INTRODUCTION

Bone is a composite tissue,whose matrix consists of proteins and minerals,and which constantly undergoesmodelling and remodeling through the coordination of osteoclasts,osteoblasts,and osteocytes.Osteoclasts,derived from mononuclear hematopoietic myeloid lineage cells,are responsible for bone resorption.1 Osteoblasts,accounting for the(4-6)%of total resident cells in the bone,are responsible for bone formation.2Osteocytes,the most abundant cells in bone,are terminally differentiated from the osteoblasts,and are embedded in mineralized bone matrix.Osteocytes play a critical role in sensing mechanical loading and regulate functions of osteoclasts and osteoblasts.3

8月5日，国际母婴用品专家贝亲在北京BVLGARI酒店举行“My precious臻宝之旅钢琴演奏会暨贝亲&京东母婴奶爸盛典”启动仪式。这场视听盛宴吸引了300组热情前来体验的准妈妈和准爸爸家庭。

The interaction and coordination of these bone cells are important for maintaining bone homeostasis.Bone formation usually begins with the death of osteocytes.3The apoptotic osteocytes release bioactive molecules,which induce other viable osteocytes to secret receptor activator of nuclear factor κB ligand(RANKL)which is important for osteoclast differentiation.4Subsequently,osteoclastprecursorsare recruited by chemokines such as monocyte chemoattractant protein(MCP)-1,-2,and-3.5The binding of receptor activator of nuclear factor κB(RANK)-RANKLon thesurface of monocytesthen initiates osteoclastogenesis.6-7Meanwhile,osteoblasts produce bioactive molecules including macrophage colony-stimulating factor(M-CSF),MCP-1,and RANKL for the further recruitment and differentiation of osteoclast precursors.5,8 While resorbing damaged bone,osteoclastsspontaneouslysecret“coupling factors”,such as insulin-like growth factor(IGF)I and II and transforming growth factor(TGF)-β,which mediate the refill of resorbed lacunae by osteoblast.9Finally,bone formation is completed when the newly mineralized-extracellular bone matrix completely replaces the resorbed bone matrix.10

Bone-derived exosomes are considered to be essential for intercellularcommunication between bone cells.Exosomemediated transfer of nucleic acid or protein cargos between bone cells can bypass the space barriers between different cells,and plays a vital role in the crosstalk between bone cells regulating bone homeostasis.As the role of exosome is a new mechanism of bone formation and homeostasis,which has only recently emerged,we summarize the characteristics of exosomes,itemise the known functions of exosomes in bone homeostasis,and discuss their potential for clinical applications.

HISTORY OF THE EXOSOME

A general history of the vesicular nature of exosomes Exosomes,11microvesicles,12and secretory autophagosomes13are three typicalextracellularvehicles(EVs)identified recently.However,in early studies,there was no detailed classification or understanding of these extracellular vesicles.

以后，我父亲在国军参加了我们党的地下组织，后来到了延安，再后来随许世友将军的部队到了东北，直到全国解放他才回到济南，找到了居无定所的奶奶……1951年出生的我，因为我父母部队换防和职务调动，小的时候就在奶奶身边长大。六十年代初，我父母转业到地方，他们才回到了我奶奶身边，而我才回到爹妈的怀抱。

Cellular vesicular components were recognised 140 years ago.Under dark-ground illumination,serum-derived particles were first seen by Edmunds in 1877.14The main mass of these particles was then proved as fat in 1939.14Since the function of these particles was unclear,they were just seen as blood dust.14Clearer structure of cellular vesicles was then seen in microscope in 1962.15 However,the function of cellular vesicular components remains mysterious until 1969,when the finding of crystals of appetite suggested the participation of cartilage-derived matrix vesicles in calcification.16Five years later,microvesicles in fetal calf serum were detected,which was the last class of EVs detected before exosome was defined.17

Osteocyte-derived exosomes in bone homeostasis

在一定程度上，软件中用户账号需要与学生本人具有同等法律效力，特别在审批、签到等环节。这是软件能够顺利运行推广的前提条件。因此，学院需要明确告知学生该账号在学生管理工作中的重要性，提醒同学关注账号安全并签订协议。

The past decade has witnessed an acceleration of exosomal investigations,especially in studies of exosomal function.It is believed that exosomes are the most clearly defined group of secreted membrane vesicles,characteristically containing nucleic acid and proteins for cell signalling.25Physiologically,they are critical to the immune system as they are involved with both stimulatory and tolerogenic responses.26Also,it has been postulated that exosomes could be involved in regeneration,reducing tissue injury and improving tissue repair.27Moreover,they may also be involved in tumor progression28and delivery of inflammatory mediators.29Consequently,the investigation of exosomes is becoming increasingly attractive as they are now suggested to be the key regulators of various cellular and physiological functions(Fig.1).

History of bone-derived exosome

The history of bone-derived exosomes,however,is relatively recent.In 1975,extracellular membrane particles were first found in bone marrows which suggests a possible link between multiple myeloma-derived extracellular vesicles and bone tissue damage.30 Then in 1979,normal bone-derived EVs were first mentioned when alveolar bone-derived extracellular matrix vesicles were detected by microscopy.31In 1980,osteoblast-derived matrix vesicles were investigated through ultrastructural techniques.While comparing scanning electron microscopy(SEM)with transmission electron microscopy(TEM),researchers theorised that osteoblast-derived vesicles probably serve as the initial locus of calcification32(Fig.2).

The first mention of bone-derived exosomes was 20 years after the naming of the exosome.33At the beginning,bone marrow stromal cell-derived exosomes were the focus of bone-derived exosomes.However other bone cell-secreted exosomes were barely mentioned until 2013 when osteoclast precursors were reported to release exosomes.34This initiated the investigation of exosomes from other bone cells.In 2015,the proteome of osteoblast-derived exosomes was for the first time investigated.35 In 2016,the characteristics and regulatory activities of osteoclastderive exosomes were demonstrated.36Then in 2017,osteocytederived exosomes and their miRNA contents were demonstrated.37Now,the emerging data of bone-derived exosomes has established the details of exosome-based cell-to-cell interaction in bone.

更需要警惕的是“粮食银行”涉及粮食生产、流通等众多环节，缺乏规范的规章制度，甚至个别地方加工作坊也挂着“粮食银行”的招牌，发展情况良莠不齐，容易形成风险漏洞，如果出现问题或将形成连锁负面效应。

COMPOSITIONS OF EXOSOMES

The function and biological characteristic of exosomes are determined by their specific contents.Among the exosomal components,lipids,proteins,and nucleic acids are three main cargos which determine the specificity of exosomes38and distinguish them from other extracellular vesicles(Fig.3).A great variety of exosomal cargos have already been identified in exosomes and put together into a database named Exocarta,39 which was subsequently integrated into a broader database,Vesiclepedia.40Some examples of exosomal cargos are summarized in(Table 1).

(1)胡家庄碳酸岩型稀土矿流体包裹体以气液两相H2O包裹体为主，均一温度和盐度变化范围与微山稀土矿极具相似性，表明与其具有相似的成矿物理环境。

Lipids

Exosomal structure and cargo sorting are largely dependent on lipid composition.Variouslipidsin exosomeshave been investigated in the past decades.In a study of cancer cellderived exosomes,more than 520 lipids from 36 different classes were identified.41Lipids generally are enriched in exosomal membranes.The major non-polar lipids in plasma membrane are sterols,which are highly enriched in multivesicular bodies(MVBs)from late endosomes.42Sphingolipids are also important for exosomal membrane construction,in which sphingomyelin is the dominant component,and is also involved in cargo sorting.43 Among exosomal membrane phospholipids,phosphatidylserine is of importance for being the activator of negative charge and the recruiter of signalling proteins.44-45

草莓的品种比较繁多，目前种植较多的品种主要有明旭、春旭、星都号、石莓号草莓等。这些品种具有一定的抗病性，如石莓号的休眠期很短，平均产量超过330克，适合加工生产和直接鲜食。由此可见，在实际的栽培过程中，品种的选择以早熟、大果、丰产、果实硬度为主，同时结合所在地区的气候条件等，选择具有针对性的抗病品种。

Besides contributing to the composition of the exosomal bilayer membranes,lipids in exosomes also play important roles in exosomal trafficking.During the formation of exosomes,the enrichment of sphingomyelin is found in membrane lipid rafts.46 As a result of increased sphingomyelin,down-regulation of ceramide and diacylglycerol occurs and finally reaches a balanced proportion in exosomes.47Moreover,lipids play multiple roles in the sorting of nucleic acids and protein.In miRNA sorting,neutral sphingomyelinase 2 is the first molecule suggested to be associated with this mechanism.48Other lipids such as sphingomyelin,ceramide,and sphingosine 1-phosphate have been proved to play important roles in protein sorting mechanisms.49 On the other hand,endosomal sorting complex required for transport(ESCRT)-independentexosomesecretion islargely dependent on lipids,which are reported to significantly participate in the release of proteolipid-positive exosomes50and Aβpeptide-bearing exosomes.51Although lipids are not the main participators of exosomal intercellular communication,their roles for maintaining the biological characteristics of exosomes are of importance.

Proteins

Through proteomic analysis,many proteins have been found in all mammalian exosomes,such as cytoskeletal components(tubulin,actin,co filin,pro filin),annexins(annexins I,II,IV,V,and VII),and the small GTPase family members rab7 and rab11.Among all these exosomal proteins,cytosolic exosomal enriched proteins such as Alix and TSG101,tetraspanins like CD9 and CD63 are the markers for distinguishing exosomes from other extracellular particles(Table 1).The detection of the proteins listed above allows researchers to quickly assess the characteristic of exosomes.52Recent studies have also suggested that the heat-shockproteins(Hsp)are highly prevalent in exosomes.Among them,Hsp40 can improve the protein-folding environment in recipients,and Hsp70 is the up-regulator of pro-inflammatory cytokines.53 Protein composition is also crucially involved in ESCRT-dependent cargo sorting during the formation of exosomes.In ESCRT(-0,-I,-II,-III),recombinant human vacuolar protein sorting proteins(VPS proteins)play a major role functioning as membrane binders and cargo recognisers.54

Besides the various proteins mentioned above,there are also several other proteins in exosomes that reflect the specificity of cell origin and distinct exosomal functions.For example,latent membrane protein 1(LAMP1)is highly expressed in exosomes released from nasopharyngeal cancer(NPC)-derived malignant epithelial cells.55Similarly,a specific cell surface proteoglycan,glypican-1(GPC1),was detected in exosomes from pancreatic cancer.56Collectively,to maintain the specificity of exosomes derived from different donors,various protein cargos must be sorted into exosomes before their release.

Nucleic acids

Nucleic acids are also enriched in exosomes.Coding RNAs,noncoding RNAs,single-stranded or double-stranded DNAs are all found in exosomes.57-59

It is reported that more than 1 600 mRNAs and 700 miRNAs are detected in mammalian cell-derived exosomes.mRNAs contained in exosomes are usually related to cytogenesis,protein synthesis,and RNA posttranscriptional modification.57Exosomal mRNA have been used as biomarkers since they are specific cargos.60In patients with kidney diseases,downregulation of exosomal CD2AP mRNA has been detected in Urine,which can be used for early diagnosis.61Exosomal mRNAs are also suggested to be involved in drug resistance of tumors.Therefore,the detection of exosomal mRNA level may be used to predict optimal treatment options as well as prognosis.62Another recent report suggests that synthetic exosomal mRNA triggers exogenous protein expression,this may be a novel approach for treatment of genetic protein deficiencyrelated diseases.63

某土质心墙堆石坝，建基面高程为2 930.00 m，坝高150 m，顶宽14 m，坝体内设置黏土心墙防渗体，坡度均1∶0.25，顶宽6 m。大坝上下游坡面均在3 040.00 m高程处设4 m宽马道，上游马道以上坡比为1∶2.5，以下为1∶2.25，下游坡比均为1∶2.0。坝基以下为深度为500 m左右的深厚覆盖层，大致分为4层，在心墙底部设1.3 m厚、150 m深的混凝土防渗墙作为坝基防渗措施，插入心墙深度15 m，另在上游坝脚设置1.3 m厚、50 m深的副防渗墙，上游坝基面设水平防渗层。上游正常蓄水位3 070.00 m，对应的尾水位为2 940.00 m。

2016年，倦怠发生比例最高的是重症医学(55%)、泌尿医学(55%)和急诊医学(55%)；2017年，倦怠比例发生最高的是急诊医学(59%)、妇科医学(56%)和家庭医学(55%)；2018年倦怠发生比例最高的是重症医学(48%)、神经医学(48%)和家庭医学(47%)。见表1。

Exosomal trafficking involves three distinct mechanisms:cargo sorting,exosome release and exosome uptake(Fig.4).During the generation of the endosomal machinery,ILVs,the early stage of exosomes,are formed through inward budding.Together with the sorting of specific proteins,lipids and nucleic acids into ILVs,the formation of MVEs results.72Subsequently,MVE fuse with the cell membrane leading to the secretion of exosomes.Following that,the surface binding protein activates the uptake of exosomes in the recipient cells.73Finally,as endocytosis progresses,exosomes release their contents which may influence regulatory processes or they may be degraded in lysosomes.74

EXOSOMAL TRAFFICKING

Exosomal DNA studies began much later than that of RNA,consequently,there is less information available in the literature.To the best of our knowledge,both single-stranded and doublestranded DNAs are contained in exosomes.58Evidence has suggested that carrying cytoplasmic DNAs in exosomes protects against cell senescence and cell death caused by DNA injury.Cells can secret exosomes and remove harmful DNAs to extracellular matrix.68-69However,there is only limited data to elucidate the function of double-stranded DNA in exosomes,and only little is known about the contribution of single-stranded DNAs.In a study of cancer cells,double-stranded DNA is reportedly being used to identify the mutations in cancer cells.58Intriguingly,the expression of DNA cargo in tumor cell-derived exosomes is much higher than that in normal cell-derived exosomes,suggesting that tumor cells can modify target cells via the transfer of DNAs.70There is still long way to go for the complete understanding of the role of exosomal DNA since the mechanism of chromosomal DNA sorting within intralumenal vesicles(ILVs)is still largely unknown.71

Sorting cargos into exosomes

Sorting of protein into exosomes relies on specialized mechanisms,which ensure the specificity of exosomes for various intracellular communication purposes.Here,the ESCRT system,constituted of four multiprotein subcomplexes(ESCRT 0,I,II,and III)appears to be the main mechanism for exosomal formation.75 ESCRT 0,I,and II are responsible for recognizing and sequestering ubiquinated membrane proteins at the endosomal membrane,and ESCRT III is responsible for membrane budding and repartition of intraluminal vesicles.76Categorizing of exosomes,however,appears to be a part of cargo ubiquination and only specific ESCRT segments are involved.77The sorting of membrane proteins of the syndecan family into exosomes is regulated by an ESCRT accessory protein Alix through the cytosolic adaptor syntenin.74 Alix then binds to ESCRT III which is in control of ILV formation at the MVEs.78Lateral involvement of heparin sulfate polysaccharide chains was reported to determine syndecan complex formation,which are degraded into shorter ones by heparinase activity in endosomes,favoring clustering of syndecans.79Heparinaseinduced recruitment is also believed to incite the binding of syndecan cytoplasmic domains to PDZ domains of syntenin,leading the sorting of proteins via Alix-ESCRT pathway.78,80

Bone homeostasis is of critical importance and relies on the transfer of active molecules between cells,which are summarized in(Table 2).Previous studies have suggested a direct interaction with secretion exchange among bone cells to occur.110-111 Recently,however,compelling evidence has emerged to show the regulatory activities that exosomes exert in bone remodelling.Almost all bone cells have been suggested to secret exosomes,and the relationship between bone remodelling and bone-cell derived exosomes is now well documented.Published reports have suggested that transfer of exosomal specific proteins,mRNA and miRNA is the main mechanism for exosome-mediated bone remodelling.This crosstalk establishes a novel network for cell-tocell interaction during bone homeostasis.112

Exosomes also contain abundant miRNAs.In the immune system,miRNA-enriched exosomes are released from T-lymphocyte cells,B-lymphocyte cells and DCs,and the miRNAs are involved in the interaction between T-lymphocytes and antigen-presenting cells.64-65In several tumors,exosomal miRNAs participate in tumor growth,66metastasis,and drug resistance.67 Since specific variation of exosomal miRNAs can be detected in some diseases,exosomal profiling can be used as a tool for disease detection.

Nucleic acid sorting,however,relies on a different mechanism.While DNA sorting is still largely unknown,RNA sorting is concluded previously.Loading RNA into exosomes begins with the formation of the raft-like region.84Subsequently,anionic phospholipids are enriched in the raft-like region of exosomes,which then recruits neutral sphingomyelinase 2 to produce ceramide molecules,an indispensable factor for RNA sorting.85-87Binding of RNAs to the raft-like region is dependent on differential af fi nity of RNA motifs,88and randomly structured RNAs can bind to rafted domains with a 20-fold higher af fi nity.Once binding to the budding in the raft-like region,the RNA becomes encapsulated into ILVs and then released into the extracellular space within this vesicle.84

Release of exosomes

The greatest difference in the exocytosis pathway between exosomes and other extracellular vesicles(autophagosomes and microvesicles)is that exosomes are dependent on late endosomes for their release,74and fusion of the MVBs,the late endosomes containing ILVs,with the plasma membrane is the last step before the exosomes are secreted to extracellular matrix.During this phase SNARE proteins and synaptotagmin family members are the main mediators.89Exosomal exocytosis requires SNARE complexes,consisting of syntaxin 7,synaptotagmin 7,and VAMP 7.The SNARE complex is activated by upregulation of intercellular calcium which is Rab protein-dependent.90Subsequently,vesicle(v)-SNAREs and target(t)-SNAREs promote the apposition of budding vesicles and cell membranes.91After the coupling of v-SNAREs and t-SNAREs,the chaperone ATPase N-ethylmaleimidesensitive factor(NSF)and soluble NSF attachment proteins(SNAPs)catalyze the disassembling of SNARE complexes,leading to the release of exosomes.91

Another key factor for exosomal release involves Rab proteins.They are a family of more than 60 proteins which participate in vesicle budding,cytoskeleton interaction and tethering of the receptor compartment to the membrane.92 Several examples revealed their participation in exosomal release.In oligodendroglia,Rab 35 was found to participate in PLP(genuine myelin proteins)-bearing exosomal secretion.93Moreover,Rab 27A and Rab27B have been linked to MVBs interaction with the plasma membrane.94These Rab proteinsare thoughtto participate in the eventual fusion of the membranes of exosomes and the plasmalemma of donor cells,resulting in the exocytosis of exosomes.95

Uptake of exosomes

The fusion of exosomes with recipient cells relies on the interaction of vesicular ligands with cellular receptors,such as tetraspanins,integrins,and intercellular adhesion molecules(ICAMs),which induce the binding of exosomes to the surface of target cells.The recognition of surface proteins is the first step during exosomal internalization.96Compelling evidence has proved that exosomal uptake is highly dependent on the signalling statusoftargetcellsand ofexosomalsurface proteins.11,97-98

During exosomal internalization,various pathways,including those ofendocytosis,phagocytosis,micropinocytosis,and membrane fusion,are shown to participate.99Among them,endocytosis seems to be the commonest way for exosomal uptake.This is a quick process occurring within 15 min.100The most distinctive part of exosomal endocytosis is inward budding of the plasmalemma,which is dependent on the participation of caveolin101and clathrin.102By contrast,during macropinocytosis,exosomes are attached to a highly ruffled region on the cell surface and then taken in via the internalization of the whole region.103This process is similar to phagocytosis.104

A breakthrough in exosomal investigation took place in 1996 when peptide-major histocompatibility complex(MHC)class II complexes-enriched exosomes released from B cells targeting T cells were detected.This finding first described the role of exosome in cell-to-cell communication.22Following that,dendritic cell(DC)-derived exosomes23and tumor-derived exosomes24were investigated one after the other.These two studies showed the interactions and crosstalk between DCs and tumor cells.DC-derived exosomes could suppress the growth of tumors,and tumor cell-derived exosomes which contained tumor-rejection antigens could be carried by DCs for cross-protection from tumors.23-24These findings were appealing to tumor investigators,and resulted in the generation of numerous reports associated with the tumor-derived exosomes.

Moreover,exosomes can also directly fuse their membrane with the plasma membrane of target cells.105This is dependent on two steps of intermediates:hemifusion structures and fusion pores.106-107In most cases hemifusion structures are suggested to be lipid mixture without content mixing which represents the content of outer leaflets but not the inner leaflets of the two bilayers.106Fusion initiates from the formation of fusion stalk,a point-like membrane protrusion of outer leaflet that establishes an hourglass-like connection between the apposed monolayers.108 Then an immediate contact of proximal leaflets leads to the formation of hemifusion stalk where leaflets fused and distal leaflets unfused.Finally,a fusion pore opens in the hemifusion diagram dependent on the expansion of stalk,109where a connection between apposed membranes leads to the release of secretions.107

EXOSOMES IN BONE HOMEOSTASIS

ESCRT-independent protein sorting is another important pathway for exosomal formation.This process requires the formation of a tetraspanins-associated dynamic membrane platform,where cytosolic and transmembrane proteins exert their ability to accept specific proteins into ILVs.81Examples can be seen in CD63-induced endosomal sorting in melanocytes,82 and in tetranspanins-dependent recruitment of cholesterol-contained cone-like structures for inward budding.83Although ESCRT-independent protein sorting is different to its counterpart,they both undergo cargo clustering and membrane budding.

Exosome induces osteogenic differentiation of mesenchymal stem cells(MSCs)and osteogenesis

Bone remodelling is a complex process,which is mainly associated with two steps:osteoclastogenesis(for clearance of damaged bone tissues)and osteogenesis(for bone formation).It has been shown that exosomes are crucially involved in these two steps(Fig.5).

During the process of bone formation,exosomes are suggested to involve in osteogenic differentiation of MSCs.Monocyte-derivedexosomes are important stimulators for osteoblast differentiation.34 Fusion of these exosomes with MSCs can trigger the up-regulation of two osteogenic markers:RUNX2 and BMP-2.34Intriguingly,newly formed osteoblasts can also secret exosomes to affect their progenitor cells.A group of researchers113found that mature osteoblast-derived exosomes could trigger variation of miRNA expression pro fileswhich,in turn,cooperatively inhibitthe expression of Axin1,a central component of Wnt signalling pathway.As a result,β-catenin was up-regulated leading to the enhancement of osteogenic differentiation.

Osteogenesis is also dependent on exosomal functions.Before differentiating into osteoblasts,osteoblast precursors secret exosomes to promote osteogenesis.114During fracture healing,bone marrow stem cell-derived exosomes express MCP-1,MCP-3,SDF-1,angiogenicfactors,mRNAsand miRNAsand cooperatively contribute to bone remodelling.114They probably also enhance osteoblastproliferation and differentiation by upregulating osteogenesis-related proteins(RUNX-2,ALP,OCN,and OPN),as well as several genes(miRNA-196a,miRNA-27a,and miRNA-206.115 Enhancement of osteoblast proliferation induced by MSC-derived exosomes has also been reported and that the MAPK pathway may be a key factor in exosome-mediated osteoblast activity.116

In addition,exosomes derived from osteoblasts and osteoclasts are also involved in osteogenesis.Osteoblastscan secret exosomes to enhance osteogenesis and Let-7-enriched exosomes derived from osteoblastshavebeen reported to enhance osteogenesis by regulating AT-hook 2(HMGA2)and AXIN2.113,117 By contrast,osteoclast-derived exosomes act as inhibitors of osteogenesis.Exosomal miR-214-3p was suggested to be involved in the inhibition ofosteoblastactivity by targeting the 3′-untranslated region(UTR)of ATF4 mRNA.The exosomal transfer of miR-214-3p from osteoclasts to osteoblasts was also detected in vitro and thus triggered the reduction of bone mass in mice models.36,118-119

Exosome induces osteoclastogenesis and bone resorption

It is widely accepted that osteoclastogenesis is the basis for bone resorption.The classical osteoclastogenesis model is based on the direct interaction between different bone cells.However,recent studies have suggested a novel mechanism dependent on crosstalk phenomena.Initially,osteoblasts secret RANKL-enriched exosomes which targets monocytes.The RANKL-RANK binding on the monocyte surface then activates osteoclastogenesis.120This process can be augmented by MSC-derived exosomes that can upregulate the expression of Nfatc1,Trap,and Ctsk.While osteoclast differentiation is initiated,the mechanism that controls the number of osteoclasts is initiated.This can be mediated either by osteoclast-derived exosomes or osteoblast-derived exosomes.121-123Newly formed osteoclasts release RANK-enriched exosomes,and these exosomes can either directly fuse to osteoblasts or competitively bind RANKL in the extracellularmatrix to regulate the formation of osteoclasts121(Fig.5b).Additionally,osteoblasts can release exosomes containing miR-503-3p to inhibit osteoclastogenesis by inactivating the RANK-RANKL signalling pathway.113,122-123 Alternately,large numbers of monocytes can secret exosomes to promote osteoclast differentiation.121The end result is thatosteoclasts are rapidly recruited during thisphase,even though osteoclastogenesis-inhibiting exosomes are constantly released.

During bone resorption,the resorbing ability of osteoclasts can be also affected by exosomes.For example,exosomes derived from serum of osteoporotic,osteopenic or aged patients enhance bone resorption.124When bone resorption is close to completion,abundant RANK-enriched exosomes derived from osteoclasts impede osteoclastogenesis.Finally,RANKL-enriched exosomes that are secreted from osteoblasts can inhibit bone resorption via the induction of osteoclast apoptosis.125

In 1981,the term exosome was first used for extracellular vesicles ranging from 50 to 1 000nm.18In 1983,the Stahl group and the Johnstone group reported that exosomes derived from reticulocytes could fuse with the plasma membrane and release their contents through exocytosis.19Then in 1985,the same group provided the electron microscopic evidence for externalization ofexosomes.20In 1987,the formation ofexosomeswas described,and was the first time that the intraluminal vesicles of multivesicular endosomes(MVEs)were mentioned.21The analysis of exosomal characteristic developed quickly in first decade after the exosome was defined.However,the function of exosomes remained largely unknown.

Compared to the investigation of osteoblast and osteoclastderived exosomes,studies focusing on osteocyte-derived exosomes are few.Available data show that osteocytes also have the ability to release exosomes,37and there appears to be a link between osteocyte-derived exosomes and bone homeostasis.

第二，管辖制度。管辖是指确定同级人民法院以及上下级人民法院之间受理第一审案件的分工和权限。管辖制度解决的是违法行为发生后，有起诉权的主体（原告）向何地何级法院起诉的问题。建立水资源保护公益诉讼管辖制度，首先要科学合理确定有管辖权的法院，不能出现同一个案件在流域不同地区法院皆有管辖权或皆无管辖权的情况；其次要便利起诉（方便当事人起诉、应诉），并保证案件能够得到公正审理。

A group of researchers have shown that myostatin-modified osteocytic exosomes can regulate osteoblastic differentiation via exosomal miRNA-218,by targeting the wnt/β-catenin-signalling pathway.126Wnt/β-catenin signaling is of great importance in bone homeostasis,involving both bone formation and bone resorption,and is widely believed to be orchestrated by the osteocyte.127-128Previous studies have revealed that Sclerostin and DKK1 were the inhibitors of Wnt signalling by binding to the Wnt co-receptors LRP5/6,thereby contributing to bone loss.127 Interestingly,induction ofexosomescontaining miRNA-218 derived from myostatin-modified osteocyte was also inhibited.These exosomes were then found accepted by osteoblast leading to the up-regulation of sclerostin,DDK1,and RANKL.

Another interesting finding is that osteocytes can secret exosomes in response to mechanical loading.Initially,mechanical stimulation triggers immediate contraction of the actin network which results in Ca2+transients.Simultaneously,mechanical stimulation induces the secretion of osteocytic exosomes,shown by immunostaining with the secretory vesicle marker,lysosomalassociated membrane protein 1(LAMP-1).This process can also be enhanced by the upregulation of intercellular Ca2+.Finally,released exosomes which contain sclerostin,RANKL,and osteoprotegerin target osteoblasts to activate osteogenesis.129

Exosomes derived from tumor cells in bone homeostasis

Exosomes can be released from a variety of cell types.The tumorcell derived exosomes130can affect bone homeostasis.These effects of cancer cells on bone remodelling provides a new perspective for understanding bone diseases in the course of malignancy.

白煞留下和铁卫连夜清理黑旗会分坛，因为与黑旗会博弈须要充裕的银子作基础，所以萧飞羽临走时吩咐将所有值钱的东西带去安和庄，余下的事情交给樊虎带领的伙计。白煞和铁卫搜索发现天问大师和紫阳道长要找的少林俗家弟子之首的白云飞竟然囚禁在地牢里。

Tumor cells can spontaneously secret exosomes,and fusion of exosomes to bone cells may trigger either inhibition or abnormal enhancement of bone cell function.Exosomes released from multiple myeloma cells have been proved to support the survival of osteoclast precursors via the down-regulation of TRAP mRNA expression induced by inhibition of caspase-3 activity.Further,enhanced differentiation of osteoclast precursor was observed,which explains the increased bone resorption in myeloma patients.130Enhanced osteoblast activity has been observed as well.It can be induced by the transfer of exosomal miRNA-214-3p which facilitates osteoblastic metastases.131As bone is the initial site for tumor metastases,132Exosomes can also participate in the establishment of bone metastasis,leading to tumor-induced osteolysis.133In the process of metastasis,exosomes play an important role as they are carrier of miRNA-192,a pivotal factor in tumor induced angiogenic activity.134This is likely to influence pathways involved in the generation of proteases,adhesion molecules,and chemokine ligands,contributing to the metastatic spread of the tumors.

Exosomes-based clinical applications in fracture healing

企业社会责任从内容的争论到企业社会责任报告的发布，经历了从理论到实践的飞跃。一旦一种理论成为实践的指导就获得了理论证实的合法地位。“我们知道，科学界利用规范的一个收获是，只要接受了这种规范，就有了一个标准来选择那些可以肯定有解的问题。”〔1〕30“有了一种规范，有了规范所容许的那种更深奥的研究，这是任何一个科学部门达到成熟的标志。”〔1〕9企业社会责任的提出、内容的界定以及最终形成共识被确定为评判企业非经济层面行为的规则和标准，实现了企业伦理从宽泛的而又抽象的甚至有点空洞的企业文化研究转变到务实的社会责任研究上，渡过了企业伦理研究对象模糊不清的阶段，这是它成熟的一种标志。

Recent studies have shown the therapeutic potential of exosomes in different stage of fracture healing,suggesting that individualized strategies can be used to promote bone tissue repair.The initial step of fracture repair is the establishment of new vessels and formation of hematoma at the fracture site where inflammatory cells are being recruited.135Prolonged activation or attenuation of inflammation may lead to excessive bone tissue damage or accumulation of necrotic bone respectively.136-138 MSC-derived exosomesare supposed to ideally attenuate inflammation-based delay of fracture healing.By using MSC-derived exosomes,proinflammation factors TNF-α and IL-1β are significantly suppressed,while anti-inflammatory factor TGF-β is increased139(Fig.6a).Moreover,exosomes are stable carriers for antiinflammation drug delivery.When encapsulated in exosomes,curcumin,an anti-inflammation drug,is more highly concentrated in blood.Moreover,as the drug is more accurately delivered to inflammatory cells due to target specificity of exosomes,there is obvious reduction of unwanted side effects.However,in a certain period of bone healing,inflammation is suggested to be indispensable.Over inactivation of inflammation may lead to delay of fracture healing or even non-union.140Thus,timing of intervention is important.Exosomes also play a role in proinflammatoryprocesses.There isevidence to suggestthat acrophage-derived exosomes induce the differentiation of naive monocytes into macrophages.141In this way,recruitment of macrophages,which contain approximately 29 cytokines for tissue repair and inflammation,will relieve an inflammation deficiencybased fracture healing delay.140

Tissue repair is the second stage of bone healing when exosomes act as promoters of angiogenesis and bone regeneration(Fig.6b).MSC-derived exosomes have been reported to contain abundant angiogenesis-related proteins.142The latter enable endothelial cell proliferation and vessel formation.143 Interestingly,pro-angiogenesis effectand tissue repairare detected contemporaneously in vitro.144In vivo,MSC-derived exosomes are seen to promote angiogenesis and osteogenesis.Eightweeksafterimplantation ofMSC-derived exosomes,strong formation of vessels and bone tissue is detected in osteoporotic rats compared to untreated controls.145These findings provide a novel approach for enhancing early tissue repair when revascularization and fibroblast proliferation in soft callus occur.135Also,the wide range of exosomal functions may allow the use of MSC-exosomes throughout the whole period of fracture healing.145

Bone remodelling at its final stage generally is long-lasting(Fig.6c).It reaches a degree of homeosteosis between different bone cells.Bone-derived exosomes have been proposed to have a regulatory function on each bone cell type.Osteoclast precursors together with osteoblast-derived exosomes have been detected to promote osteoclastogenesis in vivo,121,125thus,could be used to boostthe clearance ofdamaged tissue.During bone remodelling,MSC-derived exosomeshavebeen shown to promote this process.146In a femur fracture model of CD9-/-mice,which is suppressed in exosome production,there is obvious delay of callus formation leading to retardation of bone union.By local injection of exosomes,however,this retardation is rescued.114Enhancement of cell proliferation and protection from cell death,MSC-derived exosomes could then serve as a powerful tool in bone remodelling.147Such data support the concept that MSC-exosomes-based therapy is ideal for fracture healing for the repair of large bone defects.148

CONCLUSION

The past decade has witnessed significant progress in the investigation of exosomes as regulators of bone homeostasis,although the function of each of their single molecular species requiresfurtheranalysis.Whetherexosomesare,however,dominant factors in bone homeostasis needs to be further addressed in the future.Such studies will help to better understand the nosogenesis of several exosome-associated bone diseases.Although the introduction of exosomes into clinical practice is not likely to be soon,the perceived power of exosomes in bone homeostasis provides the possibility of novel approaches in the treatment of bone damage and disease.

在对赵甲的出场介绍中，莫言完全隐退了作者的叙事视角，他通过旁观者赵小甲的态度，烘托出赵甲的权势地位和冷酷的性格。同时，莫言让当事人赵甲自己发声，进一步补充他的人生经历，使得赵甲的形象丰满、立体。

ACKNOWLEDGEMENTS

We would like to thank the support from Perron Institute for Neurological and Translational Science,Department of Orthopaedics,The Second Af filiated Hospital and Yuying Children's Hospital of Wenzhou Medical University and Department of Orthopaedics,Shanghai Sixth People's Hospital of Shanghai Jiaotong University.

ADDITIONAL INFORMATION

Competing interests:The authors declare no competing interests.

检索模块是面向检索用户的Web，接收并解析用户键入的搜索关键词，在后台匹配索引并反馈满足搜索条件的结果。

REFERENCES

1.Cappariello,A.,Maurizi,A.,Veeriah,V.&Teti,A.The great beauty of the osteoclast.Arch.Biochem.Biophys.558,70-78(2014).

2.Capulli,M.,Paone,R.&Rucci,N.Osteoblast and osteocyte:games without frontiers.Arch.Biochem.Biophys.561,3-12(2014).

3.Compton,J.T.&Lee,F.Y.A review of osteocyte function and the emerging importance of sclerostin.J.Bone Jt.Surg.Am.96,1659-1668(2014).

4.Kennedy,O.D.et al.Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations.Bone 50,1115-22(2012).1115-cell 1122.

5.Graves,D.T.,Jiang,Y.&Valente,A.J.The expression of monocyte chemoattractant protein-1 and other chemokines by osteoblasts.Front.Biosci.4,D571-D580(1999).

6.Lacey,D.L.et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell 93,165-176(1998).

7.Nakagawa,N.et al.RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis.Biochem.Biophys.Res.Commun.253,395-400(1998).

8.Yoshida,H.et al.The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.Nature 345,442-444(1990).

9.Martin,T.J.&Sims,N.A.Osteoclast-derived activity in the coupling of bone formation to resorption.Trends Mol.Med.11,76-81(2005).

10.Raggatt,L.J.&Partridge,N.C.Cellular and molecular mechanisms of bone remodeling.J.Biol.Chem.285,25103-25108(2010).

11.Thery,C.,Ostrowski,M.&Segura,E.Membrane vesicles as conveyors of immune responses.Nat.Rev.Immunol.9,581-593(2009).

12.Tricarico,C.,Clancy,J.&D'Souza-Schorey,C.Biology and biogenesis of shed microvesicles.Small GTPases 8,220-232(2017).

13.Ponpuak,M.et al.Secretory autophagy.Curr.Opin.Cell Biol.35,106-116(2015).

14.Frazer,A.C.&Stewart,H.C.Ultramicroscopic particles in normal human blood.J.Physiol.90,18-30(1937).

15.Barland,P.,Novikoff,A.B.&Hamerman,D.Electron microscopy of the human synovial membrane.J.Cell Biol.14,207-220(1962).

16.Anderson,H.C.Vesicles associated with calcification in the matrix of epiphyseal cartilage.J.Cell Biol.41,59-72(1969).

17.Dalton,A.J.Microvesicles and vesicles of multivesicular bodies versus“viruslike”particles.J.Natl Cancer Inst.54,1137-1148(1975).

18.Trams,E.G.,Lauter,C.J.,Salem,N.Jr.&Heine,U.Exfoliation of membrane ecto-enzymes in the form of micro-vesicles.Biochim Biophys.Acta 645,63-70(1981).

19.Harding,C.,Heuser,J.&Stahl,P.Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.J.Cell Biol.97,329-339(1983).

20.Pan,B.T.,Teng,K.,Wu,C.,Adam,M.&Johnstone,R.M.Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.J.Cell Biol.101,942-948(1985).

21.Johnstone,R.M.,Adam,M.,Hammond,J.R.,Orr,L.&Turbide,C.Vesicle formation during reticulocyte maturation.Association of plasma membrane activities with released vesicles(exosomes).J.Biol.Chem.262,9412-9420(1987).

22.Raposo,G.et al.B lymphocytes secrete antigen-presenting vesicles.J.Exp.Med.183,1161-1172(1996).

23.Zitvogel,L.et al.Eradication of established murine tumors using a novel cellfree vaccine:dendritic cell-derived exosomes.Nat.Med.4,594-600(1998).

24.Wolfers,J.et al.Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming.Nat.Med.7,297-303(2001).

25.Lai,R.C.,Yeo,R.W.,Tan,K.H.&Lim,S.K.Exosomes for drug delivery-a novel application for the mesenchymal stem cell.Biotechnol.Adv.31,543-551(2013).

26.Bobrie,A.,Colombo,M.,Raposo,G.& Thery,C.Exosome secretion:molecular mechanisms and roles in immune responses.Traf fi c 12,1659-1668(2011).

27.Lo Cicero,A.,Stahl,P.D.&Raposo,G.Extracellular vesicles shuf fl ing intercellular messages:for good or for bad.Curr.Opin.Cell Biol.35,69-77(2015).

28.Hoshino,D.et al.Exosome secretion is enhanced by invadopodia and drives invasive behavior.Cell Rep.5,1159-1168(2013).

29.Gasser,O.& Schifferli,J.A.Activated polymorphonuclearneutrophils disseminate anti-inflammatory microparticlesby ectocytosis.Blood 104,2543-2548(2004).

30.Bernier,G.M.,Duca,V.D.Jr,Brereton,R.&Graham,R.C.Jr Multiple myeloma with intramedullary masses of M-component.Blood 46,931-935(1975).

31.Bab,I.A.,Muhlrad,A.&Sela,J.Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats.Cell Tissue Res.202,1-7(1979).

32.Ornoy,A.,Atkin,I.&Levy,J.Ultrastructural studies on the origin and structure of matrix vesicles in bone of young rats.Acta Anat.(Basel)106,450-461(1980).

33.Peche,H.,Heslan,M.,Usal,C.,Amigorena,S.&Cuturi,M.C.Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomesmodulatesallograftrejection.Transplantation 76,1503-1510(2003).

34.Ekstrom,K.et al.Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells.PLoS ONE 8,e75227(2013).

35.Ge,M.,Ke,R.,Cai,T.,Yang,J.&Mu,X.Identification and proteomic analysis of osteoblast-derived exosomes.Biochem.Biophys.Res.Commun.467,27-32(2015).

36.Li,D.et al.Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation.Nat.Commun.7,10872(2016).

37.Sato,M.,Suzuki,T.,Kawano,M.&Tamura,M.Circulating osteocyte-derived exosomes contain miRNAs which are enriched in exosomes from MLO-Y4 cells.Biomed.Rep.6,223-231(2017).

38.Villarroya-Beltri,C.,Baixauli,F.,Gutierrez-Vazquez,C.,Sanchez-Madrid,F.&Mittelbrunn,M.Sorting it out:regulation of exosome loading.Semin.Cancer Biol.28,3-13(2014).

39.Mathivanan,S.,Ji,H.&Simpson,R.J.Exosomes:extracellular organelles important in intercellular communication.J.Proteom.73,1907-1920(2010).

40.Kalra,H.et al.Vesiclepedia:a compendium for extracellular vesicles with continuous community annotation.PLoS Biol.10,e1001450(2012).

41.Lydic,T.A.et al.Rapid and comprehensive‘shotgun'lipidome profiling of colorectal cancer cell derived exosomes.Methods 87,83-95(2015).

42.Mobius,W.et al.Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway.Traf fi c 4,222-231(2003).

43.van Meer,G.,Voelker,D.R.&Feigenson,G.W.Membrane lipids:where they are and how they behave.Nat.Rev.Mol.Cell Biol.9,112-124(2008).

44.Moser von Filseck,J.et al.Intracellular transport.Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.Science 349,432-436(2015).

45.Brouwers,J.F.et al.Distinct lipid compositions of two types of human prostasomes.Proteomics 13,1660-1666(2013).

46.Simons,K.&Toomre,D.Lipid rafts and signal transduction.Nat.Rev.Mol.Cell Biol.1,31-39(2000).

47.Luberto,C.&Hannun,Y.A.Sphingomyelin synthase,a potential regulator of intracellular levels of ceramide and diacylglycerolduring SV40 transformation.Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C? J. Biol. Chem. 273,14550-14559(1998).

48.Kosaka,N.et al.Neutral sphingomyelinase 2(nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis.J.Biol.Chem.288,10849-10859(2013).

49.Theos,A.C.et al.A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis.Dev.Cell 10,343-354(2006).

50.Trajkovic,K.et al.Ceramide triggers budding of exosome vesicles into multivesicular endosomes.Science 319,1244-1247(2008).

51.Yuyama,K.,Sun,H.,Mitsutake,S.&Igarashi,Y.Sphingolipid-modulated exosome secretion promotes clearance of amyloid-beta by microglia.J.Biol.Chem.287,10977-10989(2012).

52.Keerthikumar,S.et al.ExoCarta:A Web-Based Compendium of Exosomal Cargo.J.Mol.Biol.428,688-692(2016).

53.Takeuchi,T.et al.Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level.Proc.Natl Acad.Sci.USA 112,E2497-E2506(2015).

54.Henne,W.M.,Buchkovich,N.J.&Emr,S.D.The ESCRT pathway.Dev.Cell 21,77-91(2011).

55.Keryer-Bibens,C.et al.Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9.BMC Cancer 6,283(2006).

56.Melo,S.A.et al.Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.Nature 523,177-182(2015).

57.Valadi,H.et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat.Cell Biol.9,654-659(2007).

58.Thakur,B.K.et al.Double-stranded DNA in exosomes:a novel biomarker in cancer detection.Cell Res.24,766-769(2014).

59.Li,S.P.,Lin,Z.X.,Jiang,X.Y.&Yu,X.Y.Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools.Acta Pharmacol.Sin.39,542-551(2018).

60.Januszyk,K.&Lima,C.D.The eukaryotic RNA exosome.Curr.Opin.Struct.Biol.24,132-140(2014).

61.Lv,L.L.et al.CD2AP mRNA in urinary exosome as biomarker of kidney disease.Clin.Chim.Acta 428,26-31(2014).

62.Shao,H.et al.Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.Nat.Commun.6,6999(2015).

63.Liu,R.,Liu,J.,Ji,X.&Liu,Y.Synthetic nucleic acids delivered by exosomes:a potential therapeutic for generelated metabolic brain diseases.Metab.Brain Dis.28,551-562(2013).

64.Mittelbrunn,M.et al.Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.Nat.Commun.2,282(2011).

65.Luo,S.S.et al.Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.Biol.Reprod.81,717-729(2009).

66.Liu,H.et al.Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility.Oncogene:https://doi.org/10.1038/s41388-018-0309-x(2018).

67.Wang,J.et al.Bone marrow stromal cell-derived exosomes as communicators in drug resistance in multiple myeloma cells.Blood 124,555-566(2014).

68.Takahashi,A.et al.Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.Nat.Commun.8,15287(2017).

69.Hoeijmakers,J.H.DNA damage,aging,and cancer.N.Engl.J.Med.361,1475-1485(2009).

70.Kahlert,C.et al.Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer.J.Biol.Chem.289,3869-3875(2014).

71.Kalluri,R.&LeBleu,V.S.Discovery of Double-Stranded Genomic DNA in Circulating Exosomes.Cold Spring Harb.Symp.Quant.Biol.81,275-280(2016).

72.Raiborg,C.&Stenmark,H.The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins.Nature 458,445-452(2009).

73.Svensson,K.J.et al.Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.J.Biol.Chem.288,17713-17724(2013).

74.van Niel,G.,D'Angelo,G.&Raposo,G.Shedding light on the cell biology of extracellular vesicles.Nat.Rev.Mol.Cell Biol.19,213-228(2018).

75.Henne,W.M.,Stenmark,H.&Emr,S.D.Molecular mechanisms of the membrane sculpting ESCRT pathway.Cold Spring Harb.Perspect.Biol.5,https://doi.org/10.1101/cshperspect.a016766(2013).

76.Stoorvogel,W.Resolving sorting mechanisms into exosomes.Cell Res.25,531-532(2015).

77.Colombo,M.et al.Analysis of ESCRT functions in exosome biogenesis,composition and secretion highlights the heterogeneity of extracellular vesicles.J.Cell Sci.126,5553-5565(2013).

78.Baietti,M.F.et al.Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.Nat.Cell Biol.14,677-685(2012).

79.Ramani,V.C.,Pruett,P.S.,Thompson,C.A.,DeLucas,L.D.&Sanderson,R.D.Heparan sulfate chains of syndecan-1 regulate ectodomain shedding.J.Biol.Chem.287,9952-9961(2012).

80.Roucourt,B.,Meeussen,S.,Bao,J.,Zimmermann,P.&David,G.Heparanase activates the syndecan-syntenin-ALIX exosome pathway.Cell Res.25,412-428(2015).

81.Charrin,S.,Jouannet,S.,Boucheix,C.&Rubinstein,E.Tetraspanins at a glance.J.Cell Sci.127,3641-3648(2014).

82.van Niel,G.et al.The tetraspanin CD63 regulates ESCRT-independent and-dependent endosomal sorting during melanogenesis.Dev.Cell 21,708-721(2011).

83.Zimmerman,B.et al.Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket.Cell 167,1041-1051(2016).e1011.

84.Janas,T.,Janas,M.M.,Sapon,K.&Janas,T.Mechanisms of RNA loading into exosomes.FEBS Lett.589,1391-1398(2015).

85.Wu,B.X.et al.Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.J.Biol.Chem.286,22362-22371(2011).

86.Record,M.,Carayon,K.,Poirot,M.&Silvente-Poirot,S.Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies.Biochim.Biophys.Acta 1841,108-120(2014).

87.Kosaka,N.et al.Secretory mechanisms and intercellular transfer of microRNAs in living cells.J.Biol.Chem.285,17442-17452(2010).

88.Janas,T.,Janas,T.&Yarus,M.Specific RNA binding to ordered phospholipid bilayers.Nucleic Acids Res.34,2128-2136(2006).

89.Kozlov,M.M.&Chernomordik,L.V.Membrane tension and membrane fusion.Curr.Opin.Struct.Biol.33,61-67(2015).

90.Savina,A.,Fader,C.M.,Damiani,M.T.&Colombo,M.I.Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.Traf fi c 6,131-143(2005).

91.Wei,Y.et al.Pyruvate kinase type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23.Nat.Commun.8,14041(2017).

92.Colombo,M.,Raposo,G.&Thery,C.Biogenesis,secretion,and intercellular interactions of exosomes and other extracellular vesicles.Annu.Rev.Cell.Dev.Biol.30,255-289(2014).

93.Fruhbeis,C.,Frohlich,D.,Kuo,W.P.&Kramer-Albers,E.M.Extracellular vesicles as mediators of neuron-glia communication.Front.Cell.Neurosci.7,182(2013).

94.Ostrowski,M.et al.Rab27a and Rab27b control different steps of the exosome secretion pathway.Nat.Cell Biol.12,19-30(2010).sup pp 11-13.

95.Kowal,J.,Tkach,M.&Thery,C.Biogenesis and secretion of exosomes.Curr.Opin.Cell Biol.29,116-125(2014).

96.Christianson,H.C.,Svensson,K.J.,van Kuppevelt,T.H.,Li,J.P.&Belting,M.Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.Proc.Natl Acad.Sci.USA 110,17380-17385(2013).

97.Cocucci,E.,Racchetti,G.&Meldolesi,J.Shedding microvesicles:artefacts no more.Trends Cell Biol.19,43-51(2009).

98.Keller,S.et al.Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes.Cancer Lett.278,73-81(2009).

99.Mulcahy,L.A.,Pink,R.C.&Carter,D.R.Routes and mechanisms of extracellular vesicle uptake.J.Extracell.Vesicles 3,https://doi.org/10.3402/jev.v3.24641(2014).

100.Feng,D.et al.Cellular internalization of exosomes occurs through phagocytosis.Traf fi c 11,675-687(2010).

101.Boscher,C.&Nabi,I.R.Caveolin-1:role in cell signaling.Adv.Exp.Med.Biol.729,29-50(2012).

102.Kirchhausen,T.Clathrin.Annu.Rev.Biochem.69,699-727(2000).

103.Tian,T.et al.Exosome uptake through clathrin-mediated endocytosis and macropinocytosisand mediating miR-21 delivery.J.Biol.Chem.289,22258-22267(2014).

104.Swanson,J.A.Shaping cups into phagosomes and macropinosomes.Nat.Rev.Mol.Cell Biol.9,639-649(2008).

105.Parolini,I.et al.Microenvironmental pH is a key factor for exosome traf fi c in tumor cells.J.Biol.Chem.284,34211-34222(2009).

106.Chernomordik,L.V.&Kozlov,M.M.Mechanics of membrane fusion.Nat.Struct.Mol.Biol.15,675-683(2008).

107.Chernomordik,L.V.&Kozlov,M.M.Protein-lipid interplay in fusion and fi ssion of biological membranes.Annu.Rev.Biochem.72,175-207(2003).

108.Kozlov,M.M.&Markin,V.S.[Possible mechanism of membrane fusion].Bio fi zika 28,242-247(1983).

109.Kozlov,M.M.,Leikin,S.L.,Chernomordik,L.V.,Markin,V.S.&Chizmadzhev,Y.A.Stalk mechanism of vesicle fusion.Intermixing of aqueous contents.Eur.Biophys.J.17,121-129(1989).

110.Helfrich,M.H.et al.Beta 1 integrins and osteoclast function:involvement in collagen recognition and bone resorption.Bone 19,317-328(1996).

111.Crockett,J.C.,Mellis,D.J.,Scott,D.I.&Helfrich,M.H.New knowledge on critical osteoclast formation and activation pathways from study of rare genetic diseases of osteoclasts:focus on the RANK/RANKL axis.Osteoporos.Int.22,1-20(2011).

112.Li,J.et al.Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity.Nat.Immunol.14,793-803(2013).

113.Cui,Y.,Luan,J.,Li,H.,Zhou,X.&Han,J.Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of micro-RNA expression.FEBS Lett.590,185-192(2016).

114.Furuta,T.et al.Mesenchymal stem cell-derived exosomes promote fracture healing in a mouse model.Stem Cells Transl.Med.5,1620-1630(2016).

115.Qin,Y.,Wang,L.,Gao,Z.,Chen,G.&Zhang,C.Bone marrow stromal/stem cellderived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo.Sci.Rep.6,21961(2016).

116.Zhao,P.,Xiao,L.,Peng,J.,Qian,Y.Q.&Huang,C.C.Exosomes derived from bone marrow mesenchymal stem cells improve osteoporosis through promoting osteoblast proliferation via MAPK pathway.Eur.Rev.Med.Pharmacol.Sci.22,3962-3970(2018).

117.Wei,J.et al.let-7 enhances osteogenesis and bone formation while repressing adipogenesis of human stromal/mesenchymal stem cells by regulating HMGA2.Stem.Cells Dev.23,1452-1463(2014).

118.Wang,X.et al.miR-214 targets ATF4 to inhibit bone formation.Nat.Med.19,93-100(2013).

119.Sun,W.et al.Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity.Cell Discov.2,16015(2016).

120.Deng,L.et al.Osteoblast-derived microvesicles:a novel mechanism for communication between osteoblasts and osteoclasts.Bone 79,37-42(2015).

121.Huynh,N.et al.Characterization of regulatory extracellular vesicles from osteoclasts.J.Dent.Res.95,673-679(2016).

122.Chen,C.et al.MiR-503 regulates osteoclastogenesis via targeting RANK.J.Bone Miner.Res.29,338-347(2014).

123.Xie,Y.,Chen,Y.,Zhang,L.,Ge,W.&Tang,P.The roles of bone-derived exosomes and exosomal microRNAs in regulating bone remodelling.J.Cell.Mol.Med.21,1033-1041(2017).

124.Xie,Y.et al.Involvement of serum-derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone-related proteins.Aging Cell 17,e12758(2018).

125.Cappariello,A.et al.Osteoblast-derived extracellular vesicles are biological tools for the delivery of active molecules to bone.J.Bone Miner.Res.33,517-533(2018).

126.Qin,Y.et al.Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218:a novel mechanism in muscle-bone communication.J.Biol.Chem.292,11021-11033(2017).

127.Bonewald,L.F.The amazing osteocyte.J.Bone Miner.Res.26,229-238(2011).

128.Kramer,I.et al.Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis.Mol.Cell.Biol.30,3071-3085(2010).

129.Lu,X.L.,Huo,B.,Park,M.&Guo,X.E.Calcium response in osteocytic networks under steady and oscillatory fluid flow.Bone 51,466-473(2012).

130.Raimondi,L.et al.Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation.Oncotarget 6,13772-13789(2015).

131.Ye,Y.et al.Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.Oncotarget 8,94834-94849(2017).

132.Mundy,G.R.Metastasis to bone:causes,consequences and therapeutic opportunities.Nat.Rev.Cancer 2,584-593(2002).

133.Guise,T.A.et al.Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis.J.Clin.Invest.98,1544-1549(1996).

134.Valencia,K.et al.miRNA cargo within exosome-like vesicle transfer influences metastatic bone colonization.Mol.Oncol.8,689-703(2014).

135.Claes,L.,Recknagel,S.&Ignatius,A.Fracture healing under healthy and inflammatory conditions.Nat.Rev.Rheumatol.8,133-143(2012).

136.Grundnes,O.&Reikeraas,O.Effects of macrophage activation on bone healing.J.Orthop.Sci.5,243-247(2000).

137.Yu,B.,Zhang,X.&Li,X.Exosomes derived from mesenchymal stem cells.Int.J.Mol.Sci.15,4142-4157(2014).

138.Schmidt-Bleek,K.et al.In flammatory phase of bone healing initiates the regenerative healing cascade.Cell Tissue Res.347,567-573(2012).

139.Chen,W.et al.Immunomodulatory effects of mesenchymal stromal cellsderived exosome.Immunol.Res.64,831-840(2016).

140.Kolar,P.et al.The early fracture hematoma and its potential role in fracture healing.Tissue Eng.Part B Rev.16,427-434(2010).

141.Ismail,N.et al.Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.Blood 121,984-995(2013).

142.Anderson,J.D.et al.Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogenesis via nuclear factor-kappaB signaling.Stem Cells 34,601-613(2016).

143.Salomon,C.et al.Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.PLoS ONE 8,e68451(2013).

144.Shabbir,A.,Cox,A.,Rodriguez-Menocal,L.,Salgado,M.&Van Badiavas,E.Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts,and enhance angiogenesis in vitro.Stem.Cells Dev.24,1635-1647(2015).

145.Qi,X.et al.Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporotic rats.Int.J.Biol.Sci.12,836-849(2016).

146.Narayanan,R.,Huang,C.C.&Ravindran,S.Hijacking the cellular mail:exosome mediated differentiation of mesenchymal stem cells.Stem Cells Int.2016,3808674(2016).

147.De Jong,O.G.,Van Balkom,B.W.,Schiffelers,R.M.,Bouten,C.V.&Verhaar,M.C.Extracellular vesicles:potential roles in regenerative medicine.Front.Immunol.5,608(2014).

148.Ferreira,E.&Porter,R.M.Harnessing extracellular vesicles to direct endochondral repair of large bone defects.Bone Jt.Res.7,263-273(2018).

149.Nazarenko,I.et al.Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation.Cancer Res.70,1668-1678(2010).

150.Escola,J.M.et al.Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes.J.Biol.Chem.273,20121-20127(1998).

151.Yanez-Mo,M.,Barreiro,O.,Gordon-Alonso,M.,Sala-Valdes,M.&Sanchez-Madrid,F.Tetraspanin-enriched microdomains:a functional unit in cell plasma membranes.Trends Cell Biol.19,434-446(2009).

152.Tamai,K.et al.Exosome secretion of dendritic cells is regulated by Hrs,an ESCRT-0 protein.Biochem.Biophys.Res.Commun.399,384-390(2010).

153.Mathew,A.,Bell,A.&Johnstone,R.M.Hsp-70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes.Biochem.J.308(Pt 3),823-830(1995).

154.Hsu,C.et al.Regulation of exosome secretion by Rab35 and its GTPaseactivating proteins TBC1D10A-C.J.Cell Biol.189,223-232(2010).

155.Gutierrez-Vazquez,C.,Villarroya-Beltri,C.,Mittelbrunn,M.&Sanchez-Madrid,F.Transfer of extracellular vesicles during immune cell-cell interactions.Immunol.Rev.251,125-142(2013).

156.Laulagnier,K.et al.PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes.FEBS Lett.572,11-14(2004).

157.Shinozaki,K.&Waite,M.A novel phosphatidylglycerol-selective phospholipase A2 from macrophages.Biochemistry 38,1669-1675(1999).

158.Beh,C.T.,McMaster,C.R.,Kozminski,K.G.&Menon,A.K.A detour for yeast oxysterol binding proteins.J.Biol.Chem.287,11481-11488(2012).

159.Xu,C.et al.CD82 endocytosis and cholesterol-dependent reorganization of tetraspanin webs and lipid rafts.FASEB J.23,3273-3288(2009).

160.Carayon,K.et al.Proteolipidic composition of exosomes changes during reticulocyte maturation.J.Biol.Chem.286,34426-34439(2011).

161.Subra,C.,Laulagnier,K.,Perret,B.& Record,M.Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies.Biochimie 89,205-212(2007).

162.Falguieres,T.et al.In vitro budding of intralumenal vesicles into late endosomes is regulated by Alix and Tsg101.Mol.Biol.Cell 19,4942-4955(2008).

163.Yang,M.et al.Microvesicles secreted by macrophages shuttle invasionpotentiating microRNAs into breast cancer cells.Mol.Cancer 10,117(2011).

164.Ibrahim,A.G.,Cheng,K.&Marban,E.Exosomes as critical agents of cardiac regeneration triggered by cell therapy.Stem Cell Rep.2,606-619(2014).

165.Fujita,Y.et al.Intercellular communication by extracellular vesicles and their microRNAs in asthma.Clin.Ther.36,873-881(2014).

166.Liu,S.et al.MSC Transplantation Improves Osteopenia via Epigenetic Regulation of Notch Signaling in Lupus.Cell.Metab.22,606-618(2015).

167.Zhang,J.et al.Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.Stem Cell Res.Ther.7,136(2016).

168.Morrell,A.E.et al.Mechanically induced Ca(2+)oscillations in osteocytes release extracellular vesicles and enhance bone formation.Bone Res.6,6(2018).

169.Martin,P.J.et al.Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles(EVs).BMC Cell.Biol.16,10(2015).