Cerium oxide nanoparticles-carrying human umbilical cord mesenchymal stem cells counteract oxidative damage and facilitate tendon regeneration

Background Tendon injuries have a high incidence and limited treatment options. Stem cell transplantation is essential for several medical conditions like tendon injuries. However, high local concentrations of reactive oxygen species (ROS) inhibit the activity of transplanted stem cells and hinder tendon repair. Cerium oxide nanoparticles (CeONPs) have emerged as antioxidant agents with reproducible reducibility. Results In this study, we synthesized polyethylene glycol-packed CeONPs (PEG-CeONPs), which were loaded into the human umbilical cord mesenchymal stem cells (hUCMSCs) to counteract oxidative damage. H2O2 treatment was performed to evaluate the ROS scavenging ability of PEG-CeONPs in hUCMSCs. A rat model of patellar tendon defect was established to assess the effect of PEG-CeONPs-carrying hUCMSCs in vivo. The results showed that PEG-CeONPs exhibited excellent antioxidant activity both inside and outside the hUCMSCs. PEG-CeONPs protect hUCMSCs from senescence and apoptosis under excessive oxidative stress. Transplantation of hUCMSCs loaded with PEG-CeONPs reduced ROS levels in the tendon injury area and facilitated tendon healing. Mechanistically, NFκB activator tumor necrosis factor α and MAPK activator dehydrocrenatine, reversed the therapeutic effect of PEG-CeONPs in hUCMSCs, indicating that PEG-CeONPs act by inhibiting the NFκB and MAPK signaling pathways. Conclusions The carriage of the metal antioxidant oxidase PEG-CeONPs maintained the ability of hUCMSCs in the injured area, reduced the ROS levels in the microenvironment, and facilitated tendon regeneration. The data presented herein provide a novel therapeutic strategy for tendon healing and new insights into the use of stem cells for disease treatment. Graphical Abstract


Background
Tendons are the essential connective tissues that tether skeletal muscles to bones and transmit force.The tendon is easily torn because it is subjected to a high tensile strain.Tendon injuries are among the most common musculoskeletal disorders, affecting more than 32 million people in the United States [1].Fibrotic scarring is inevitable during healing, resulting in the disruption of tendon matrix continuity and disability [2].The existing effective treatment options for tendon injuries are limited.
One reason for the inferiority of healed tendons is the limited number of resident cells in the tendon tissues [3][4][5][6][7].Therefore, cell-based therapies for tendon injury have attracted increasing attention.Because of their selfrenewal and differentiation abilities, stem cells are used in multiple tissue regeneration and disease treatments [8][9][10].The human umbilical cord is an abundant source of multipotent stem cells, and human umbilical cord mesenchymal stem cells (hUCMSCs) exhibit multiple lineage differentiation potential and immune modulation capacity [11].hUCMSCs possess many advantages, such as fewer ethical issues, a painless collection process, and a lack of immunity compared to other mesenchymal stem cells (MSCs) [12].hUCMSCs have general benefits for tissue regeneration in the bone, cartilage, uterus, and brain [13][14][15].Jo et al. have reported that hUCMSCs induce rotator cuff tendon regeneration in rat models [16].Lee et al. observed partial healing in a rabbit model of tendon rupture treated with hUCMSCs [17].Therefore, hUCMSCs therapy is a potential treatment modality for tendon injuries.
Excessive reactive oxygen species (ROS) are crucial mediators of multiple pathological processes that induce cellular senescence, apoptosis, and dysfunction [18][19][20].The therapeutic activity of MSCs is permanently impaired in injured tissues owing to abnormally high oxidative stress [21].In tendons, both acute and chronic injuries can promote ROS generation, resulting in only a slight improvement in some MSC-related tendon healing studies [1,22,23].Thus, effective antioxidant therapies must be developed in conjunction with stem cell therapies to enhance the efficacy of hUCMSCs.
Cerium is a lanthanide metal element that exhibits repeatable reducibility because of the conversion of cerium ions between trivalent and tetravalent ions.Recently, cerium oxide nanoparticles (CeONPs) have emerged as antioxidants owing to their superoxide dismutase, catalase, and peroxidase activities [24].Consequently, CeONPs have been widely used in ROSassociated diseases.Du et al. suggested that injection with atorvastatin-loaded CeONPs mitigates acute kidney injury [25].Lee

Graphical Abstract
against ischemic stroke by scavenging ROS [26].Similar effects have been demonstrated in liver diseases and glaucoma [27][28][29].However, a CeONPs-based defense strategy against oxidative injury during cellular transplantation and tendon injury needs to be developed.
In the present study, we synthesized methyl polyethylene glycol 2000 distearylphosphatidylethanolamine (mPEG 2k -DSPE)-packed CeNOPs (PEG-CeONPs) and constructed PEG-CeONPs-carrying hUCMSCs.The protective effects of the PEG-CeONPs against oxidative stress were evaluated in vitro.We also assessed the therapeutic effects of the PEG-CeONPs-carrying hUCMSCs in a rat model of tendon injury.This study aimed to develop a novel stem cell modification strategy for tendon injury.

Concentration of cellular ROS
The concentration of cellular ROS was assessed using dichlorodihydrofluorescein diacetate (DCFH-DA, S0033S, Beyotime, Shanghai, China) and dihydroethidium (DHE, 50102ES02, Yeason, Shanghai, China) fluorescent probes following the manufacturer's protocol.hUCMSCs were washed with PBS and cultured in a serum-free medium supplemented with 10 µM DCFH-DA or 10 µM DHE for 30 min.After washing out the free probe with PBS, images were captured using an inverted fluorescence microscope.The mean fluorescence intensity was measured using ImageJ software (version: 1.8.0).

Mitochondrial membrane potential
The membrane potential of the isolated mitochondria was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (G1515, Servicebio) following to the manufacturer's instructions.After washing with PBS, hUCMSCs were incubated with the JC-1 staining solution at 37 °C for 20 min.The hUCMSCs were then washed three times with JC-1 staining buffer.The images were visualized using an inverted fluorescence microscope and analyzed using ImageJ software.

Colony formation assay
hUCMSCs or PEG-CeONPs-carrying hUCMSCs were exposed to H 2 O 2 and trypsinized.Five hundred hUCM-SCs were replanted in six-well plates and cultured for 3 weeks.After fixing with 4% paraformaldehyde, the plates were stained with a crystal violet solution.ImageJ software was used to test the colony expansion ability.

Immunofluorescence staining
hUCMSCs were fixed using a 2% paraformaldehyde solution for 15 min, followed by permeabilization using 0.5% Triton X-100 for 20 min at room temperature.The hUCMSCs were washed with PBS and blocked with 5% goat serum.After incubating with primary antibody Ki67 (1:50, A11390, Abclonal) at 4 °C overnight and fluorescent secondary antibody (1:250 GB21303, Servicebio) at 37 °C for 1 h, cells were stained with DAPI, and the images were analyzed under a fluorescent microscope.

Real-time quantitative PCR (RT-qPCR)
Briefly, 1000 ng of RNA was isolated using RNA isolation kits (R0032, Beyotime) and reverse transcribed using a double-stranded cDNA synthesis kit (G3331, Servicebio).The mixture incubation conditions were: 5 min at 25 °C, 55 °C for 15 min, and 85 °C for 5 s.RT-qPCR was performed in triplicate using the Universal SYBR green fast qPCR mix kit (G3320, Servicebio) on Light-Cycler ® 480 Software (Roche, Swiss Confederation).The primer sequences used in this research were as follows: β-actin, forward primer CAC CCA GCA CAA TGA AGA TCA AGA T, reverse primer CCA GTT TTT AAA TCC TGA GTC AAG C; P16, forward primer CTG CCC AAC GCA CCG AAT AG, reverse primer AGC TCC TCA GCC AGG TCC AC; P21, forward primer ACC ACT GGA GGG TGA CTT C, reverse primer CGG CGT TTG GAG TGG TAG; IL-1β, forward primer GTG CAC GAT GCA CCT GTA CG, reverse primer ACG GGC ATG TTT TCT GCT TG; IL-6, forward primer AAG CAG CAA AGA GGC ACT GG, reverse primer TGG GTC AGG GGT GGT TAT TG; TNFα, forward primer GAA CCC CGA GTG ACA AGC CT, reverse primer CCC TTG AAG AGG ACC TGG GA.

In vivo study
This study design was approved by the Laboratory Animal Welfare and Ethics Committee of the Renmin Hospital of Wuhan University (Approval No: 20230101 A) and conducted in compliance with the National Research Council's Guide for the Care and Use of Laboratory Animals.Eight-week-old male Wistar rats were obtained from SiPeiFu Biotechnology Co, Ltd. (Beijing, China) and divided into the four following groups: sham group (n = 6), tendon injury group (n = 6), tendon injury + hUC-MSCs group (n = 6), and tendon injury + PEG-CeONPcarrying hUCMSCs group (n = 6).The rats were anesthetized using 5% isoflurane inhalation, and a single defect (1 mm) was created in the patellar tendon.The latter two groups received 10 6 hUCMSCs or 10 6 PEG-CeONP-carrying hUCMSCs through intra-tendon injections once a week [32].After 4 weeks, the rats were sacrificed and the patellar tendon was collected and fixed for further experiments.

Statistical analysis
All data in this research are shown as mean ± standard deviation.Shapiro-Wilk normality test was used to perform the normality test.For the data with normal distribution, one-way analysis of variance (ANOVA) followed by Bonferroni's test (multiple groups) was administered.For the data with non-normal distribution, we performed the Kruskal-Wallis H-test, followed by Dunn's test (multiple groups).For repeated measurements, ANOVA for repeated measurements was administered.p < 0.05 was considered a statistically significant difference.
Thus, hUCMSCs used in this study possessed high purity and excellent differentiation potential.

Synthesis and antioxidant analysis of PEG-CeONPs
The hydrophobic CeONPs were synthesized by a previously reported thermal decomposition method [30].After coating with mPEG 2k -DSPE, CeONPs were transferred to the aqueous phase.As shown in Fig. 2A, the obtained PEG-CeONPs exhibited uniform morphologies with sizes of 5.45 ± 1.08 nm.Compared with hydrophobic CeONPs, there are two additional absorption peaks at 1737 and 1104 cm −1 , which could be attributed to C=O and C-O-C of DSPE-mPEG respectively [34,35], indicating the successful surface modified (Fig. 2B).XPS analysis indicates that Ce 3+ and Ce 4+ co-exist on the surface of hydrophobic CeONPs and PEG-CeONPs, which provide the chemical basis for the catalytic activities (Fig. 2C) [36].EDS results revealed a Ce:O atomic ratio of 0.50 (Fig. 2D).Moreover, CeONPs were stable in an aqueous solution for at least 2 weeks, as evidenced by their appearance and the results of the UV-visible spectra analysis (Fig. 2E, F).
The conversion of cerium ions into their trivalent and tetravalent forms typically endows CeONPs with multiple antioxidant enzyme activities [37].To substantiate this function, we performed a series of in vitro antioxidant assays.The results of SOD and CAT enzyme mimetic activity analysis revealed that PEG-CeONPs inhibited the generation of superoxide anions and hydrogen peroxide in a dose-dependent manner (Fig. 2G, H).DPPH free radical and hydroxyl radicals scavenging assays are widely used to evaluate antioxidant properties [38,39].PEG-CeONPs decreased the concentration of free radicals in a dose-dependent manner (Fig. 2I, J).The data suggested that the preparation of PEG-CeONPs with excellent antioxidant properties was successful.

PEG-CeONPs decreased the concentration of ROS in hUCMSCs
We loaded PEG-CeONPs into hUCMSCs to enhance their tolerance to high ROS levels in the injured area (Fig. 3A).CCK8 assay showed that PEG-CeONPs were safe for hUCMSCs with a concentration of less than 50 µg/mL both at 24-and 48-h exposure (Fig. 3B).Thus, we administrated 50 µg/mL PEG-CeONPs to hUCMSCs for 24 h to harvest PEG-CeONPs-carrying hUCMSCs.As shown in Fig. 3C, Cy3-PEG-CeONPs were intaken by hUCMSCs and dispersed around the nucleus.High levels of ROS are characteristic of the injured lesions and are responsible for the limited efficacy of stem cell transplants.Accordingly, hydrogen peroxide was used to mimic the oxidative stress in the injured area of tendon.DCFH-DA and DHE are cell-permeable probes that are widely used to detect the intracellular concentration of H 2 O 2 or superoxide [40,41].DCFH probe imaging suggested that PEG-CeONPs reduced the concentration of ROS after H 2 O 2 treatment; there was no significant difference between the NC and PEG-CeONPs groups (Fig. 3D).Subsequently, a DHE experiment was conducted and similar results were obtained (Fig. 3E).The results showed that PEG-CeONPs were successfully carried by hUCMSCs and reduced the intracellular ROS levels under H 2 O 2 intervention.

PEG-CeONPs-carrying hUCMSCs resisted ROS-mediated apoptosis
High ROS levels promote apoptosis through diverse signaling pathways [42].Therefore, enhancing the regenerative and anti-apoptotic capacities of stem cells before treatment is considered an interventional strategy for improving therapeutic efficacy [43].Here, we investigated the effect of PEG-CeONPs on hUCM-SCs apoptosis under oxidative stress.Intrinsic cellular apoptosis is always accompanied by loss of mitochondrial membrane potential [43].We performed a mitochondrial membrane potential assay, and the results showed that healthy polarized mitochondria (JC-1 aggregates, red) decreased and unhealthy depolarized mitochondria (JC-1 monomers, green) increased in the H 2 O 2 group (Fig. 4A).However, PEG-CeONPs treatment partially reversed this effect.Additionally, PEG-CeONPs treatment reduced the expression of BAX (a pro-apoptotic protein) and increased the expression of BCL2 (an anti-apoptotic protein) under oxidative stress (Fig. 4B).PEG-CeONPs also reduced the percentage of apoptotic hUCMSCs under oxidative stress as shown by the results of flow cytometry (Fig. 4C).These results suggest that PEG-CeONPs-carrying hUCMSCs could resist ROS-mediated apoptosis.

PEG-CeONPs-carrying hUCMSCs resisted ROS-mediated senescence
Excessive ROS levels disrupt mitochondria and trigger cellular senescence, impairing stem cell function and tissue regeneration [44].Therefore, we investigated whether PEG-CeONPs exerted their anti-senescent function by scavenging ROS.Irreversible growth arrest is a characteristic of cellular senescence [45].We performed a clonal formation assay and a CCK-8 assay to evaluate the proliferative capacity.The results showed that PEG-CeONPs counteracted the inhibitory effects of H 2 O 2 on clonal formation and proliferation (Fig. 5A, B).Moreover, PEG-CeONPs enhanced the expression of Ki-67, a proliferation maker, under oxidative stress (Fig. 5C).Compared with the H 2 O 2 group, the PEG-CeONPs + H 2 O 2 group showed decreased senescence, as revealed by the reduced level of β-galactosidase, and decreased expression of the senescence-related factors, including P16 and P21, at the mRNA and protein levels (Fig. 5D−F).Senescent cells secrete pro-inflammatory factors and proteases to alter the tissue microenvironment, which is collectively termed the senescence-associated secretory phenotype (SASP) [46].RT-qPCR and Western blot results indicated that treatment with PEG-CeONPs decreased the expression of SASP makers, including IL-1β, IL-6, and TNFα, which were enhanced by H 2 O 2 (Fig. 5E and F).In summary, PEG-CeONPs-carrying hUCMSCs counteracted senescence induced by high ROS levels.

PEG-CeONPs-carrying hUCMSCs resisted apoptosis and senescence through NFκB and MAPK signaling pathways
Given that nuclear factor kappa-B (NFκB) and mitogenactivated protein kinase (MAPK) signaling pathways are involved in the transduction of oxidative signal and cellular apoptosis, we performed further experiments to elucidate the mechanism underlying PEG-CeONPs in hUCMSCs exposed to excessive oxidative stress [47,48].As shown in Fig. 6A and B, H

Local administration of PEG-CeONP-hUCMSCs effectively promotes the recovery of patellar tendon defect
High levels of oxidative stress contribute to the limited efficacy of stem cell transplantation in injured areas.Thus, to investigate the advantages of the local injection of PEG-CeONP-hUCMSCs in tendon healing, we established a patellar tendon defect (PTD) model induced by surgery [2].hUCMSCs and PEG-CeONP-hUCMSCs were injected into the tendon once per week (Fig. 7A).After 4 weeks of treatment, tendon tissues collected from the PTD group presented a significant defect.In contrast, tendons from the hUCMSCs and the PEG-CeONP-hUCMSCs presented smaller defects, especially in the latter (Fig. 7B).HE and Masson staining showed that treatment with PEG-CeONP-hUCMSCs reversed the disorderly arrangement of collagen fibers and attenuated the formation of vacuole-like structures (Fig. 7C).To evaluate the oxidative stress levels, the ROS fluorescent probe DHE was loaded into the tissues.Fluorescent images and semiquantitative analysis showed that PEG-CeONP-hUCMSCs remarkably inhibited the increase in ROS levels evoked by the injury compared to the hUC-MSCs intervention (Fig. 7D).Moreover, PEG-CeONP-hUCMSCs enhanced tendon repair as evidenced by the higher expression of tenogenic markers SCX, TNMD, COL1A1, and COL3A1 (Fig. 7E-H).Taken together, the local administration of PEG-CeONP-hUCMSCs effectively promoted the repair of patellar tendon defects in rat models.

Discussion
Tendon injuries cause prolonged disability with high incidence [51].The repair of tendon injuries is incomplete because of fibrotic scarring.Plenty of treatment strategies have been developed to enhance tendon healing; however, their efficacy requires improvement.Stem cell transplantation holds great promise in multiple diseases, including tendon injuries, as stem cells secrete a diverse repertoire of growth factors such as vascular endothelial growth factors, fibroblast-like growth factors, and insulin-like growth factors [52][53][54].However, the poor survival of the stem cells in injured tissues impedes their therapeutic development, which is attributed to the high ROS concentrations in the injured areas.
Cerium oxide is a stable lanthanide metal element and the pale-yellow oxide form of the most abundant rareearth metal [55].Driven by a ground-state Ce 4f electron, the powerful Ce 3+ to Ce 4+ redox couple contribute to considerable reducibility [56].Possessing superoxide dismutase, catalase, and peroxidase activities, CeONPs have been considered antioxidant agents [57][58][59].CeONPs, with a higher surface-area-to-volume ratio, have better reducibility than cerium oxide with larger particles because of oxygen vacancies and Ce 3+ mostly existing on the surface [60].In our study, ultrasmall PEG-CeONPs with a size of 5.45 nm were synthesized and exhibited significant reducibility.Previous studies have been conducted to enhance the therapeutic efficacy of cell transplantation by pre-treating stem cells with natural drugs such as curcumin, Exendin-4, and resveratrol or by genetically modulating [61][62][63].The above options often face high cost, non-specificity, limited activity and consequent uncontrolled side effects and thus fail in clinical trials [28].In comparison, CeONPs, the artificial enzyme, has the advantage of low cost, high efficiency and stability, massive production, and easy handling, which attracted us to explore its role in cell transplantation [64].Nanoparticles can be actively taken up by cells via endocytosis, indicating PEG-CeONPs can be carried by hUCMSCs and transplanted to the injured areas [65].Although generally safe, concentration-dependent cytotoxicity of CeONPs has been observed in various cell types [66,67].Therefore, we explored and verified the appropriate loading conditions under which PEG-CeONPs exhibited both biocompatibility and antioxidant capacity in hUCMSCs.
Apoptosis, a form of programmed cell death, markedly affects stem cell transplantation [21].Oxidative stress can induce apoptosis in many ways, including activation of the mitochondrial pathway (intrinsic) and activation of death receptors at the cell surface (extrinsic) [68].Apoptotic stem cells possess severely compromised regenerative potential and secret fewer growth factors; therefore, improving the outcome of stem cell therapy is a novel interventional strategy, as exemplified by endowing stem cells with pro-survival and anti-apoptotic genes [43].In this work, we confirmed that PEG-CeONP-carrying hUCMSCs could resist oxidative damage-induced cellular apoptosis in vivo and in vitro.In addition to causing apoptosis in transplanted cells, high oxidative stress can damage in site tendon-derived stem cells and deteriorate tendon healing [69].Our study suggests that injection of PEG-CeONP-carrying hUCMSCs remodeled the microenvironment in the injured area by scavenging and inhibiting apoptosis.Senescence is a cellular state characterized by irreversible growth arrest and an altered epigenetic mechanism [70].Recently, the transplantation of senescent cells into young animals was shown to result in persistent physical dysfunction [71,72].Senescence can be triggered by various stressors, such as multiple generations, activation of oncogenes, and ROS-induced DNA damage [73].The accumulation of senescent cells in tendon tissue is a possible pathogenesis mechanism underlying tendinopathy [74].Our data revealed that PEG-CeONPs protected hUCMSCs from H 2 O 2 -evoked cellular senescence and decreased the expression of senescence-associated molecules P16 and P21 in rats.
NFκB is a pleiotropic, redox-sensitive, nuclear transcription factor regulating the expression of many genes and associating with multiple biological processes, including apoptosis and senescence [75,76].The NFκB signaling pathway can be activated by H 2 O 2 at different sites [77].The activation of the NFκB signaling pathway is involved in musculoskeletal diseases including tendon diseases [78].The MAPK pathway is a representative stress-responsive signaling pathway that induces cellular responses to divergent environmental stimuli [79].Increased ROS generation leads to the activation of MAPK cascades, including c-Jun NH2-terminal kinase (JNK), and p38 MAPK [80].Apoptosis and senescence are mediated by the MAPK signaling pathway [81,82].Therefore, we sought to explain the function of PEG-CeONPs by assessing the activation of NFκB and MAPK signaling pathways.Our data showed that PEG-CeONPs inhibited the activation of NFκB and MAPK signaling pathway in H 2 O 2 -treated hUCMSCs, and human recombinant TNFα and DE reversed the effect of PEG-CeONPs loading, indicating that PEG-CeONPs acted through the NFκB and MAPK signaling pathways (Fig. 8).
Various biomaterials strategies have been developed for tendon regeneration because of the poor repairs induced by low cellularity sources [83], poor blood supply [84], inflammatory microenvironment, and excessive oxidative stress [1].Currently, biomaterials strategies for tendon regeneration mainly include scaffolds to excel at mechanical stabilization and hydrogels to deliver biochemical cues or cells [85].For instance, A polymeric three-dimensional scaffold was reported to retain tendon-like mechanical properties and accelerate the healing progression [86].Ren et al. designed a high-tenacity shape-adaptive hydrogel to deliver fibroblast growth factor [87]. Ji et al. proposed a cocktail-like hydrogel to transmit bone marrow mesenchymal stem cells [88].The main issue of our research would be concerns about is the excessive oxidative stress in injured areas.In previous studies, people used antioxidative drugs such as melatonin and hormone agonists against oxidative injury in injured areas [89,90].We took advantage of the excellent and repeatable reversibility of cerium oxide nanoparticles and proposed a novel strategy that constructed cerium oxide nanoparticle-carried hUCMSCs.Our results indicate that the carriage of cerium oxide nanoparticles could against oxidative stress and enhance the effect of hUC-MSCs.In future studies, novel bioactive hydrogel-based delivery solutions are expected to join forces with stem cell modification programs to provide a more effective intervention strategy for tendon injury.Our study has some limitations.First, given the potential ethical issue, we must explore the effects of PEG-CeONP-carrying hUCMSCs in rats, and the impact of xenogeneic transplantation should be considered.Additionally, the biological behavior of PEG-CeONP-carrying hUCMSCs in vivo should be tracked using advanced experimental methods.Finally, a synthetic scheme for producing clinical-grade PEG-CeONPs should be developed for further clinical research.

Conclusion
This study indicates that PEG-CeONPs with repeatable reducibility were taken up by hUCMSCs by endocytosis.When exposed to excessive concentrations of H 2 O 2 , most cells carried with the PEG-CeONPs remained active to avoid senescence and apoptosis by inhibiting of ROS-induced NFκB and MAPK activation.In addition, to intracellular oxidative stress, high levels of ROS in the injured microenvironment were reduced by PEG-CeONP-carrying hUCMSCs.Intra-tendon injection of PEG-CeONP-carrying hUCMSCs facilitated tendon regeneration by preventing senescence and apoptosis.The results in our study provide a novel nongenetic strategy to improve the therapeutic potential of stem cells, which could be widely used in the transplantation of stem cells target various diseases.

Fig. 1
Fig. 1 Characterization of hUCMSCs.A Primary hUCMSCs were spindle-shaped under light microscopy.B Flow cytometry analysis showed that 98.5% of cells expressed CD29, 97.4% expressed CD44, 98.6% of cells expressed CD90, and 98.1% of cells expressed CD105.Meanwhile, only 0.48% and 0.28% expressed CD45 and CD34.C Lipid droplets were stained by oil red O after adipogenic differentiation induction of hUCMSCs.Mineralized nodules were stained by Alizarin Red after osteogenic differentiation induction of hUCMSCs.Acid mucopolysaccharide was stained by Alcian Blue after chondrogenic differentiation induction of hUCMSCs