M2 macrophage-derived exosomes carry miR-142-3p to restore the differentiation balance of irradiated BMMSCs by targeting TGF-β 1

Purpose Radiotherapy is essential to cancer treatment, while it inevitably injures the surrounding normal tissues, and bone tissue is one of the most common sites prone to irradiation. Bone marrow mesenchymal stem cells (BMMSCs) are sensitive to irradiation and the irradiated dysfunction of BMMSCs may be closely related to irradiation-induced bone damage. Macropahges paly important role in stem cell function regulation, bone metabolic balance and irradiation response, but the effects of macrophages on irradiated BMMSCs are still unclear. This study aimed to investigate the role of macrophages and macrophage-derived exosomes in restoring irradiated BMMSCs function. Methods The effects of macrophage conditioned medium (CM) and macrophage-derived exosomes on osteogenic and �brogenic differentiation capacities of irradiated BMMSCs were detected. The key microribonucleic acids (miRNAs) and targeted proteins in macrophage-derived exosomes were also determined. Results The results showed that X-ray irradiation signi�cantly inhibited the proliferation of BMMSCs. Additionally, it caused a differentiation imbalance of BMMSCs, with decreased osteogenic differentiation and increased �brogenic differentiation. M2 macrophage-derived exosomes (M2D-exos) inhibited the �brogenic differentiation and promoted the osteogenic differentiation of irradiated BMMSCs. We identi�ed that miR-142-3p was signi�cantly overexpressed in M2D-exos and irradiated BMMSCs treated with M2D-exos. After inhibition of miR-142-3p in M2 macrophage, the effects of M2D-exos on irradiated BMMSCs differentiation were eliminated. Furthermore, transforming growth factor beta 1 (TGF-β 1), as a direct target of miR-142-3p, was signi�cantly decreased in irradiated BMMSCs treated by M2D-exos. Conclusion This study indicated that M2D-exos could carry miR-142-3p to restore the differentiation balance of irradiated BMMSCs by targeting TGF-β 1. These �ndings pave the way for a new, promising, and cell-free therapeutic method to treat radiation-induced bone damage.


Introduction
Radiotherapy, either alone or in combination with surgery and chemotherapy, has become one of the most important methods for cancer treatment.It is estimated that approximately 10 million patients with cancer worldwide received radiotherapy annually [1,2].Radiotherapy increases the cure rate of malignant tumors, but also destroys normal tissues.Compared with other tissues, bone tissues absorb 30-40% more radiation energy, thereby making them the most commonly irradiated tissues [3].However, the prevention and treatment of radiation-induced bone damage still remains a clinical challenge.Radiationinduced bone damage includes bone loss, bone fragility, bone fracture, and osteonecrosis [4,5].As one of the most severe types of radiation-induced bone damage, the pathology of osteoradionecrosis of the jaw (ORNJ) after radiotherapy in the head and neck region is also characterized by excessive brotic accumulation, in addition to bone loss and bone necrosis [6,7].However, the mechanisms underlying this irradiation-induced bone damage and brosis are not fully understood.
BMMSCs are multidirectional differentiation-capable stem cells that are widely distributed in bone marrow, and were recently thought to be the main target of irradiation in bone tissues [8,9].BMMSCs can differentiate into osteoblasts, adipocytes, chondrocytes, and so on, and the differentiation ability of BMMSCs is greatly affected by the surrounding microenvironment.Studies showed that the osteogenic differentiation of BMMSCs signi cantly decreased after irradiation, which was an important cause of irradiation-induced bone damage [10].In the irradiation microenvironment, BMMSCs also showed a potential to differentiate into myo broblasts, the main effector cells of brosis, by a process called brogenic differentiation.The abnormal proliferation of myo broblasts after irradiation is the culprit of radiation-induced brosis [11].Mesenchymal stem cells from different origins are usually used for radiation protection.However, the potential risk of brogenic differentiation hinders stem cell therapy.In the acute in ammatory response induced by irradiation at the beginning of the initial stage, cells begin to secrete cytokines, such as broblast growth factor-β, transforming growth factor-β-1 (TGF-β1), tumor necrosis factor-α, and interleukins, thereby leading to the aggregation and transdifferentiation of broblasts and BMMSCs into myo broblasts [12], which are responsible for the characterization of brosis in many irradiation-induced bone injury diseases.In summary, restoring the normal function of BMMSCs is important to treat irradiation-induced bone injury diseases.
After irradiation damage, various immune cells were recruited to the irradiated eld sites, which started the repair process and regulated bone homeostasis [13].As a key player in the immune system, macrophages have been widely involved in regulating the in ammatory responses and promoting tissue injury and repair.Previous studies have found that macrophages play an important role in regulating the irradiation protection of speci c cells [14][15][16].In addition, macrophages displayed the strongest interaction with BMMSCs among all the immune cells and had regulatory effects on BMMSCs differentiation function [17].However, the role of differently polarized macrophages in regulating the function of irradiated BMMSCs, especially in osteogenic and brogenic differentiation ability, has not been explored.
Exosomes, double membrane-structured vesicles with 30-150 nm in diameter, are a component of paracrine secretion that contain functional messengers, such as ribonucleic acids (mRNAs), miRNAs and proteins.Furthermore, they are cytoprotective and promote tissue repair [18].Among the contents encapsulated by exosomes, miRNAs are the most widely studied, and many studies have con rmed that miRNAs in exosomes played a regulatory role in the balance of bone metabolism [19] and brogenic differentiation [20].Therefore, further identi cation of the molecular mechanisms that regulate the osteogenic and brogenic differentiation of BMMSCs after irradiation is critical for treating or preventing ORNJ and other types of radiation-induced bone damage.This study investigated the effects of different polarized macrophage-derived exosomes on irradiated BMMSCs, and the underlying mechanisms.

Isolation And Culture Of Bmmscs And Bmdms
The male Sprague-Dawley (SD) rats (2-3 weeks of age) used in the experiment were all obtained from the Laboratory Animal Center of the Fourth Military Medical University and approved by the Welfare and Ethics Committee of the Laboratory Animal Center of the Fourth Military Medical University.Femurs and tibias of SD rats were used to obtain bone marrow, and the whole bone marrow adhesion method was used to culture BMMSCs as described in previous study [21].The cells were cultured in DMEM complete medium (include 10% FBS and 1% penicillin-streptomycin) at 37°C in a 5% CO 2 incubator, and the medium was refreshed every 3 days.Cells at passages 3-5 (P3-P5) were used in this study.The multidifferentiation potential and surface makers were detected by osteogenesis-induction, adipogenesisinduction, chondrogenesis-induction and ow cytometric analysis to identify BMMSCs.
To obtain primary bone marrow derived macrophages (BMDMs), the bone marrow cells were isolated from femurs and tibias of rats, then cultured with DMEM supplemented by 10% FBS, 1% penicillin-streptomycin, and stimulated by 30 ng/mL M-CSF for 7 days.M1 and M2 macrophages polarization was initiated by 100 ng/mL LPS and 30 ng/mL IFN-γ, or 20 ng/mL IL-4 for 24 h, respectively.The macrophages were identi ed by ow cytometric analysis and Real-time Quantitative polymerase chain reaction (RT-qPCR).
The P3-P5 BMMSCs were transfered into a centrifuge tube with cell numbers of 4×10 5 , and centrifuged at 250 × g for 4 min.And then a chondrogenesis-induction kit was used to induce the BMMSCs following the manufacturer's instructions.When the induced cells formed into a cartilage pellet, it was xed and stained with alcian blue solution.
Alp Activity, Alp Staining And Alizarin Red Staining After 7 days of osteogenesis induction, the supernatant of BMMSCs was collected and the ALP activity was detected by using an ALP Detection Kit according to the manufacture's instructions.The optical density (OD) value of the supernatant was measured at 520 nm using a spectrophotometer (Epoch spectrophotometer, USA).ALP staining was performed on day 7 of osteogenesis induction by using the BCIP/NBT alkaline chromogenic phosphatase kit.After 3 weeks of the osteogenesis induction of BMMSCs, alizarin red staining was performed.BMMSCs were xed with 4% paraformaldehyde for 20 min and incubated with 1% alizarin red staining solution for 30 min.The images of stained mineralized nodules were obtained using an inverted light microscope (Olympus, Tokyo, Japan).Then 2% cetylpyridinium chloride was used to dissolve the stained mineralized nodules, and OD value of the supernatant solution was determined at 560 nm using a spectrophotometer.
For macrophages, surface makers CD11b, F4/80, CD86 and CD206 were used for M0, M1 and M2 macrophage identi cation.The cells were incubated with polyclonal antibodies of these surface makers in the dark at 4°C for 30 min.Subsequently, ow cytometry (Beckman Coulter, USA) was used to detect the positively stained cells.
Macrophage Conditioned Medium (Cm) Preparation M1 and M2 macrophages were cultured with DMEM complete medium at 37°C in a 5% CO 2 incubator for 24 h, and the medium supernatant was collected.To remove debris and cells, the medium supernatant was centrifuged at 2,000× g for 30 min at 4 ℃ and then ltered through a sterilized 0.22-µm lter.After these protocols, the obtained uid was de ned as macrophage conditioned medium (CM).The CM derived from M1 and M2 macrophages was respectively termed as CM-M1 and CM-M2.The CM-M1 and CM-M2 was stored at -80°C before use.

Puri cation, Characterization And Uptake Of Exosomes
Before exosomes collection, the culture medium of M1 and M2 macrophages was replaced with an exosome-depleted complete medium.After 24 h, the obtained cell supernatant was centrifuged at 1,000 × g for 10 min and then ltered using a 0.22-um lter.This process was followed by an ultra-centrifugation at 100,000 × g at 4°C for 70 min.Subsequently, the supernatant was discarded and the precipitates were resuspended in PBS.The medium was ultra-centrifuged at 100,000 × g for another 70 min to purify the exosomes.The isolated exosomes were resuspended in PBS and stored − 80°C for further use.For identi cation of collected exosomes, the morphology was observed by transmission electron microscopy (TEM, JEOL, Japan), the distribution size was assessed by Nanoparticle Tracking Analysis (NTA, NanoFCM, China), and the speci c surface markers (TSG 101, CD81) were detected by western blot.To con rm exosomes were uptaken by irradiated BMMSCs, PKH 26 was used to label the exosomes.Brie y, the exosomes were incubated in 1 mL of diluted C solution containing 5 µM PKH26 dye for 5 min.The labeling reaction was stopped by adding 10 mL PBS.It was followed by ultra-centrifugation at 100,000 × g for 70 min at 4°C and suspension in 100 µL PBS.The irradiated BMMSCs were treated with these labeled exosomes for 12 h and then analyzed by confocal microscope (NiKon, Japan).

Bmmscs Irradiation And Treatment
BMMSCs at 70-90% con uence were irradiated by X-rays using an RS2000 X-ray Biological Irradiator (RAD SOURCE, USA) at a voltage of 160 kV and a current of 25 mA.The radiation dose rate was 1.20 Gy/min, and the radiation dose was 2, 6, and 10 Gy, respectively.After 1 d, 3 d, 5 d, and 7 d of radiation, CCK-8 assay was used to detect cell proliferation of irradiated BMMSCs.Brie y, BMMSCs with a density of 5x10 3 cells/well were added to 96-well plates, and incubated with 10 µL of CCK-8 solution at 37 ℃ for 1 h.The OD value was measured at 450 nm using a spectrophotometer.The Nanog and octamer-binding transcription factor 4 (OCT-4) mRNA expression of irradiated BMMSCs was detected by RT-qPCR at 48 h after radiation.The most proper radiation dose was chosen according to the CCK-8 assay and the RT-qPCR results.
After treatment for 48 h, the expression of α-SMA and Col of BMMSCs in all groups were detected by western blot and RT-qPCR.After treatment and osteogenesis induction for 7 d, ALP staining and ALP activity detection were performed in all groups, and the expression of Runx-2, ALP and Col were detected by western blot and RT-qPCR.After treatment and osteogenesis induction for 21 d, alizarin red staining was performed in all groups.Notebly, the CM was mixed 1:1 with osteogenesis-induction medium.

Rt-qpcr
For mRNA detection, TRIzol Reagent was used for total RNA isolation of cells, and PrimeScript TM RT Master Mix kit was used for cDNA synthesis.For miRNA detection, Universal miRNA extraction Kit was used to extract miRNAs of cells and exosomes, followed by cDNA synthesis using GoldenstarTM RT6 cDNA Synthesis Kit, based on the manufacture's instructions.RT-qPCR was conducted using the TB Green Premix Ex Taq Kit.PCR reactions were performed by Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scienti c,USA).All results were calculated by 2 ( −ΔΔCT ), and normalised to GAPDH or U6.All primer sequences were listed in the Supplementary Table S1.

Western Blot
The total protein of cells was extracted using the RIPA buffer containing 1% protease inhibitor.

Statistical Analyses
All experiments were performed at least thrice, the number of experimental replicates is denoted by n. Results were analyzed using the GraphPad Prism 8.0.2 software (GraphPad Software Inc.) and were presented as the mean ± standard deviation.The differences between two groups and multiple groups were analyzed by the t-test and one-way analysis of variance (ANOVA).A value of p < 0.05 was considered statistically signi cant.
The ow cytometry results showed that the positive rate of cell surface markers in BMDM, was 82.2% (F4/80) and 90.1% (CD11b) (Fig. S1E).The positive rate of CD86 was signi cantly increased after 24 h of treatment with LPS and IFN-γ (Fig. S1F).The positive rate of CD206 was also signi cantly increased after 24 h treatment of IL-4(Fig.S1G).RT-qPCR results showed that TNF-α and IL-1β were signi cantly increased after LPS and IFN-γ treatment, while Arg-1 and CD206 were signi cantly increasedafter IL-4 treatment (Fig. S1H).These results showed that M1 and M2 macrophages were successfully cultured and polarized.

Irradiation Induced Differentiation Imbalance Of Bmmscs
In order to select the appropriate radiation dose, BMMSCs were irradiated with different radiation doses (0, 2, 6, 10Gy).The CCK-8 results showed that the proliferation rate of irradiated BMMSCs with 6 and 10 Gy radiation doses was signi cantly lower than that of 0 Gy (Fig. 1A).However, BMMSCs showed negative growth after 5 days of irradiation at 10 Gy (Fig. 1A).The RT-qPCR results showed that the expression levels of stemness maintenance markers, OCT-4 and Nanog, were decreased with increasing doses of radiation (Fig. 1B-C).Based on these results, 6 Gy radiation dose was chosen in follow-up experiment, because this dose achieved a certain degree of radiation damage to BMMSCs without completely destroying cell proliferation and stemness.
Alpha-smooth muscle actin (α-SMA) is a marker protein for myo broblasts, and COL III is a common marker of brosis.The mRNA and protein expression levels of α-SMA and COL III in BMMSCs were signi cantly increased after irradiation (Fig. 1D-F).The mRNA and protein expression levels of Runx-2, ALP and COL I in BMMSCs were signi cantly decreased after irradiation.The results con rmed that an irradiation dose of 6 Gy inhibited the osteogenic differentiation of BMMSCs (Fig. 1G-I), which was also con rmed by ALP staining, ALP activity and alizarin red staining (Fig. 1J-M).These results were in accordance with the pathological phenomenon that radiation-induced necrotic bones were surrounded with myo broblasts and brotic matrix.In a word, irradiation caused a differentiation imbalance of BMMSCs, with increased brogenic differentiation and decreased osteogenic differentiation.

Cm-m1 And Cm-m2 Couldn't Reverse The Differentiation Imbalance Of Irradiated Bmmscs
The effects of CM-M1 and CM-M2 on irradiation-induced differentiation imbalance of BMMSCs were investigated.CM-M1 treatment signi cantly reduced the expression of α-SMA and COL in irradiated BMMSCs, whereas CM-M2 treatment signi cantly promoted the expression of α-SMA and COL in irradiated BMMSCs (Fig. 2A-C).Western blot and RT-qPCR results showed that the expression levels of osteogenesis-related genes in irradiated BMMSCs, including COL I, RUNX2, and ALP, were signi cantly increased after CM-M2 treatment (Fig. 2D-G).The CM-M1 treatment had no signi cant effect on the expression levels of these osteogenesis-related genes.Furthermore, compared with irradiation group, the ALP activity of the CM-M2 group was signi cantly increased (Fig. 2H-I), and the formation and staining degree of calcium nodules were also increased in the CM-M2 treatment group (Fig. 2J-K).These results indicated that CM-M2 treatment promoted the osteogenic differentiation and brogenic differentiation capacity of irradiated BMMSCs, whereas CM-M1 treatment inhibited the brogenic differentiation capacity without affecting the osteogenic differentiation capacity.In one word, both CM-M1 and CM-M2 couldn't completely reverse the differentiation imbalance of irradiated BMMSCs.

Macrophage-derived Exosomes Were Internalized By Irradiated Bmmscs
TEM results showed that the extracted M1D-exos and M2D-exos had a cup or ball shape (Fig. 3A).The results of the NTA showed that the diameters of these extracted particles were ranged from 40 to 150 nm (Fig. 3B).Western blot further proved that the isolated exosomes were positive for speci c surface protein markers, such as TSG101 and CD81 (Fig. 3C).To test whether the irradiated BMMSCs could internalize these exosomes, the PKH26-labeled exosomes (Red) were co-cultured with irradiated BMMSCs.The nucleus and cytoskeleton of irradiated BMMSCs were stained with DAPI (Blue) and ghostly cyclopeptide (Green).After 12 h, exosomes labeled with red uorescence were mainly distributed in the cytoplasm surrounding the nucleus of irradiated BMMSCs under confocal microscopy, which indicated that irradiated BMMSCs exhibited e cient uptake of M1D-exos and M2D-exos (Fig. 3D).

M2d-exos Improved The Differentiation Imbalance Of Irradiated Bmmscs
The irradiated BMMSCs were co-cultured with M1D-exos and M2D-exos for 48 h to detect the effects of these exosomes on irradiated BMMSCs.Western blot and RT-qPCR results showed that both M1D-exos and M2D-exos signi cantly inhibited the expression of α-SMA and COL in irradiated BMMSCs (Fig. 4A-C).After 7 days of osteogenesis induction, the expression of COL I, Runx-2 and ALP were signi cantly increased in the M2D-exos group compared to the irradiation group (Fig. 4D-F).However, M1D-exos treatment had no obvious effect on the expression of these osteogenesis-related genes.ALP staining, ALP activity and alizarin red staining also showed the same trends as the expression of osteogenesisrelated genes (Fig. 4G-J).These results con rmed that the M2D-exos inhibited the brogenic differentiation capacity and promoted the osteogenic differentiation capacity of irradiated BMMSCs, which successfully restored the differentiation function of the BMMSCs suffered from radiation damage.

Discussion
Irradiation-induced bone damage is a common complication after radiotherapy for cancer treatment, and it has become an increasingly important clinical challenge with the prolonged survival of cancer patients.
However, there is still a lack of effective strategies for the prevention and treatment of irradiation-induced bone damage, leading to a high risk of fragility bone fractures and osteonecrosis [23,24].BMMSCs were previously thought to be the main targets of irradiation, which caused the inhibition of bone formation [25].Macrophages have been proven to reduce the radiosensitivity of cells [26].However, the interaction between macrophages and irradiated BMMSCs remains unclear.In the current study, we investigated the effects of macrophages and macrophages-derived exosomes on irradiated BMMSCs, and the ndings con rmed that M2D-exos could carry miR-142-3p to restore the differentiation balance of irradiated BMMSCs through promoting osteogenic differentiation and inhibiting brogenic differentiation via targeting TGF-β1.Our ndings provided a novel therapeutic strategy for irradiation-induced bone damage and offered a reference for further studies.
BMMSCs are self-renewal and multi-directional differentiation pluripotent stem cells, which are widely distributed in bone marrow and have the ability to regulate bone metabolism and promote bone regeneration [27,28].As main stem cells with active proliferative capacity in bone tissues, BMMSCs were sensitive to irradiation and the inhibition of osteoblast differentiation of irradiated BMMSCs was thought to be the key elements of irradiation-induced bone damage, including irradiation-induced bone loss, bone fracture and osteonecrosis [29,30].Except for this, BMMSCs were usually used as stem cell therapy in many diseases.Based on the regenerative and anti-in ammatory functions, BMMSCs and mesenchymal stem cell (MSCs) from other origins were previously proved to have positive effects on the prevention and treatment of irradiation-induced diseases, such as irradiated pneumonia, irradiated pulmonary brosis, irradiated liver injury and so on [31][32][33].Similarly, BMMSCs were also used to treat brotic diseases, such as idiopathic pulmonary brosis, non-alcoholic fatty liver brosis, skin brosis and so on, due to the immunomodulatory function of BMMSCs to the recipient cells [34][35][36].However, in recent years, researchers found that BMMSCs could differentiate into myo broblasts which is the key effector cell in brosis, in particular microenvironment including irradiation injury [37,38].This brogenic differentiation phenomenon makes MSCs a potential danger to aggravate brosis stimulated by irradiation or other factors [39].In this study, we found that X-ray irradiation suppressed the proliferation of BMMSCs in a dose-dependent manner.Furthermore, at a dose of 6 Gy, irradiation signi cantly inhibited the osteogenic differentiation and mineralization, while signi cantly promoted the brogenic differentiation with high expression levels of COL and α-SMA mRNA and protein after irradiation.These ndings of differentiation imbalance in irradiated BMMSCs in our study con rmed the concerns of brotic stimulation potential of BMMSCs in irradiated microenvironment, which were proposed by other researchers in previous studies.Therefore, it is essential to restore the differentiation balance in BMMSCs after irradiation to treat irradiation-induced bone damage, other irradiation-induced diseases or brosis.
Macrophages play a key role in the immune system and have been widely used to regulate in ammatory responses and promote the repair of tissue damage.Generally, M1 macrophages help to clean wounds and play an in ammatory role.In contrast, M2 macrophages are anti-in ammatory and promote tissue repair.In the past few years, several studies have con rmed the regulatory role of macrophages in MSCs [40].In the other way, studies also found that BMMSCs had a signi cant impact on macrophage polarization in the process of in vitro culture.To avoid the in uence of this interaction between BMMSCs and macrophages, M1 and M2 macrophages conditioned medium (CM-M1, CM-M2) were used in most previous studies to investigate effects of macrophages polarization on the in vitro cell behavior of BMMSCs.In CM-M1 and CM-M2, cytokines secreted by macrophages are major contributor to regulate function of BMMSCs.The effects of macrophages on irradiated BMMSCs haven't been studied further, while the effects of macrophages on normal BMMSCs have been studied a lot.Previous studies have shown that M2 macrophages can promote osteogenic differentiation of BMMSCs.It has also been studied that M2 macrophages promoted the differentiation of myo broblasts through secreting TGF-β1.
Similarly, the current study found that CM-M2 signi cantly promoted the osteogenic differentiation and brogenic differentiation of irradiated BMMSCs.And for M1 macrophages, many studies showed that it could inhibit osteogenesis and osteogenic differentiation of BMMSCs, and it could reduce brotic accumulation [41,42].In our study, we found that CM-M1 had no signi cant effects on the osteogenic differentiation of irradiated BMMSCs, but it could inhibit brogenic differentiation of irradiated BMMSCs.
So, neither CM-M1 nor CM-M2 could reverse the differentiation imbalance of irradiated BMMSCs, other effective methods should be searched further.
Exosomes are small vesicles secreted by a range of cells and acted as important intercellular and interorgan communication tools through transferring small molecules including proteins and nucleic acids.
Therefore, it is popular to use exosomes as ideal nanomaterials for delivering the regulatory substances to the targets to treat diseases in recent years [43].In addition, exosomes derived from macrophages are key regulatory factors to adjacent cells, including BMMSCs [44].Therefore, macrophage-derived exosomes should be considered to better explain the mechanism of crosstalk between macrophages and BMMSCs.According to previous studies, M1D-exos could inhibit osteogenesis [45], while we found that M1D-exos had no effects on the osteogenic differentiation of irradiated BMMSCs.And M1D-exos previously have been proved to be effective anti-brotic therapy [46].Consistently, the current study showed that M1D-exos signi cantly reduced the differentiation of BMMSCs into myo broblasts after irradiation.These ndings indicated that M1D-exos could not completely restored the differentiation balance of irradiated BMMSCs, as M1D-exos had positive effects on inhibiting brogenic differentiation, while had negative effects on promoting osteogenic differentiation.M2 macrophages play a key role in tissue damage repair.M2D-exos were used to be effective bone regeneration and bone repair tools through promoting osteogenic differentiation of BMMSCs, proliferation of osteoblasts and osteocytes and bone mineralization [45].Our study found that M2D-exos could signi cantly promote osteogenesis of irradiated BMMSCs, which indicated that M2D-exos could also act as effective bone regenerative tools in irradiation microenvironment.However, the role of M2D-exos in brosis was ambiguous and even contradictory in previous studies.Some studies showed that M2D-exos stimulated brogenic differentiation of broblasts, stem cells and endothelial cells into myo broblasts, promoted myo broblasts proliferation and secretion, and accelerated extracellular matrix accumulation, nally leading to tissue brosis [47][48][49].On the contrary, some studies found that M2D-exos acting as immunomodulatory factors, could migrate in ammation, reduce the pro-in ammatory factors, and restore the normal function of recipient cells, nally promoting tissue repair in a regular way but not a brotic way [50].These different roles of M2D-exos in brosis may be related to different pro-brotic irritants, different organs and tissues, different microenvironment and different action timing.Our study found that M2D-exos could signi cantly inhibit the differentiation of BMMSCs into myo broblasts after irradiation, which indicated that M2D-exos may play an anti brotic role in irradiation microenvironment.
The ndings in our study showed that M2D-exos successfully restored the differentiation function of irradiated BMMSCs with promoting osteogenic differentiation and inhibiting brogenic differentiation, which provided a cell-free and less harmful way to treat irradiation-induced bone damage.Nevertheless, we only performed in-vitro study, the role of M2D-exos in irradiation-induced brosis and irradiationinduced bone damage needed to be studied further in vivo.
Among the components of macrophage-derived exosomes, miRNA is the most important factor.miRNA regulates target cell-related genes by combining with mRNA in target cells.The results of this study found that miR-142-3p was abundant in M2D-exos and could be transferred into irradiated BMMSCs.And we also found that inhibition of miR-142-3p could inhibit the effects of M2D-exos on irradiated BMMSCs, which indicated that miR-142-3p played a key role in M2D-exos restoring irradiated BMMSCs function.In previous studies, miR-142-3p was proved to have effective anti-brotic role in hypoxia/reoxygenationinduced cardiac brosis, liver brosis, idiopathic pulmonary brosis, nonalcoholic fatty liver disease and skin scars, through inhibiting myo broblasts differentiation and proliferation, suppressing TGF-β1 expression and so on [51][52][53][54].Previous studies in bone diseases found that miR-142-3p could inhibit osteoclastogenesis, promote osteoblast activity and matrix mineralization in bone healing process, and induce osteogenic differentiation of BMMSCs, which indicated that miR-142-3p had a positive role in bone regeneration and bone repair [55][56][57].Although the results of miR-142-3p inhibiting brogenic differentiation and promoting osteogenic differentiation of irradiated BMMSCs in our study were in consistent with previous cognition of miR-142-3p effects, we believe that mi-RNA in M2D-exos were more abundant than miR-142-3p.Therefore, further in-vivo and in-vitro studies are needed to determine the speci c mechanism of M2D-exos in preventing and treating irradiation-induced bone damage.

Conclusion
In conclusion, this study indicated that M2 macrophage-derived exosomes (M2D-exos) restored the differentiation balance of irradiated BMMSCs by promoting osteogenic differentiation and inhibiting broblastic differentiation.This positive effects of M2D-exos were mediated partially by delivering miR-142-3p into irradiated BMMSCs and suppressing the expression of TGF-β1 (Fig. 7).Our ndings provide new insight into the prevention and treatment of irradiation-induced bone damage.

Declarations
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FundingThis work was supported
by the National Natural Science Foundation of China (81903249) and Shaanxi Provincial Natural Science Basic Research Program project (2022JZ-50).Con The authors have no relevant nancial or non-nancial interests to disclose in connection with this manuscript.

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Figure 3 The
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