mo-scaffold) was printed, and its ability to regulate pro-osteogenic and antiosteoclastogenic cellular responses was evaluated in vitro and in vivo. We found that extracts derived from Mo-scaffold(Moextracts) strongly stimulated osteogenic differentiation of bone marrow mesenchymal stem cells and inhibited differentiation of osteoclast progenitors. The identified comodulatory effect was further demonstrated to arise from Mo ions in the Mo-extract,wherein Mo ions suppressed osteoclastic differentiation by scavenging reactive oxygen species(ROS) and inhibiting mitochondrial biogenesis in osteoclasts. Consistent with the in vitro findings, the Mo-scaffold was found to significantly promote osteoblastmediated bone formation and inhibit osteoclast-mediated bone resorption throughout the bone healing process, leading to enhanced bone regeneration. In combination with our previous finding that Mo ions participate in material-mediated immunomodulation, this study offers the new insight that Mo ions facilitate bone repair by comodulating the balance between bone formation and resorption. Our findings suggest that Mo ions are multifunctional cellular modulators that can potentially be used in biomaterial design and bone tissue engineering.

Specifc inflammatory osteoclast precursors induced during chronic inflammation give rise to highly active osteoclasts associated with inflammatory bone loss

Elevated osteoclast(OC)activity is a major contributor to inflammatory bone loss(IBL)during chronic inflammatory diseases.However,the specific OC precursors(OCPs)responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood.We identified two distinct OCP subsets:Ly6C^(hi)CD11b^(hi) inflammatory OCPs(iOCPs)induced during chronic inflammation,and homeostatic Ly6C^(hi)CD11b^(lo)OCPs(hOCPs)which remained unchanged.Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions,they expanded during chronic inflammation and generated OCs with enhanced activity.In contrast,hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment.Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs,highlighting that different osteoclast populations are formed by distinct precursors.We further identified the TNF-αand S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation.Furthermore,we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α^(-/-)mice,in correlation with attenuated IBL.Our findings suggest a central role for iOCPs in IBL induction.iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

Biodegradation of Tricalcium Phosphate Ceramics by Osteoclasts

Biodegradation of tricalcium phosphate (TCP) ceramics was observed through mixed culture of osteoclasts and TCP discs in vitro in this study. Osteo-clasts were isolated from newborn SD rat's marrow of long bone and cultured on TCP discs. The culture terminated at the 48th h and 96th h respectively. Under an inverted microscope, the osteoclasts imparted round or oval body with multinu-clear and many thin processes. These cells were positively stained for tartrate-re-sistance acid phosphatase (TRAP). Scanning electron microscope showed that many resorption lacunae on TCP disc surface and their diameters were smaller than 20 μm. Osteoclasts were located in the lacunae. At the 96th h, the resorption lacunae become larger and osteoclasts showed degeneration. It is suggested that osteoclasts possess ability to re-absorb TCP ceramics under in vitro culturing condition.

BMP-IHH-mediated interplay between mesenchymal stem cells and osteoclasts supports calvarial bone homeostasis and repair

Calvarial bones are connected by fibrous sutures. These sutures provide a niche environment that includes mesenchymal stem cells(MSCs), osteoblasts, and osteoclasts, which help maintain calvarial bone homeostasis and repair. Abnormal function of osteogenic cells or diminished MSCs within the cranial suture can lead to skull defects, such as craniosynostosis. Despite the important function of each of these cell types within the cranial suture, we have limited knowledge about the role that crosstalk between them may play in regulating calvarial bone homeostasis and injury repair. Here we show that suture MSCs give rise to osteoprogenitors that show active bone morphogenetic protein(BMP) signalling and depend on BMP-mediated Indian hedgehog(IHH) signalling to balance osteogenesis and osteoclastogenesis activity. IHH signalling and receptor activator of nuclear factor kappa-Β ligand(RANKL) may function synergistically to promote the differentiation and resorption activity of osteoclasts. Loss of Bmpr1a in MSCs leads to downregulation of hedgehog(Hh) signalling and diminished cranial sutures. Significantly, activation of Hh signalling partially restores suture morphology in Bmpr1a mutant mice, suggesting the functional importance of BMP-mediated Hh signalling in regulating suture tissue homeostasis. Furthermore, there is an increased number of CD200+ cells in Bmpr1a mutant mice, which may also contribute to the inhibited osteoclast activity in the sutures of mutant mice. Finally, suture MSCs require BMPmediated Hh signalling during the repair of calvarial bone defects after injury. Collectively, our studies reveal the molecular and cellular mechanisms governing cell–cell interactions within the cranial suture that regulate calvarial bone homeostasis and repair.