The role of exosomes in bone remodeling and osseointegration of implants

ZENG Xiao-mei, LI Peng-cheng, FU Qi-ya,2

1.Schoolof Stomatology, Hainan Medical University, Haikou 570216, China

2.Department of Stomatology, The First Affiliated Hospital of Hainan Medical University, Haikou 570216, China

Keywords:

ABSTRACT Dental implant is an effective method in the treatment of missing teeth.The process of osseointegration of implant teeth involves the coordinated operation of immune system and bone system.The interaction between cells is closely related to bone formation and repair.Exosomes are important intercellular communication molecules.They were originally found in the supernatant of sheep erythrocytes cultured in vitro.They are micro vesicles with a diameter of 40～150 nm.They exist in a variety of cells and body fluids.They enter the target cells by endocytosis and transport, affecting the expression of cell genes and changing the fate of cells.It has an important regulatory function in the microenvironment of implant bone binding.It plays a role in bone remodeling through small molecular RNA, specific proteins and other growth factors secreted by different cells.This article reviews the role of bone derived cellderived exosomes in bone remodeling and their function in implant osseointegration.

Extracellular vesicles (EVS) are membranous vesicles with the size of 30～150 nm released by cells.As intercellular communication molecules, EVs have attracted much attention.Cells can secrete a variety of extracellular vesicles, which can be divided into exosomes(exos), microbubbles, apoptotic bodies, etc.according to biogenesis,size, density and major protein markers[1].The diameter of exosomes is generally 50～150 nm, which comes from the intracellular body of cells, while the diameter of microbubbles is 50～500 nm, even up to 1 μm.It usually comes from the cell membrane.Apoptotic bodies are generally produced after apoptosis, and their diameters are about 1～ 5 μm or so.

As a kind of extracellular vesicles, exosomes with a diameter of about 50～150 nm can be secreted and ingested by endothelial cells,immune cells, tumor cells, mesenchymal stem cells (MSc) and other cells, and exosomes from different sources have specificity in morphology, content and function.As small vesicles secreted into circulation, exosomes can be internalized by proximal or distal cells.Small molecules in these exosomes (including proteins and nucleic acids) can regulate the function of receptor cells during internalization, so as to communicate between various cells and organs.In addition, exosomes have been proved to be one of the ideal nanomaterials for delivering regulatory substances to target cells, and play a protective role in this process.In the process of dynamic balance between bone resorption and bone formation, bone tissue cells include bone marrow mesenchymal stem cells (BMMSC), osteoblast, osteoclast,osteocyte and macrophages (Mφs) It plays an important role in bone tissue.The interaction between bone cells and cell functions is the key to maintain normal bone structure[2].The imbalance between osteogenesis and osteoclasty is an important cause of dental implant failure.This study focuses on bone remodeling and bone derived exosomes, summarizes the different roles of exosomes in the whole body bone tissue remodeling, and takes exosomes related to bone binding as the starting point, summarizes the application and Prospect of exosomes involved in bone binding.

1.The role of exosomes in bone remodeling

The dynamic balance of bone remodeling is the premise of maintaining normal bone structure and plays an important role in maintaining bone homeostasis.Exosomes exist in various cellular microenvironments and play a mediating role in intercellular communication.Recent studies have focused on the role of exosome mediated intercellular communication in bone remodeling, and have studied the therapeutic effects of exosomes in various disease models.Exosomes can provide genetic information for recipient cells, affect their characteristics and paracrine factors, and lead to tissue remodeling.Compared with cells, exosomes have the advantages of good stability, no immunogenicity, easy transformation and easy access.Exosomes from different bone derived cells secrete specific proteins, small molecular RNA and other growth factors.Observe the contents of exosomes and understand the mechanism of their mediating bone remodeling.

1.1 Effect of exosomes derived from mesenchymal stem cells on bone remodeling

There is a kind of stromal stem cells with the ability of self renewal in bone marrow, which is called bone marrow mesenchymal stem cells.They can differentiate into osteoblasts, chondrocytes and adipocytes, so they can affect the formation, maintenance and reconstruction of bone.Many researchers believe that MSCs can achieve self proliferation and differentiation, which can be used to replace damaged tissues, and change the microenvironment of damaged tissues by paracrine regulation of important microenvironment regulators[4].Functionally, MSCs regulate cell proliferation, differentiation, apoptosis and other biological functions, and indirectly play a therapeutic role in repair.

According to previous studies, the gene expression and function of osteoblasts can be affected under the stimulation of mitogen activated protein kinase (MAPK) signal.At the same time, mesenchymal stem cell derived exosomes (MSc exos) can increase the expression of MAPK pathway related proteins[5].Relevant studies have confirmed that MSC exos has an effect on transforming growth factor β1(TGF β1) The expression of bone morphogenetic protein 9 (BMP9) and growth factor can regulate[6], which plays an inducing role in the osteogenic differentiation of bone marrow mesenchymal stem cells.The exosomes contain miRNAs, which mediate the communication between cells by transmitting genetic information.In addition,miRNAs are involved in the gene regulatory networks of various signal pathways, play different roles in regulating osteogenesis, and have been studied as drugs for target gene therapy.A large number of studies have shown that miRNAs in exosomes can promote osteoblast differentiation.Lai Yu et al.[7] showed that three osteogenic related miRNAs (mir-196a, mir-27a and miR-206) that promote bone regeneration in extracellular vesicles secreted by MSCs were highly up-regulated, and mir-196a was also considered to be one of the most important regulatory factors in osteogenesis.Differentially expressed miRNAs such as miR-21, mir-4532, mir-125b-5p and mir-338-3p in the exosomes secreted by MSCs may contribute to promoting osteogenesis and angiogenesis.Specific binding protein 2 (Satb2) rich in at sequence is a specific immunohistochemical biomarker, which is mainly differentiated from osteoblasts and has been proved to have a certain effect on bone and soft tissue tumors[8].It has been reported that the exocrine lung adenocarcinoma metastasis related transcripts (malat1) from MSCs can enhance the osteoblast activity of osteoporosis mice, and the mir-34c/satb2 axis plays a major mediating role in this process[9].In addition, some scholars have shown that miRNAs in exosomes can inhibit osteoblast differentiation.Studies have found that mir-424-5p is overexpressed in exosomes derived from MSCs, and the overexpression of mir-424-5p weakens osteogenic differentiation[10], indicating that exosomes derived from bone marrow mesenchymal stem cells mediate Wnt by up regulating mir-424-5p/ β- Catenin axis, thus inhibiting osteoblast differentiation.

MSc exos may promote osteogenesis by promoting angiogenesis.Angiogenesis contributes to the progress of osteogenesis, in which blood supply induces osteoblast migration and bone mineralization.Vascular endothelial cells play a stimulating role in the maturation and activity of bone cells, and bone regeneration is closely related to angiogenesis.Vascular endothelial growth factor (VEGF) is a specific growth factor.It mainly acts on vascular endothelial cells, promotes vascular regeneration, regulates the formation of capillaries, the proliferation and migration of vascular endothelial cells, and further enhances the activity of osteoblasts, thus promoting bone regeneration.Liunana[11] et al.Found that MSC exos not only enhanced the expression of osteogenesis related genes, but also enhanced the expression of angiogenesis related genes such as VEGF,angiopoietin 1 (ang 1) and angiopoietin 2 (Ang2).This indicates that MSC exos provides recipient cells with some information related to angiogenesis and bone regeneration, which leads to the activation of vascular endothelial growth factor and osteogenic related paracrine factor secretion by recipient cells.Therefore, MSc exos may partially help to control the cell niche in the local environment and activate paracrine factors including vascular endothelial growth factor in bone regeneration.Current studies have shown that bone marrow mesenchymal stem cell-derived exosomes (BMMSC exos) can accelerate the proliferation and migration of endothelial cells and osteoblasts, and further promote angiogenesis and osteogenesis to promote fracture healing.As a intercellular communicator, exosomes can activate HIF-1α/VEGF and bmp-2/smad1/runx2 signaling pathway play an important role in the process of fracture healing.Long noncoding RNA (lncrnas) is becoming a new regulator in the osteogenesis of MSCs [12].

1.2 Effect of osteoblast derived exosomes on bone remodeling

Osteoblasts come from bone marrow mesenchymal stem cells.Because bone marrow mesenchymal stem cells have multidirectional differentiation potential, they can secrete collagen and glycoprotein, which is conducive to the synthesis, secretion and mineralization of bone matrix.Bone marrow mesenchymal stem cells first differentiate into osteoblasts, then into osteoblast precursors, and finally into osteoblasts.This process is regulated by miRNAs, proteins, signal pathways and so on.The communication between osteoblasts and osteoblasts is thought to be carried out through small membrane encapsulated cystic particles called exosomes, which can fuse with the surrounding cell membrane in the circulation pathway.In addition, some studies have shown that osteoblast derived exosomes play a role in bone microenvironment and bone metabolism by regulating the activity of osteoclasts.

Recent studies have shown that many miRNAs of bone derived exosomes are involved in the regulation of bone remodeling[13].There are 43 miRNAs highly expressed in the exosomes of mouse embryonic osteoblast precursor cells (MC3T3-E1 cells), including Mir-140-3P, Mir-133b-3P and Mir-30d-5p.Previous studies have confirmed that they play a regulatory role in bone tissue remodeling,and also participate in a variety of pathways for osteoblast differentiation and function, such as calcium signaling pathway,Wnt and TGF β And insulin, etc.In the process of osteogenic differentiation, nine miRNAs, including Mir-135B, Mir-148A, Mir-199b, Mir-203, Mir-218, Mir-219, Mir-299-5p, Mir-302B and let-7a, were up-regulated in the exosomes of human bone marrow mesenchymal stem cells (hBMSC).In contrast, five miRNAs(MIR -155, Mir-181a, Mir-221, Mir-320C and Mir-885-5p) were significantly down regulated in hBMSC exosome samples.These downregulated miRNAs and their co target genes are rich in insulin signaling pathway, inositol phosphate 3-kinase /akt pathway and mitogen activated protein kinase, which play an important role in osteoblast differentiation.As a communicator in bone remodeling,miRNAs are mainly derived from the exosomes of osteoblast lineage, which contain targeted key osteoclast differentiation factors.The exosomes of mineralized osteoblasts containing Mir-503-3P can regulate the expression of rank and can be used to inhibit the differentiation of osteoclasts induced by rank.Wang Lu et al.[14] found that circ_ 0008542 is a novel circular RNA contained in MC3T3-E1 cell-derived exosomes under tension stimulation(Flexcell culture at 20% amplitude /1 hz/24 h), CIRC_ 0008542 gradually up regulates osteoclast differentiation and bone resorption through mir-185-5p/rank axis.

The exosomes of mature osteoblast cell lines (mineralized MC3T3-E1 cells) can promote the osteogenic differentiation of ST2 osteoblast precursor cell lines, which is manifested by the upregulation of the expression of osteogenic markers, runt related transcription factor 2 (Runx2) and alkaline phosphatase, and the enhancement of matrix mineralization[15].Let-7 exists in the exosomes of mineralized osteoblasts and osteoblast precursors, and enhances osteogenesis by regulating high mobility group at hook protein 2 (HMGA2) and Axin 2 antibody (AXIN2).In addition,osteoblast differentiation β-Catenin is an essential transcription factor.When the exosomes derived from mineralized osteoblasts were transferred, AXIN1 expression was inhibited and enhanced β-The expression of catenin can promote the osteogenic differentiation of osteoblast precursors.

1.3 Effect of osteoclast derived exosomes on bone remodeling

Osteoclasts are derived from giant multinucleated cells, which are precursors of mononuclear macrophages and are mainly responsible for bone resorption in living organisms.Osteoclasts are involved in the occurrence and development of many bone diseases, such as osteoporosis, rheumatoid arthritis and so on[16].The identification proteins of exosomes in osteoclasts include: cell differentiation (CD)63, epithelial cell adhesion molecule (EpCAM), tumor susceptibility gene (TSG) 101, heat shock protein (HSP) 70, and β- Actin.Hanguangli et al.[17] analyzed the regulatory activity of osteoclast derived exosomes and concluded that the number of osteoclasts in mouse bone marrow increased under the stimulation of exosomes from osteoclast precursors, while the same number of exosomes from mature osteoclasts inhibited the formation of osteoclasts.

Osteoclasts inhibit osteoblasts through miRNA containing exosomes.Bone remodeling is a delicate process, and the physiological coupling between osteoblasts and osteoclasts is essential.The sun w study identified the mechanism of osteoclast mediated inhibition of osteoblasts through miRNA containing exosomes.Osteoclasts secrete exosomes rich in miR-214, which is 4-fold enriched in osteoclasts.These exosomes can be integrated into osteoblasts through ephrina2/epha2 molecules.Therefore, miR-214 can inhibit the activity of osteoblasts.In addition, a previous study by Garg P et al.[18] determined that miR-214 inhibits bone formation by regulating osterix and activating transcription factor (ATF) 4 in osteoblasts, which is an important transcription factor.This study further confirmed that miR-214 promotes osteoclast formation mainly through pi3k/akt targeting PTEN tensin homolog.Therefore,the exosomes containing Mir-214 have a variety of beneficial effects on bone destruction.LV py et al.Conducted a series of studies to identify that the exosome mir-214-3p derived from osteoclasts can be transferred to osteoblasts to inhibit bone formation in vitro and in living organisms, and proposed a miRNA mediated osteoclast osteoblast communication paradigm to participate in the steadystate mechanism of local bone environment.The results showed that mir-214-3p in exosomes could act as an intercellular messenger to mediate the communication between osteoclasts and osteoblasts,thus inhibiting the bone formation of osteoblasts.Other studies have shown that the exosomes containing mir-23a-5p in osteoclasts can effectively inhibit Runx2 and increase the metallothionein 1D pseudogene (mt1dp) mediated by yes related protein 1 (Yap1) and effectively inhibit osteoblast differentiation[19].In addition, Liangling et al.[20] found that microrna-146a was enriched more than 80 times in osteoclasts, making it a candidate for regulatory molecules and biomarkers.

1.4 Effect of exosomes from bone cells on bone remodeling

Bone cells are the most abundant cells in bone, which are distributed in an interconnected network.Through this network,they sense and respond to system or local stimuli to regulate bone remodeling, and play their role through cell-cell interaction and soluble media.Luoyu et al.[21] found that bone cells secrete exosomes and then circulate in the blood.This is the first study on exosomes from bone cells combined with circulating exosomes:compare the plasma of osteopenic mice with that of normal mice.The former plasma circulates in exosomes, A total of 12 miRNAs(mir-3473a, mir-3473b, mir- 3473e, mir-5128, mir-6244, mir-6239,mir- 5132-5p, mir-705, mir- 208a-5p, mir-3104-5p, mir-1224-5p, mir-5621-5p) showed a downward trend.The reason may be related to the reduction or leakage of exosomes from bone cells.Exosomes derived from bone cells can transfer bioactive molecules to target cells and play a key role in bone remodeling, especially in the differentiation of osteoclasts and osteoblasts[22].Current studies have shown that exosomes produced by bone cells may circulate throughout the body, and then transfer their constituent signal molecules, including miRNA and pre miRNA, by binding to the cell surface, and then be fused and internalized by receptor cells in other organs or tissues [23].Exosomes derived from bone cells have been shown to regulate muscle bone communication.Sunjinsheng et al.[24]confirmed that exosomes with down regulated expression of mir-218 derived from osteoblasts can be incorporated into osteoblasts after myostatin treatment, and inhibit osteoblast differentiation by down regulating Wnt signal; This process can be reversed by the expression of exogenous mir-218, indicating that mir-218 in exosomes derived from osteoblasts can promote osteogenesis and has the potential to treat bone diseases.

1.5 Effect of macrophage derived exosomes on bone remodeling

Macrophages are a cell type that can differentiate into a series of effector subtypes according to local cell and secretory signals.Angiogenesis is a prerequisite for implant osseointegration, which can transport oxygen, nutrients, metabolites and cytokines, so it is very important for bone repair and bone regeneration[25].Macrophages can play an immunomodulatory role in the process of implant osseointegration.So the interaction between macrophages and osteocytes is very important for bone formation and repair.Exosomes can actively transport between microRNAs and proteins,and transfer information to target cells, thus affecting their behavior and changing the entire microenvironment[26].

Some scholars have studied the role of M0, M1 and M2 polarized macrophage derived EVs in the paracrine function of bone repair[27].The functional examination of miR-155 rich in M1 macrophage EVs showed that by reducing the expression of BMP2, bone morphogenetic protein 9 and Runx2, miR-155 simulated the reduction of osteogenic differentiation of MSC.On the contrary,the treatment of bone marrow mesenchymal stem cells with mir-378a rich in M2 macrophage EVs simulated the increase of bone induced gene expression of bone marrow mesenchymal stem cells.These findings suggest that polarized macrophage derived exosomal microRNA is involved in the positive or negative regulation of bone regeneration observed in vivo, M0 and M2 macrophage EVs promote repair and regeneration, and M1 macrophage EVs inhibit bone repair.

MiRNA rich in exosomes is a key post transcriptional regulator.A recent study shows that copper ion can make m S polarization is a pro-inflammatory M1 phenotype, which can promote angiogenesis by secreting vascular endothelial growth factor (VEGF) [28].In addition to VEGF, M S can also secrete exosomes, target recipient cells, including endothelial cells, and regulate its function by delivering RNA, proteins and other components.M φS secreted exosomes contain mir-361-5p, which can negatively regulate the transcription of vascular endothelial growth factor A (VEGFA)mRNA[29, 30].In conclusion, copper ion stimulated M Φ It can down regulate the expression of anti angiogenic RNA in exosomes and up regulate the expression of Pro angiogenic RNA in exosomes,and finally promote angiogenesis.Exosomes derived from M1 macrophages highly express miR-155 and can be taken up by endothelial cells (ECS), which simultaneously target two signal pathways, rac1-pak2 and sirt1/ampkα2-enos, thereby inhibiting angiogenesis and cardiac repair.Jeppesen DK et al.[31] tried to study the effect of exosomal microRNA derived from M2 macrophages on the differentiation of bone marrow mesenchymal stem cells.The study showed that mir-5106 was highly expressed in exos of M2 macrophages, and it could be transferred to bone marrow mesenchymal stem cells.Among them, it targeted hydrochloric acid-induced kinase 2 (sik2) and hydrochloric acid-induced kinase 3 (sik3) genes to induce osteoblast differentiation in vitro and in vivo.Other studies have reported that macrophage derived exosomes contain mir-184, which can inhibit VEGF expression through negative regulation of gata2 transcription regulator (Fog-2)[2].

2.The function of exosomes in implant osseointegration

Studies have shown that ta-speek coating in vitro can promote the direct osteogenic differentiation of bone marrow mesenchymal stem cells.More importantly, exo TA speek implantation has the characteristics of immune regulation and direct osteogenesis, and can ultimately promote bone binding and new bone formation in living organisms.In conclusion, the research of tengronglin et al.[33]shows that exosomes are effective and powerful additives to produce advanced immunomodulatory and bone regeneration materials.In addition, exosomes derived from bone marrow mesenchymal stem cells bind and bind extracellular matrix proteins such as fibronectin and type I collagen to biomaterials and bone surfaces.This function also allows exosomes as bionic tools to induce bone marrow mesenchymal stem cells to differentiate into osteogenic lineages.

Research findings, CIRC_ 0008542 gradually up regulates osteoclast differentiation and bone resorption through mir-185-5p/rank axis[34, 35, 36].This phenomenon is a new molecular mechanism in response to the unbalanced stress in the implant neck in the bone binding microenvironment.In addition, Wang et al.Found that overexpression of alkB homologue 5 (alkbh5) in exosomes significantly saved circ_ 0008542 induced bone loss.This study provides a method to enhance immediate implant resistance by releasing alkbh5 exosomes from MC3T3-E1 cells.

Angiogenesis is crucial for implant bone bonding[37].It is well known that macrophages play an immunomodulatory role in angiogenesis.Macrophages can affect the secretion of exosomes through the ions released from implants.Co ions can enter cells through divalent metal transporter 1 (DMT1), stimulate the production of reactive oxygen species, activate pi3k/akt/mtorc1 and mek/erk signaling pathways, and regulate hypoxia inducible factor-Iα(HIF-1α) And thus promote angiogenesis.Current research shows that proper surface treatment of implants 200 μ The Co ion concentration of M can stimulate macrophages to secrete angiogenesis promoting exosomes, which can play the potential of angiogenesis[38].In addition, exosomes can also be loaded onto the implant surface to promote angiogenesis and bone binding around the implant[39].

3.Summary and Outlook

The regulation of bone remodeling by exosomes is a complex process, which is affected by many factors.The effect of exosomes derived from bone related cells on the activity and function of bone cells can be regulated by Wnt/ β- Catenin signaling pathway, osteogenic suppressor genes (RANKL, SOST, DKK1)and osteogenic differentiation genes (Runx2, ALP) are realized.In this paper, the effects of exosomes derived from mesenchymal stem cells, osteoblasts, osteoclasts, osteoblasts and macrophages on bone remodeling and the functions of exosomes in implant osseointegration were described.It is hoped that this article will help readers understand the role of exosomes in the regulation of bone remodeling, so as to provide a new idea for improving the success rate of implant bone binding, such as regulating the exosomes secreted by bone derived cells through the ions released by implants,and effectively regulating the M2 polarization of macrophages to form a local bone remodeling environment.