The role and mechanism of exosomes from umbilical cord mesenchymal stem cells in inducing osteogenesis and preventing osteoporosis

Mesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the inuence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of the osteogenic differentiation microenvironment on the functions of human umbilical cord MSCs (HucMSC)-derived exosomes. We explored the role and mechanism by which exosomes promote osteogenic differentiation and prevent osteoporosis, and propose a method to treat and prevent osteoporosis. HucMSCs were isolated from human umbilical cords, and after osteogenic differentiation and normal culture for 48 h, cell supernatants were collected to isolate exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, and alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-decient osteoporosis model mice were established, and treated with the two exosomes preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatics.


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Background Mesenchymal stem cells (MSCs) have the potential for multidirectional differentiation, immune regulation, and tissue repair and regeneration, and have therapeutic effects on a variety of orthopedic diseases [1]. However, studies have found that after MSC transplantation, the number of cells remains very small, and the substitution effect of local cell differentiation is insu cient to support complex pathological changes [1,2], suggesting that local proliferation and differentiation of MSCs might not be the mechanism by which MSCs exert their therapeutic effects. Recent studies have found that exosomes play an important role in intercellular communication [3,4]. MSC-derived exosomes contain a variety of bioactive substances, and have the functions of low immunogenicity, immune regulation, promoting proliferation, and promoting tissue repair. For example, MSC-derived exosomes have therapeutic effects on diabetes, renal disease, and ischemic stroke [5,6]. MicroRNAs (miRNAs) are one of the main bioactive substances in exosomes, and comprise endogenous non-coding single-stranded small RNAs (18)(19)(20)(21)(22)(23)(24)(25) nucleotides in length) that regulate gene expression at the post-transcriptional level by binding to the 3' untranslated region (UTR) region of target mRNAs [7]. Experimental studies have con rmed that miRNAs in MSC-derived exosomes play an important role in regulating cell function and treating various diseases [8]. For example, by targeting WNT5A (Wnt family member 5A), high expression of exosome-derived miR-92a-3p in MSCs could promote cartilage regeneration [9]. However, the secretion and function of exosomes are greatly in uenced by the cell state and microenvironment. For example, in the microenvironment of tumor formation, MSC-derived exosomes promote the disappearance of normal functions of tissue repair, and changes to the exosome cargoes can promote the proliferation and invasion of tumor cells, and lead to the transformation of normal stromal cells to tumor broblasts [10].
Human umbilical cord MSCs (HucMSCs) can be collected in a non-invasive manner, and have a strong regenerative ability [11], resulting in them having broad application prospects in regenerative medicine.
However, the effect of the osteogenic differentiation microenvironment on the function of exosomes remains unclear. In the present study, we aimed to analyze the effects of exosomes secreted by HucMSCs in the microenvironment of osteogenic differentiation on the proliferation and differentiation of osteoblasts, and to explore the changes of exosome function in the microenvironment. The exosomes were applied to treat an osteoporosis animal model. High-throughput gene sequencing and bioinformatics techniques were used to analyze the differential expression of exosomal miRNAs, to reveal the relevant molecular mechanisms of exosome action, and provide new targets to treat and prevent osteoporosis.

Methods
Isolation and characterization of hucMSCs Umbilical cords were obtained from healthy newborn fetuses, and Wharton's jelly tissues were isolated under sterile conditions. Tissues were cut to 1-2 mm 3 size tissue pieces and inoculated in culture asks containing 10% fetal bovine serum (FBS) in a cell incubator at 37 °C and 5% CO 2 saturated humidity.
Collected cell supernatants were subjected to 2000 ´ g gradient centrifugation (Eppendorf, Hamburg, Germany) at 4 °C for 20 min and the pellet was discarded. The supernatant was removed to a new centrifuge tube and centrifuged at 10,000 ´ g at 4 °C for 40 min. The pellet was discarded and the supernatant was removed to a new centrifuge tube. The pellet was collected by centrifugation at 100,000ǵ for 60 min, resuspended by PBS and centrifuged at 100,000 ´ g (Beckman, USA) for 60 min at 4 °C. The pellets were resuspended in PBS, and then ltered and sterilized through 0.22 μm sterile lter membrane, and stored in a freezer (Panasonic, Osaka, Japan) at -80 °C After adjusting the exosome suspension to the appropriate concentration with PBS, the exosomes were dripped on special carbon-lm copper mesh for electron microscopy observation. The exosomes on the mesh were stained with 2% phosphotungstic acid for 2-3 min, air-dried naturally, and photographed using transmission electron microscopy (Hitachi, Tokyo, Japan). Western blotting was used to detect three exosome-derived proteins, CD9, CD81, and HSP70 (ProteinTech, Rosemont, IL, USA). Vesicle diameter in the exosome suspensions was measured using particle size analyzer (Malvern Nanosight NS300, Malvern, UK).

Isolation of osteoblasts
Osteoblasts (OBs) were derived from the skulls of suckling SD rats. OBs in the skull were isolated using an appropriate amount of 0.2% type II collagenase (Gibco NY USA) and inoculated into T75 culture asks (Corning, NY, USA) containing DMEM medium (Gibco NY USA) (containing 10% FBS, penicillin 100 U/ml, streptomycin 0.1 mg/ml).
Cell Counting Kit-8 (CCK-8) The isolated OBs were seeded into 96-well plates at a concentration of 3 ´ 10 3 /ml, and 100 μL of cell suspension was added into each well. After 12 h, Exo1 and Exo2 were co-cultured with the OBs at three concentration of 0, 0.05, 0.1, and 0.2 mg/ml. Four control duplicate wells were set at each concentration.
After incubation for 36 and 60 h, 10 μl of CCK-8 (MCE, Monmouth Junction, NJ, USA) reagent was added to each well, and after incubation at 37 °C for 2 h in the dark, the absorbance at 450 nm wavelength was detected using a microplate reader (Tecan, Männedorf, Switzerland).

Alkaline phosphatase (ALP) and alizarin red staining
The isolated OBs were seeded in 24-well plates, and after 24-48 h, when the cells had grown to 80% con uence, osteogenic induction was performed using osteogenic induction medium. Co-cultures with Exo1 and Exo2, at 0.2, 0.1, and 0 mg/ml were set, respectively. Each concentration gradient had four duplex holes, and the solution was changed every 3 days. Alkaline phosphatase kit(Beyotime, Shanghai, China) was used for analysis after 10 days. After 21 days, alizarin red (Solarbio, Beijing, China) staining was performed according to the manufacturer's instructions. The results for the groups were compared and analyzed according to the depth of staining.

Western Blotting
OBs were co-cultured with Exo1 and Exo2 in osteogenic induction medium conditions, respectively. Three concentration of 0.1, 0.2, and 0 mg/ml were set. After 7 days of culture, the cells were collected and 150 μL of Radioimmunoprecipitation assay (RIPA) Lysis Buffer (Beyotime, Shanghai, China) was added to each well of the 6-well plate for lysis. All protein concentrations were determined using a bicinchoninic acid (BCA) protein concentration determination kit (Beyotime). The protein concentrations in the samples were adjusted so that the protein content was the same in the same volume. The SDS-PAGE gel preparation kit (Beyotime) was used for constant voltage protein electrophoresis at 80 V for 40 min, followed by 120 V for 30 min. The separated proteins were transferred to PVDF membranes (0.2 μm; Thermo Scienti c, Rockford, IL. USA) at 300 mA for 80 min. The membranes were the incubated in 10% skimmed milk (BD) for 1 h at room temperature, incubated overnight with primary antibodies at 4 °C, followed by incubation with horseradish peroxidase-labeled secondary antibody (Beyotime) at room temperature for 1 h. Immunoreactive proteins on the membranes were visualized using a chemiluminescent imager (Bio-Rad, Hercules, CA, USA) using extremely hypersensitive ECL luminescent reagents. The mouse-derived primary antibodies recognized target proteins of osteogenic differentiation including RUNX family transcription factor 2 (RUNX2), osteopontin (OST), collagen type I alpha 1 Chain (COL1A1), and ALP (Abcam, Cambridge, MA, USA), and the internal reference was detected using mousederived anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ProteinTech). The protein expression intensity was compared and analyzed according to the ratio of the gray value of target protein to the gray value of the internal reference protein.

Animal model of osteoporosis
This experimental study was approved by the Medical Laboratory Animal Ethics Committee, and all experimental procedures were in accordance with the procedures of the Medical Laboratory Animal Center of Shantou University Medical College. All C57BL/6J mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd (Beijing, China). Forty 10-week-old C57 female mice were divided into ve groups with eight mice in each group: Sham group, ovariectomized (OVX) group, OVX+Exo1 group, OVX+Exo2 group, and OVX+E2 (estradiol) group. Referring to experimental studies such as Zhen Lian [12], all mice in the experimental group were anesthetized with 2% pentobarbital sodium.
Except for the sham group, ovariectomy was performed to simulate the osteoporotic disease model caused by estrogen de ciency. The sham group underwent a sham operation as the control group. A small incision was made on the back of mice to remove the ovaries, including part of the fallopian tube, and the incision was sutured with 5-0 arti cial absorbable sutures. After 1 week of recovery time, the experiment was carried out. The OVX+Exo1 group, OVX+Exo2 group, and OVX+E2 group were intraperitoneally injected with Exo1 (0.5 mg/kg), Exo2 (0.5 mg/kg), and E2 (0.15 mg/kg), respectively. The OVX group was injected with the same volume of PBS. Injection every 3 days for 6 weeks.
Six weeks later, the tibia of mice were taken, and ve groups were subjected to micro computed tomography (microCT) scanning, to detect and analysis various data indicators: Bone volume (BV), relative bone volume (BV/trabecular volume (TV)), cortical bone area (Ct.Ar), cortical bone thickness (Ct.Th), bone surface (BS), ratio of bone surface area to bone volume (BS/TV), bone mineral content (BMC), bone mineral density (BMD), trabecular number (Tb.N), trabecular separation/Spacing(Tb.Sp), and trabecular thickness (Tb.Th). After microCT (SCANCO, Wangen-Brüttisellen, Switzerland) scanning, mouse tibias were xed with 4% paraformaldehyde and decalci ed with 10% EDTA for 4 weeks, and para n-embedded sectioning was performed. After hematoxylin and eosin (HE) staining, the sections were observed and analyzed under a microscope.

MicroRNA Sequencing Analysis
When the P3 generation HucMSCs grew to 80% con uence, they were divided into three groups, and each group had three samples. The control group was replaced with conditioned medium without exosome serum, and the supernatant was collected after 48 h of culture; osteogenic group 3 and osteogenic group 7 were replaced with osteogenic induction differentiation medium containing 10% exosome-free serum, and the supernatant was collected after 48 h and 7 days of culture, respectively. The cell supernatants of the three groups were separated by ultracentrifugation. RNA sequencing uses next generation sequencing (NGS) technology was used to obtain the sequences of miRNAs (18-30 nt or 18-40 nucleotides) in exosomes. Sequencing data were then compared with databases to identify and analyze the small RNA sequences.
The sequencing results of three groups of miRNAs analyzed statistically for differentially expressed miRNAs. The p-value was corrected by multiple hypothesis tests using the Q value. Genes with two or more coincidence differences and a Q-value less than or equal to 0.001 were considered as signi cant differentially expressed genes. The domain value of p-value was determined by controlling the FDR (False Discovery Rate). The FDR value of the difference test was obtained, and the multiple of differential expression of a gene between different samples was calculated according to the expression amount of the gene, as calculated using the FPKM value (Fragments Per Kilobase of transcript per Million mapped reads). The smaller the FDR value, the greater the difference multiples, indicating more signi cant expression differences. Differentially expressed genes were de ned as those with an FDR < 0.001 and > 2fold expression difference.
RNAhybrid [13], miRanda [14], and TargetScan [15] were used to predict the potential target genes of the miRNAs, and then the intersections of the results predicted by three software were noted. According to the results of differential miRNA detection, hierarchical clustering analysis was performed using the pheatmap function in the R software to form a clustering heat map of differentially expressed miRNAs between the groups. The biological functions of genes were investigated using Kyoto Encyclopedia of Genes and Genome (KEGG) Pathway analysis. According to KEGG Pathway [16] public database, pathway signi cance enrichment analysis was carried out to identify those pathways that were signi cantly enriched in candidate genes compared with the whole genome background. Pathways with a Q value < 0.05 were de ned as pathways that were signi cantly enriched in differentially expressed genes. Pathway signi cant enrichment can identify the most important biochemical metabolic pathways and signal transduction pathways in which the candidate genes participate.

Typical features of hucMSCs and hucMSC-derived exosomes
HucMSCs isolated from Wharton's Kelly's tissue of the umbilical cord are typically fusiform, triangular, or brous. When they approach maturity, they appear to from a whirlpool or broom shape. When HucMSCs differentiated into osteoblasts, Alizarin red could stain the mineralized nodules red or orange red; when they differentiated into adipocytes, oil red dye could dye the fat drops in adipocytes orange red; after 30 days of induction, chondrocytes were formalin-xed and para n-embedded, and nally stained with alixin blue, which partly showed the endo-acidic mucopolysaccharides in cartilage tissue (see Figure 1A). Flow cytometry showed that the hucMSCs were positive for CD73, CD90, and CD105; and negative for CD45, CD34, CD11b, CD19, and HLA-DR (see Figure 1B). Electron microscopy showed that the exosomes were typical vesicles(see Figure 1C). The particle size range of exosomes suspension was 20-200 μm (see Figure 1D). Western blotting analysis indicated that the hucMSC-derived exomes contained exosomal markers such as CD9, CD81, and tumor susceptibility 101 (TSG101).(see Figure 1E) Effect of exosomes on the proliferation and differentiation of osteoblasts After co-culture of Exo1 with OB cells for 36 h and 60 h, the absorbance of OD450 was measured by CCK-8, and it was found that Exo1 signi cantly promoted OB proliferation in a concentration-dependent manner (see Figure 2A), but the concentration gradients of Exo2 were not signi cantly different from those of the control group (see Figure 2B).
When the concentration of exosomes was 100 μg/ml, staining for OB differentiation was deepest in the Exo2 group, which indicated that Exo2 could promote osteogenesis better than Exo1 (see Figure 2C-D). Compared with the control group, the cells in the Exo1 and Exo2 groups were stained deeply, and there were many mineralized deposits. When the concentration of exosomes was adjusted to 200 μg/ml, the staining depth and mineralized deposits increased with the increase in concentration (see Figure 2E-H), which showed that exosomes could promote osteogenic differentiation in a concentration dependent manner. After coculture of exosomes and osteoblasts, western blotting showed that the levels of RUNX2, COL1A1, ALP, and OST in the cells of the Exo1 and Exo2 groups were higher than those of the control group, and the levels of RUNCX2, ALP and OST in Exo2 group showed the most signi cant increase (see Figure 3A-B).
The effect of exosomes on osteoporosis in an animal model Six weeks after exosomes application, three-dimensional reconstruction images of tibia and cancellous bone were scanned using micro CT in OVX+Exo1 group, OVX+Exo2 group, OVX group, OVX+E2 group, and sham group (see Figure 4A). After statistical analysis of BV/TV, BMD, Conn.D, tb.n, tb.sp and SMI, it was con rmed that exogroup 1 and Exo2 had a certain therapeutic effect on osteoporosis compared with the OVX group (see gure 4B). Para n sections of mouse tibia and cancellous bone were stained with HE.Compared with the OVX group, the degree of osteoporosis was signi cantly reduced in the Exo1 and Exo2 groups (see Figure 3C).

Expression of microRNA in exosomes
As can be seen from the differential miRNA heat map (see Figure 5A-B), the changes of differentially expressed miRNAs in each group, and the number of differentially expressed miRNAs gradually increased with prolonged osteogenic differentiation.
The predicted target genes of the differentially expressed miRNAs were analyzed by KEGG Pathway enrichment (see Figure 6A-B). We found that target genes in the control vs. osteogenic 3 comparison were enriched in the osteoclast differentiation pathway and peroxisome proliferator activated receptor (PPAR) signaling pathway. The PPAR signaling pathway is important for MSC to differentiate into adipocytes. Therefore, the regulation by miRNAs of the PPAR signaling pathway might be one of the mechanisms by which exosomes regulate cell differentiation and treat osteoporosis.
Osteoclast differentiation, osteoblast differentiation, and the PPAR signaling pathway are closely related to the occurrence and development of osteoporosis. Three miRNAs related to the above signal pathways were screened out: Hsa-mir-328-3p, hsa-mir-2110, and hsa-let-7c-5p. The target genes related to the above signal pathways were screened miRNA target gene prediction. The target of hsa-mir-328-3p was CHRD (chordin). Hsa-mir-2110 was predicted the target TNF, and hsa-let-7c-5p was predicted to target PPARG (peroxisome proliferator activated receptor gamma). The expression these miRNA in each group and the base complementary pairing sequence of miRNA 3´-UTR region with target gene are shown in Figure 6C Discussion Exosomes are carriers and transfer agents of intercellular information, and the study of exosomes is important to reveal the mechanism of intercellular information exchange. As physiological vesicles, exosomes can be targeted to alter speci c regulatory cell function materials they contain to improve the accuracy and effectiveness of treatment. The use of cell-derived vesicles can reduce the risks associated with transplantation and minimize the possible immune response and ectopic tissue development problems caused by MSC transplantation [1,9,17]. Experimental studies showed that exosomes derived from MSCs have therapeutic effects on many diseases, and their cargoes are important to regulate cell function and play a therapeutic role [17]. Therefore, the present study aimed to explore the mechanism of exosomes-related osteogenic differentiation, and identify targets related to the proliferation and differentiation of osteoblasts.
Previous studies have proved that the exosomes from HucMSCs have therapeutic effects in a variety of diseases, such as immune diseases, Alzheimer's disease, in ammatory bowel disease, spinal cord nerve injury, cancer, and ischemia, which are similar to the therapeutic effects of HucMSCs [5,18]. To further explore the effect of exosomes on osteogenic differentiation, we isolated the exosomes produced by HucMSCs. Preparations Exo1 and Exo2 could promote osteogenic differentiation, as observed using ALP and alizarin red staining; however, the effect of Exo2 was more obvious. Western blotting showed that the levels of osteogenic differentiation-related proteins RUNX2, COL1A1, ALP, and OST were higher in the two exosome preparation than that in the blank control group, and was concentration dependent. [19]. The results of CCK-8 showed that Exo1 could promote the proliferation of osteoblasts, while Exo2 did not. These results showed that in the microenvironment of osteogenic differentiation, the regulatory function of exosomes had changed, and the effect of exosomes on osteogenic differentiation was enhanced; the microenvironment reduced the promoting effect on OB proliferation. Osteoporosis is caused by an imbalance of the differentiation ratio of MSCs to osteoblasts and adipocytes, decreased numbers of osteoblasts, enhanced osteoclast activity, and the increase in bone absorption caused by changes of the microenvironment, such as ageing and estrogen de ciency [19,20]. The differentiation direction of MSCs is affected by the microenvironment, and the expression and inhibition of various related genes in cells will also change markedly. Therefore, changes in exosome function in different microenvironments might play an important role in the regulation of osteoporosis.
According to the analysis of BV/TV, BMD, BS / BV, conn.d, TB. N, TB. SP, TB. Th, SMI, and the 3D reconstruction images, Exo1 and Exo2 appeared to have therapeutic effects on osteoporosis. Although Exo 2 strongly promoted osteogenic differentiation, there was no statistical difference between Exo 1 and Exo 2 data in the animal models. It is possible that some exosomes lose their activity when they enter the body. In addition, MSCs affected by the microenvironment of osteogenic differentiation gradually differentiate into osteoblasts, and the expression of proliferation information is reduced; therefore, the exosomes may lack material information to promote proliferation or regulate other functions. For example, studies have con rmed that HucMSC-derived exosomes have the functions of local regulation of osteogenic differentiation, promotion of cell proliferation, promotion of angiogenesis, and regulation of immune response [5,18].
The core mechanism of microRNAs is to induce mRNA degradation or inhibit the expression of their target genes through complementary pairing with mRNA base sequences, which is a key mechanism of biological processes such as cell differentiation, growth, migration, and apoptosis [7]. Exosomes are carriers of information between cells or tissues; therefore, microRNAs in exosomes play an important role in the regulation of cell function [21]. It can be seen from the differential miRNA thermogram that in an osteogenic differentiation environment, with the extension of time, the type and expression of miRNAs are constantly changing, indicating that changes in the microenvironment can affect the cargoes of exosomes. In the bubble diagram of KEGG pathway enrichment, some miRNA target genes were enriched in two metabolic pathways of Osteoclast differentiation and PPAR signaling pathway related to fat differentiation, which was consistent with the pathological changes observed in the process of osteoporosis [19,20].
In the prediction of microRNA targeting genes, it was found that CHRD was the target of hsa-mir-328-3p. Chordin binds to bone morphogenetic protein (BMP) and inhibits the activation of BMP related signaling pathways [29]. The results suggested that the inhibition of CHRD expression led to the signi cant increase in ALP expression and extracellular mineral deposition. Chordin might represent a new target to promote bone regeneration [22,23]. The target gene of hsa-mir-2110 was TNF (tumor necrosis factor). The TNF superfamily is mainly secreted by macrophages. It can bind to and act through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. TNFs are involved in a wide range of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. In the process of osteoclast differentiation, TNF plays an important role in the differentiation and formation of osteoclasts. It is a necessary inducting factor of almost all osteoclast differentiation signaling pathways [20]. Hsa-let-7c-5p was predicted to target PPARG. PPARgamma is a member of the PPAR subfamily. There are three types of PPAR: PPARalpha, PPARdelta, and PPARgamma. Among them, PPARgamma is the key regulator of adipocyte differentiation. In addition, PPARgamma is involved in the pathology of many diseases, including obesity, diabetes, atherosclerosis, and cancer [7]. A study of bone marrow MSCs in the elderly with osteoporosis showed that the osteogenic differentiation ability of MSCs decreases, their ability to differentiate into adipocytes increases, and the osteoclast activity increases. Therefore, hsa-mir-2110 and hsa-let-7c-5p could be developed as targets to alleviate osteoporosis [24].
The expression levels of hsa-mir-328-3p and hsa-mir-2110 were signi cantly higher in the experimental groups of osteogenic 3 and 7 than in the controls. Moreover, hsa-mir-2110 targets members of osteoclast differentiation related signaling pathways. Hsa-let-7c-5p showed no signi cant difference in expression between osteogenic 3 and the control, but increased signi cantly in experimental group of osteogenic 7. Therefore, microRNAs in exosomes might target genes to inhibit adipose differentiation and osteoclast activity in the early stage of osteogenic differentiation.

Conclusions
In the microenvironment of osteoblast differentiation culture, hucMSC-derived exosomes can signi cantly promote the differentiation of osteoblasts, and displayed certain therapeutic and preventative effects on an OVX mouse osteoporosis model. It is suggested that exosomes are in uenced by the cells in the differentiation stage and carry related substances to promote osteoblast differentiation. Bioinformatic analysis showed that osteogenic differentiation changed the microRNA pro le in exosomes, and the target genes of these miRNAs might not only act on osteogenic differentiation, but also on the pathways related to adipogenic differentiation and osteoclastic differentiation of MSCs. Therefore, the prevention and treatment of osteoporosis may be subjected to multifaceted regulation.

Consent for publication
Not applicable.

Con icts of Interest
The authors declare that they have no competing interests.

Figure 1
MiRNA target enrichment and KEGG analysis for HucMSC exosomes. A. Control vs. osteogenic 3 differentially expressed miRNA target gene enrichment bubble map. B. Control vs. osteogenic 7 differentially expressed miRNA target gene enrichment bubble map. The X-axis is the enrichment ratio (the ratio of the number of genes annotated to an item in a selected gene set to the total number of genes annotated to that item by the species), and the Y-axis is KEGG Pathway; the size of the bubble represents the number of genes annotated to a KEGG Pathway. The color represents the enrichment Q value, and a darker color represents a smaller the Q value. Compared with control group, osteogenic 3 and osteogenic 7 were signi cantly enriched in Osteoclast differentiation and PPAR signaling pathways in the KEGG Pathway analysis, indicating that exosome function is closely related to osteoclast differentiation and adipose differentiation. The bubble map in B indicated that the target genes enriched in Osteoclast differentiation and PPAR signaling pathway increased greatly in the osteogenic group7. C. The left is the differential analysis of hsa-mir-328-3p and hsa-mir-2110 in each group, and the right is the base complementary pairing sequence of the miRNA 3´-UTR region with the target gene.

Figure 2
The osteogenic microenvironment affects the miRNA pro le of HucMSC exosomes. A. Control vs. osteogenic 3 differentially expressed genes miRNA clustering heat map. B. Control vs. osteogenic 7 differentially expressed genes miRNA clustering heatmap, showing the expression level of differentially expressed genes by color, with log2 (expression value + 1) of the sample on the horizontal axis and genes on the vertical axis. The redder the color of the block, the higher the expression level; the bluer the color, the lower the expression level. Compared with the control group, the changes of the differentially expressed genes of the miRNAs in the osteogenic 3 group were obvious, indicating that the changes of the exosomal miRNAs were affected by the microenvironment of osteogenic differentiation. Compared with the control group, the number of differentially expressed genes in the Osteogenic 7 group increased, and the expression changes were more obvious, indicating that with the prolongation of induction differentiation time, the microenvironment had a greater impact on exosomes.  HucMSC exosomes promote osteoblast differentiation, affect osteoblast differentiation-related protein levels, and reduce osteoporosis. A. Western blotting validation of the effects of Exo1 and Exo2 on the levels of RUNX2, COL1A1, ALP, and OST proteins in OB cells. The exosomes promoted the differentiation of osteoblasts in a concentration-dependent manner, and Exo2 showed a stronger effect on promoting osteogenic differentiation than Exo1. B. Statistical analysis of the expression of osteoblast differentiation-related proteins showed that the levels of RUNX2, COL1A1, ALP, and OST in the Exo1 group and Exo2 group were signi cantly higher than that in the control group. C. HE staining of bone tissue sections of mice, compared with OVX group, showed that osteoporosis was reduced in the Exo1 group and Exo2 group (shown at original magni cation, × 50). HucMSC exosomes promote osteogenic proliferation and differentiation. A. After co-culture of Exo1 with OB cells, the results of CCK-8 assays showed a signi cant promoting of proliferation in a concentrationdependent manner. B. Exo2 did not promote proliferation after co-culture with OB cells. Exo1 and Exo2 concentrations were 100 g/ml. C. Images show ALP staining, in which the color in the Exo2 image was darker than that for Exo1. D. Images show alizarin red staining, in which Exo2 produced the most mineralized nodules, indicating that Exo2 promotes stronger osteogenic differentiation than Exo1. The concentration of Exo1 was set at 200, 100, and 0 g/ml, and the concentration of Exo2 was set at 200, 100, and 0 g/ml. E and G show ALP staining, and F and H Alizarin red staining of exosomes. The staining increased with increasing concentration, indicating that Exo1 and Exo2 promoted osteogenic differentiation in a concentration-dependent manner (shown at the original magni cation, × 100).