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. 2024 Jun 8;76(5):547–558. doi: 10.1007/s10616-024-00635-0

Melatonin enhances therapeutic outcomes of adipose tissue-derived mesenchymal stem cell therapy in rat osteoarthritis by reducing TNF-α and IL-1β-induced injuries

Hao Jiang 1,3, Jiafang Chen 1,3, Zhangya Lin 2,3,✉, Naishun Liao 4,✉
PMCID: PMC11344747  PMID: 39188645

Abstract

Although adipose tissue-derived mesenchymal stem cell (ADSC) transplantation has been effectively used to treat osteoarthritis (OA), the low cell survival rate induced by the inflammatory and oxidative stress, severely affects the therapeutic efficiency of ADSC transplantation in OA. This study was designed to evaluate whether melatonin pretreatment could improve ADSC survival and its therapeutic efficacy in OA. The papain-induced OA rats were pretreated with melatonin via intra-articular injection and then intra-articular injected with indocyanine green (ICG)-labeled ADSCs (3 × 106/rat). Afterward, ADSC retention was evaluated by NIR-II fluorescence imaging. The tibia and synovial fluid were collected for histopathological examination and ELISA assay, respectively. To confirm the anti-inflammatory effect of melatonin, a TNF-α and IL-1β-induced cell model was used to evaluate the protective effects of melatonin on ADSC viability, cell apoptosis, and migration. Our results showed that melatonin pretreatment enhanced ADSC survival and improved the therapeutic effects of ADSC transplantation on cartilage repair, and anti-inflammation by reducing TNF-α, IL-6, IL-1β, and IL-12 in vivo. In particular, we also found that melatonin promoted ADSC viability and migration, and reduced cell apoptosis in vitro. In conclusion, this study supports that melatonin pretreatment can effectively improve ADSC survival and therapeutic efficiency in OA by reducing inflammatory injuries, which provides a novel strategy for enhancing ADSC therapy.

Keywords: Adipose tissue-derived mesenchymal stem cells, Melatonin, Osteoarthritis, Inflammation, Stem cell therapy

Introduction

Osteoarthritis (OA), a most common progressive joint disorder, is the leading cause of chronic joint pain and disability worldwide among the elderly populations (Baker et al. 2023). The typical pathological alterations of OA, including progressive cartilage degeneration, intra-articular inflammation, and subchondral sclerosis (Bernabei et al. 2023). At present, most attempts at OA treatment has been only effective on symptomatic treatment (e.g., acetaminophen, non-steroidal anti-inflammatory drugs, opioids, corticosteroids, hyaluronic acid, etc.), and it still lacks alternative strategy for cartilage regeneration (Richard et al. 2023; Waluyo et al. 2023). Regenerative medicine using ADSC transplantation has been effectively used to alleviate OA progression both in preclinical and clinical status, providing a promising strategy for OA treatment (Carneiro et al. 2023; Kim et al. 2022; Chen et al. 2021). However, low cell survival heavily affects the therapeutic efficiency and the widespread application of ADSC therapy in OA (Chae et al. 2018).

Oxidative stress and inflammation are the two typical characteristics (in OA) that affect cell survival and biological features after ADSC transplantation (Prakash et al. 2022; Fan et al. 2020; Zhao et al. 2021). To overcome this obstacle, pretreatment with some antioxidants or anti-inflammatory drugs has been used to enhance ADSC survival and engraftment efficiency both in vitro and in vivo (Liao et al. 2019, 2020; Wu et al. 2017; Gupta et al. 2018). Melatonin, a neurohormone in the pineal gland, involves in the regulation of circadian rhythms, immune modulation, and anti-inflammatory activity (Zhou et al. 2022). We previously found that melatonin could enhance ADSC survival, promote cell migration and proliferation in vitro (Liao et al. 2020), and could improve ADSC therapy by reducing oxidative injuries (Wu et al. 2017). Recently, accumulative evidences suggest that melatonin plays an important role in the control of inflammation in various diseases (Carrascal et al. 2018; Ke et al. 2022; Zhang et al. 2022). In particular, melatonin has also been used to prevent cartilage degeneration in the early-stage of OA (Zhou et al. 2022). Given the anti-inflammatory and anti-oxidative function of melatonin, melatonin pretreatment may benefit to enhance cell engraftment and therapeutic efficiency of ADSC transplantation in OA.

In the current study, the therapeutic functions of melatonin pretreatment on ADSC transplantation in OA rats, including histopathological characteristics and the anti-inflammation were carefully evaluated in vivo. Meanwhile, a TNFα and IL-1β-induced model (in vitro) was also used to confirm the potential anti-inflammation mechanism of melatonin pretreatment on ADSCs. To our knowledge, this study represents the first report on melatonin pretreatment for enhancing ADSC survival and improving ADSC therapeutic efficiency in OA.

Materials and methods

Animals

Thirty-five male SD rats (weight 180–200 g) were obtained from the Shanghai Slack Laboratory Animal Center (License No. SCXK hu 2022-0005). All animals (3–5 rats/cage) were housed in SPF-graded laboratory barrier facilities at 22–25 °C and 55–60% relative humidity. All animal procedures were approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University.

ADSC isolation and culture

ADSC isolation was performed following our previous reports (Liao et al. 2019, 2020). Briefly, adipose tissues were aseptically collected from the inguinal region of male SD rats, and then cut into small pieces. Afterwards, the tissues were digested with 0.1% type I collagenase (C0130, Sigma, USA) at 37 °C for 60 min. Next, the digestive solutions were filtered through a 100-μm cell strainer. After washing with PBS three times, the solutions were incubated with osmotic lysates (C3702, Biyuntian Biological Co., Ltd., Shanghai, China) to eliminate the red blood cells. Finally, the collected cells were seeded at a density of 1 × 106/mL with a complete medium (α-MEM containing 10% FBS) in the incubator. Passages 3–5 of ADSCs were used in the current study.

OA model establishment and ADSC therapy

OA model was established by intra-articular injection of 25 μL 4% papayotin (G8430, Solarbio, Beijing, China) in a lower limb, twice/week, for 4 weeks. After the development of OA, the mice were randomly divided into four groups (by use of randomized table): the model group (n = 5), intra-articular injection with PBS (100 μL/rat), once/week for 2 weeks; melatonin group (n = 5), intra-articular injection with melatonin (100 μL, 10 mg/mL; ST1497, from Biyuntian Biological Co., Ltd., Shanghai, China), once/week for 2 weeks; ADSC group (n = 5), intra-articular injection with ADSCs (3 × 106 cells/rat), once/week for 2 weeks; melatonin + ADSC group (n = 5), intra-articular injection with melatonin (100 μL, 10 mg/mL) for 1 h, and then intra-articular injection with ADSCs (3 × 106 cells/rat), once/week for 2 weeks. The normal rats (n = 5) was used a negative control. To minimise potential confounders, rats were housed in the same cage that was marked with the label. For animal care purposes, all rats were anesthetized by intraperitoneal injection of 40 mg/kg 3% pentobarbital sodium before intra-articular injection of ADSCs, PBS, or papayotin. Humane endpoints were conducted once OA rats were unable to walk (in the lower limb), and euthanatized with 150 mg/kg 3% pentobarbital sodium (intraperitoneal injection).

After ADSC transplantation for 2 weeks, all rats were euthanatized by intraperitoneal injection with 150 mg/kg 3% pentobarbital sodium. The synovial fluid and the tibial plateau were collected for further investigation.

Histological examination

The femoral specimens were fixed in 4% paraformaldehyde (P1110, Solarbio, Beijing, China) for 48 h and decalcified in 10% disodium edentate for 16 weeks. The tissues were followed by the gradual dehydration of ethanol and embedding in paraffin, and sectioned into slices. Finally, 5-μm thick sagittal sections were prepared for hematoxylin–eosin (HE) or Alcian blue staining, respectively.

Enzyme linked immunosorbent assay

The level of TNF-α, IL-6, IL-1β, and IL-12 in synovial fluid was measured using rat enzyme linked immunosorbent assay (ELISA) quantification kit (EK0526, EK0412, EK0393 and EK1652; all from BOSTER Biological Technology Co. Ltd, Wuhan, China) following the manufacturer’s protocol.

ICG labeling

The click chemistry was used for ADSC labeling as in our previous descriptions (Liao et al. 2021). Briefly, the ADSCs were firstly incubated with 25 μM Ac4ManNAz (GC60038, Glp Bio, USA) for 24 h, and then the ADSCs were collected and incubated with DBCO-ICG at 37 ℃ for 30 min. After washing with PBS three times, the ICG-labeling ADSCs were collected for in vivo cell transplantation. To further confirm the ICG labeling, the ICG-labeling ADSCs were 4% paraformaldehyde at RT for 20 min, and were stained by DAPI. Finally, a confocal laser scanning microscope (CLSM) (Zeiss, Germany) was used to observe ICG labeling.

In vivo NIR-II imaging

OA rats were transplanted with 3 × 106 ICG-labeled ADSCs (in 50 μL PBS buffer) via intra-articular injection. Afterward, rats were anesthetized with 2% isoflurane, and mixed with oxygen using an anesthesia system (throughout the NIR-II imaging), and the NIR-II fluorescence images were obtained using a NIR-II in vivo imaging system (808 nm laser, exposure time: 800 ms).

Cell viability assay

To investigate the anti-inflammatory effect of melatonin on ADSCs, a TNF-α and IL-1β-induced model was established. Briefly, ADSCs were cultured in a 96-well micro-plate (Corning, USA) at a density of 1 × 104 cells/well with 150 μL complete medium for 24 h. Then, the ADSCs are incubated with or without 10 μM melatonin for 3 h, and were following incubated with 100 ng/mL TNF-α (510-RT-010, R&D, USA) and 100 ng/mL IL-1β (501-RL-010, R&D, USA) for another 24 h. After that, the cell viability was assessed by using a CCK-8 cell proliferation kit (FC101, TransGen Biotech, Beijing, China) following the manufacturer’s instructions, and the absorbance at 450 nm was analyzed using a micro-plate reader (Spectra Max M5).

Cell apoptosis assay

ADSCs were cultured in a 6-well plate at a density of 1 × 105 cells/well overnight, followed by the incubation with 10 μM melatonin in fresh complete medium for 3 h and then incubated with 100 ng/mL TNF-α and 100 ng/mL IL-1β for another 24 h. Finally, the ADSCs were collected and measured by a commercial Annexin V-FITC apoptosis assay kit (FA101, TransGen Biotech, Beijing, China) in Flow cytometry (BD, USA).

Cell migration assay

To determine the protective effects of melatonin on ADSC migration Trans-well migration assay was used. Briefly, ADSCs were pretreated with 10 μM melatonin for 3 h, and then incubated with 100 ng/mL TNF-α and 100 ng/mL IL-1β for another 24 h. Afterwards, the ADSCs (105 cells/unit) were collected and seeded into the upper compartment of the Transwell units in α-MEM containing 2% FBS, while the lower compartment was filled with α-MEM containing 10% FBS. After cell migration for 24 h, the filters were fixed with 4% paraformaldehyde for 20 min, followed by staining with crystal violet for another 180 min at RT. The migrated cells were observed using an inverted microscope (Zeiss, Germany).

To further confirm the effect of melatonin on ADSC migration, a cell wound scratch assay was also used. In brief, ADSCs were pretreated with 10 μM melatonin for 3 h, and then incubated with 100 ng/mL TNF-α and 100 ng/mL IL-1β for another 24 h. After that, a “reference line” was scratched at the bottom of the plate using a sterile 10 μL pipette tip. Next, the cells were washed with α-MEM containing 10% FBS three times. The migration rate of ADSCs into the cell-free area was observed using an inverted microscope (Zeiss, Germany).

Western blot analysis

The ADSCs were lysed with RIPA lysis buffer (P0013B, Beyotime Biotech, Beijing, China) and ADSC protein was quantified using a commercial BCA assay kit (DQ111, TransGen Biotech, Beijing, China). Afterward, about 30 μg protein lysate was separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (PALL, USA) in transfer buffer (96 mM glycine, 12 mM Tris base, pH 8.3, and 20% methanol). Then, the membranes were blocked with 5% BSA at RT for 2 h. Subsequently, the membranes were co-incubated with the Cyclin-D1, Bax, Bcl-2, and CXCR4, antibodies (PB0403, BA0315-2, M00040-3, A00031-2, all from Wuhan Boster Biological Technology Co., Ltd, Wuhan, China) at 4 °C overnight. After that, the membranes were washed with TBST buffer three times (10 min/time), and incubated with an anti-rabbit-conjugated secondary antibody (HS101, TransGen Biotech, Beijing, China) for 1 h at RT. The protein expression levels were evaluated using a chemiluminescence system, and the quantitative analysis was performed using Image J software.

Statistical analysis

Data were expressed as the mean ± standard deviation (SD). All the statistical analyses were performed with GraphPad Prism version 9.0 (GraphPad Software, CA, USA). The ANOVA was used to determine the significant differences between three or more independent groups; the two-tailed paired sample Student’s t-tests were used to determine the significant differences between the two groups. P < 0.05 was considered a statistical difference.

Results

Melatonin pretreatment could enhance the cartilage repair function of ADSC therapy for OA

To determine the therapeutic effect of melatonin pretreatment on ADSC transplantation for OA, the OA model was established by a papayotin-induced method (Fig. 1a) and transplanted with ADSCs using intra-articular injection (Fig. 1b). As shown in Fig. 1c, the cartilage surface was smooth with complete cartilage and subchondral structure in the normal group, while the typical cartilage degeneration, and serious inflammation among the junction of cartilage and subchondral bone, as well as the subchondral destruction were all observed in the model group, implying that the OA model was successfully established. Although a slight decrease of cartilage degeneration, subchondral destruction was also observed in the melatonin group. Importantly, the obvious recovery of subchondral structure and cartilage layer in the ADSC group, indicates that ADSC transplantation could promote cartilage and subchondral bone repair in OA. Compared with ADSC or melatonin group, the complete cartilage layer and subchondral structure were observed in the combined (melatonin and ADSCs) group, which suggests that melatonin pretreatment could enhance ADSC transplantation for cartilage and subchondral recovery.

Fig. 1.

Fig. 1

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Melatonin enhances cartilage repair function of ADSC therapy for OA in vivo. A Schematic illustration of ADSC therapy for OA. B Schematic illustration of experimental period in ADSC therapy for OA. C Representative images of tissue sections stained with HE staining after ADSC transplantation with or without melatonin pretreatment (scale bar, 200 μm). D Chondrocytes of Alcian blue positive stained tissue sections (scale bar, 200 μm)

Because Alcian blue is an alkaline dye for stains acidic proteoglycan staining in chondrocytes, we further used Alcian blue staining to observe the distribution of chondrocytes. As shown in Fig. 1d, melatonin pretreatment could markedly increase chondrocyte regeneration compared with those in ADSC, melatonin, or model groups, which indicates that melatonin pretreatment could improve cartilage regeneration of ADSC therapy.

Melatonin pretreatment could enhance the anti-inflammation of ADSC therapy for OA

To confirm the anti-inflammation of ADSC therapy for OA, the joint fluid was collected, and the level of TNF-α, IL-1β, IL-6, and IL-12 were carefully examined by ELISA. Compared with the normal group, the articular level of TNF-α, IL-1β, IL-6, and IL-12 were all significantly increased in the OA model group, implying the inflammatory stress in the OA joint; while a slight decrease in melatonin group compared with the model group; after ADSC transplantation, the level of TNF-α, IL-1β, IL-6, and IL-12 were significantly decreased as compared to the model group; more importantly, melatonin pretreatment could further reduce the level of TNF-α, IL-1β, IL-6 and IL-12 (Fig. 2). These data suggested that melatonin pretreatment could enhance the anti-inflammation of ADSC therapy for OA.

Fig. 2.

Fig. 2

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Melatonin pretreatment enhances anti-inflammation of ADSC therapy for OA in vivo. The level of TNF-α (A), IL-1β (B), IL-6 (C) and IL-12 (D) after ADSC transplantation (n = 5 per group; *p < 0.05; **p < 0.01; ***p < 0.001)

Melatonin pretreatment could enhance ADSC retention in vivo

Given the excellent protective effect of melatonin pretreatment, we investigated ADSC retention in vivo after melatonin pretreatment in OA rats. ADSCs were first labeled with ICG via bioorthogonal click chemistry (Fig. 3a) as in our previous descriptions (Liao et al. 2021). As shown in Fig. 3b, the ADSCs were successfully labeled with ICGs. Compared with the control group, melatonin pretreatment could effectively promote ADSC retention in the joint (Fig. 3c), and the quantitative data of mean fluorescence intensity (MFI) further confirm the significant improvement of melatonin pretreatment for ADSC retention in OA (Fig. 3d).

Fig. 3.

Fig. 3

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Melatonin enhances ADSC retention in vivo. A Schematic illustration of bioorthogonal click chemistry for ADSC labeling with ICG. B Representative CLSM images of ICG-labeled ADSCs (scale bar, 20 μm). C NIR-II fluorescence images of joint area after ADSC transplantation with or without melatonin pretreatment. D Relative MFI of ADSCs in vivo (n = 3 per group)

Melatonin could promote ADSC survival and reduce cell apoptosis in vitro

Considering that low cell retention or cell survival in OA is closed related to oxidative stress and inflammation in vivo, the protective effect of melatonin on ADSCs within oxidative stress or inflammatory conditions should be further confirmed. Using a hydrogen peroxide-induced cell model to mimic the oxidative stress condition, we previously confirmed that melatonin could reduce ROS-induced injuries (Liao et al. 2020). In this study, we further confirm whether melatonin could reduce inflammatory injuries using a TNF-α and IL-1β-injured model to mimic inflammation in vivo. As shown in Fig. 4a, ADSC cell viability was significantly decreased by the co-incubation of TNF-α and IL-1β, while ADSC viability was significantly increased by melatonin pretreatment. Meanwhile, Cyclin D1, a cell proliferation marker, was significantly increased by melatonin pretreatment in TNF-α and IL-1β-injured model (Fig. 4b, c). These data suggested that melatonin could promote ADSC survival in vitro.

Fig. 4.

Fig. 4

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Melatonin promotes cell survival in vitro. A Cell viability of ADSCs with or without melatonin pretreatment in TNF-α and IL-1β-injured model (n = 5 per group; *p < 0.05; **p < 0.01). B Western blotting of Cyclin-D1 in ADSCs. C Quantative data of Cyclin-D1 expression in vitro (n = 3 per group; **p < 0.01; ***p < 0.001). D Cell apoptosis was evaluated by flow cytometry. E Western blotting of Bcl-2 and Bax in ADSCs. F Quantative data of Bcl-2 and Bax expression in vitro (n = 3 per group; *p < 0.05; **p < 0.01)

Next, we investigated the protective effects of melatonin on cell apoptosis. As shown in Fig. 4d, compared with normal ADSCs, the combined treatment of TNF-α and IL-1β promoted cell apoptosis, while melatonin could reduce ADSC apoptosis in vitro. In particular, the melatonin increased Bcl-2 protein expression and reduced BAX protein expression in vitro (Fig. 4e, f). Taken together, melatonin could reduce ADSC apoptosis in vitro.

Melatonin could promote ADSC migration in vitro

In light of the promotion of ADSC retention by melatonin pretreatment, we next investigate whether melatonin could enhance ADSC migration in vitro. As shown in Fig. 5a, b, c, the cell migration capability was significantly decreased by TNF-α and IL-1β induction, while melatonin could promote ADSC migration in TNF-α and IL-1β-induced model. CXCR4 is the typical surface marker that is closely related to ADSC migration. We further investigated the effect of melatonin on CXCR4 expression in vitro. As shown in Fig. 5d, e, melatonin could promote CXCR4 expression in ADSCs. Taken together, these data suggested that melatonin could promote ADSC migration in vitro.

Fig. 5.

Fig. 5

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Melatonin promotes ADSC migration in vitro. A Trans-well assay of ADSC migration (scale bar, 100 μm). B Quantification of ADSC migration in trans-well assay (n = 5 per group; **p < 0.01; ***p < 0.001). C Scratch wound assay of ADSC migration (scale bar, 100 μm). D Western blotting of CXCR4 in ADSCs. E Quantification of CXCR4 expression (n = 3 per group; *p < 0.05; **p < .001)

Discussion

Considering that inflammation is the key factor during the development and progression of OA (Liu-Bryan and Terkeltaub 2015), developing novel strategies that can inhibit inflammation holds promise for OA therapy. It has been proved that ADSC transplantation provides an alternative strategy for OA treatment by anti-inflammation, reducing chondrocyte cell apoptosis and dedifferentiation (Manferdini et al. 2013; Jiang et al. 2016). Recently, the benefic effects of ADSC transplantation have also been confirmed by animal models and clinical trials (Carneiro et al. 2023; Kim et al. 2022, 2023; Chen et al. 2021; Sadri et al. 2023). Although ADSC transplantation has achieved promising results for OA treatment, the long-term therapeutic efficiency is still limited due to the low cell survival or retention in the joint (Bhattacharjee et al. 2022). The large cell doses and multiple injections could partly avoid this obstacle. However, these approaches are associated with the risk of excessive expansion in vitro, and are not economically viable in practice (Bhattacharjee et al. 2022). Therefore, practical strategies to improve ADSC survival or retention are still urgently needed.

Given the fact that ADSC survival or retention is closely related to the interactions of transplanted cells and the host environment (such as oxidative stress and inflammation), we used melatonin pretreatment to reduce oxidative stress and inflammation for ADSC transplantation to improve cell retention in this study. As predicted, in the current study, we also found that melatonin could reduce inflammatory induced injuries in vitro. It has been proved that the inducement of TNF-α and IL-1β promotes cell apoptosis in endothelial progenitor cells (Henrich et al. 2007), inhibits cell stemness and differentiation in mesenchymal stem cells (Wang et al. 2019; Gao et al. 2018; Hu et al. 2023; Lian et al. 2016). In the current study, we found that melatonin promotes cell proliferation and inhibits cell apoptosis by increasing expression of Cyclin D1 and Bcl-2, and by down regulation of Bax in the injured ADSCs. Notably, both melatonin membrane receptors and nuclear receptors played an important role in the regulation of melatonin on cell apoptosis and proliferation (Liu et al. 2024). In particular, it has been found that the melatonin-MT1 (a melatonin receptor) signal regulates cell proliferation and apoptosis via the JNK signal pathway (Cui et al. 2021). Therefore, the melatonin-receptors signal plays a key role in the regulation of melatonin on cell proliferation and apoptosis.

Previous studies have been confirmed the anti-inflammation of melatonin on rheumatoid arthritis, OA and intervertebral disc degeneration. Zhang et al. proved that melatonin could reduce inflammatory cell aggregation and the release of the inflammatory factors IL-1β, IL-6, TNF-α in rats with intervertebral disc degeneration (Zhang et al. 2019). Guo et al. demonstrated that melatonin could reduce IL-1β-induced injuries in human chondrocytes (Guo et al. 2017). Huang et al. found that melatonin could inhibit IL-1β and TNF-α expression in synovial fibroblasts (Huang et al. 2019). It has been shown that multiple signaling pathways, including PI3K/AKT, ERK, NF-κB signaling pathway, are involved in the anti-inflammation of melatonin (Huang et al. 2019; Qiu et al. 2022). Further studies should be confirmed the detail anti-inflammatory mechanism of melatonin on TNF-α and IL-1β-injured ADSCs.

Additionally, several studies have been confirmed that the anti-inflammation of melatonin is in a melatonin-receptors depending manner (Xu et al. 2024; Qiu et al. 2022; Yang et al. 2020; Liu et al. 2022). For instance, the melatonin membrane receptors (MTNR1A/B) could activate Gαi2 protein, and subsequently upregulated the yes-associated protein (YAP) level in the TNF-α-induced cells; significantly, melatonin reduced TNF-α-induced cell injuries by targeting MTNR1B/Gαi2/YAP axis, IκBα expression, and NF-κB signal pathway (Qiu et al. 2022). Huang, et al. confirmed that the MT1 receptor is needed for the anti-inflammatory activities of melatonin (Huang et al. 2019). Considering the important role of melatonin-receptors, further study should focus on the detail role of melatonin-receptors signal in the protective effect of melatonin on the injured ADSCs.

Together with our previous result (melatonin could suppress ROS-induced injuries (in vitro) to enhance ADSC therapy for liver fibrosis (Liao et al. 2020)), we proved that melatonin could effectively reduce oxidative stress and inflammation in the host environment. Importantly, the enhanced ADSC retention and therapeutic outcomes including pathological alterations and inflammation were effectively improved by melatonin pretreatment. Therefore, melatonin pretreatment provides an alternative strategy to enhance ADSC therapy for OA.

Melatonin is a healthcare product that is commonly used to treat insomnia (Zafari Zangeneh 2022; Edemann-Callesen et al. 2023; Xia et al. 2023). Interestingly, extensive evidences have also been suggested that melatonin benefit to alleviating OA progression in some animal models (Gupta et al. 2018; Liang et al. 2023; Zhao et al. 2022; Qin et al. 2022). However, we found only a slight improvement in pathological changes and inflammation of OA rats in the current study. This may attribute to the low frequency of drug administration (twice/week, for 2 weeks in this study). Nevertheless, melatonin could enhance ADSC therapy for OA. The subsequent studies, including the comparative efficacy of melatonin (normal administration) and ADSC transplantation, as well as the combined therapeutic efficacy, need fully confirmed before further application.

Based on the clinical potential of melatonin and ADSC transplantation for OA treatment, melatonin pretreatment provides a new promising method for improving ADSC therapy in OA.

Conclusion

In summary, we demonstrated that melatonin could effectively enhance ADSC engraftment efficiency, therefore improving therapeutic outcomes of ADSC therapy for OA, and melatonin pretreatment presents a novel practical strategy to improve ADSC therapeutic efficiency for OA.

Acknowledgements

Not applicable.

Abbreviations

ADSCs

Adipose tissue-derived mesenchymal stem cells

Bcl-2

B-cell lymphoma-2

BAX

Bcl-2 associated X

CCK-8

Cell-counting-kit-8

CXCR4

C-X-C chemokine receptor type 4

ELISA

Enzyme linked immunosorbent assay

ICG

Indocyanine green

IL-1β

Interleukin-1β

IL-6

Interleukin-6

NIR

Near-infrared

OA

Osteoarthritis

TNF-α

Tumor necrosis factor-α

Author contributions

HJ, ZL and NL participated in study design and drafted the manuscript. NL participated in isolation and culture of ADSCs, and western blot analysis. HJ and NL performed the animal study, cell proliferation and cell apoptosis assay. HJ and JC performed the data analysis. HJ, ZL and NL participated in financial support and proof-read the manuscript. All authors read and approved the final manuscript.

Funding

This work was sponsored by Natural Science Foundation of Fujian Province (Grant No. 2020J01976), and Fujian Provincial Health Technology Project (Grant No.2023GGA032). The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability

The data sets supporting the results of this article are included within the article.

Declarations

Competing interests

The authors declare that they have no competing interests.

Ethical approval

All animal experiments were approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University, and all procedures were performed in accordance with the guidelines.

Consent for publication

All authors have reviewed the manuscript and approved its submission for publication.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Zhangya Lin, Email: 13799321745@139.com.

Naishun Liao, Email: liaons046@163.com.

References

  1. Baker MC, Sheth K, Lu R, Lu D, von Kaeppler EP, Bhat A, Felson DT, Robinson WH (2023) Increased risk of osteoarthritis in patients with atopic disease. Ann Rheum Dis 82:866–872 10.1136/ard-2022-223640 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernabei I, So A, Busso N, Nasi S (2023) Cartilage calcification in osteoarthritis: mechanisms and clinical relevance. Nat Rev Rheumatol 19:10–27 10.1038/s41584-022-00875-4 [DOI] [PubMed] [Google Scholar]
  3. Bhattacharjee M, Escobar Ivirico JL, Kan HM, Shah S, Otsuka T, Bordett R, Barajaa M, Nagiah N, Pandey R, Nair LS, Laurencin CT (2022) Injectable amnion hydrogel-mediated delivery of adipose-derived stem cells for osteoarthritis treatment. Proc Natl Acad Sci USA 119:e2120968119 10.1073/pnas.2120968119 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carneiro DC, Araújo LT, Santos GC, Damasceno PKF, Vieira JL, Santos RRD, Barbosa JDV, Soares MBP (2023) Clinical trials with mesenchymal stem cell therapies for osteoarthritis: challenges in the regeneration of articular cartilage. Int J Mol Sci 24:9939 10.3390/ijms24129939 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrascal L, Nunez-Abades P, Ayala A, Cano M (2018) Role of melatonin in the inflammatory process and its therapeutic potential. Curr Pharm Des 24:1563–1588 10.2174/1381612824666180426112832 [DOI] [PubMed] [Google Scholar]
  6. Chae DS, Lee CY, Lee J, Seo HH, Choi CH, Lee S, Hwang KC (2018) Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules. Biol Res 51:41 10.1186/s40659-018-0191-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen Z, Ge Y, Zhou L, Li T, Yan B, Chen J, Huang J, Du W, Lv S, Tong P, Shan L (2021) Pain relief and cartilage repair by nanofat against osteoarthritis: preclinical and clinical evidence. Stem Cell Res Ther 12:477 10.1186/s13287-021-02538-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cui L, Xu F, Jiang Z, Wang S, Li X, Ding Y, Zhang Y, Du M (2021) Melatonin regulates proliferation and apoptosis of endometrial stromal cells via MT1. Acta Biochim Biophys Sin (Shanghai) 53:1333–1341 10.1093/abbs/gmab108 [DOI] [PubMed] [Google Scholar]
  9. Edemann-Callesen H, Andersen HK, Ussing A, Virring A, Jennum P, Debes NM, Laursen T, Baandrup L, Gade C, Dettmann J, Holm J, Krogh C, Birkefoss K, Tarp S, Händel MN (2023) Use of melatonin in children and adolescents with idiopathic chronic insomnia: a systematic review, meta-analysis, and clinical recommendation. EclinicalMedicine 61:102048 10.1016/j.eclinm.2023.102048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fan C, Feng J, Tang C, Zhang Z, Feng Y, Duan W, Zhai M, Yan Z, Zhu L, Feng L, Zhu H, Luo E (2020) Melatonin suppresses ER stress-dependent proapoptotic effects via AMPK in bone mesenchymal stem cells during mitochondrial oxidative damage. Stem Cell Res Ther 11:442 10.1186/s13287-020-01948-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gao B, Gao W, Wu Z, Zhou T, Qiu X, Wang X, Lian C, Peng Y, Liang A, Qiu J, Zhu Y, Xu C, Li Y, Su P, Huang D (2018) Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells. Stem Cell Res Ther 9:162 10.1186/s13287-018-0892-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guo JY, Li F, Wen YB, Cui HX, Guo ML, Zhang L, Zhang YF, Guo YJ, Guo YX (2017) Melatonin inhibits Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes to attenuate osteoarthritis. Oncotarget 8:55967–55983 10.18632/oncotarget.18356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gupta N, Sinha R, Krasnodembskaya A, Xu X, Nizet V, Matthay MA, Griffin JH (2018) The TLR4-PAR1 axis regulates bone marrow mesenchymal stromal cell survival and therapeutic capacity in experimental bacterial pneumonia. Stem Cells 36:796–806 10.1002/stem.2796 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henrich D, Seebach C, Wilhelm K, Marzi I (2007) High dosage of simvastatin reduces TNF-alpha-induced apoptosis of endothelial progenitor cells but fails to prevent apoptosis induced by IL-1beta in vitro. J Surg Res 142:13–19 10.1016/j.jss.2006.04.011 [DOI] [PubMed] [Google Scholar]
  15. Hu Y, Xiong Y, Zha K, Tao R, Chen L, Xue H, Yan C, Lin Z, Endo Y, Cao F, Zhou W, Liu G (2023) Melatonin promotes BMSCs osteoblastic differentiation and relieves inflammation by suppressing the NF-κB pathways. Stem Cells In 2023:7638842 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang CC, Chiou CH, Liu SC, Hu SL, Su CM, Tsai CH, Tang CH (2019) Melatonin attenuates TNF-α and IL-1β expression in synovial fibroblasts and diminishes cartilage degradation: implications for the treatment of rheumatoid arthritis. J Pineal Res 66:e12560 10.1111/jpi.12560 [DOI] [PubMed] [Google Scholar]
  17. Jiang LB, Lee S, Wang Y, Xu QT, Meng DH, Zhang J (2016) Adipose-derived stem cells induce autophagic activation and inhibit catabolic response to pro-inflammatory cytokines in rat chondrocytes. Osteoarthr Cartil 24:1071–1081 10.1016/j.joca.2015.12.021 [DOI] [PubMed] [Google Scholar]
  18. Ke C, Li H, Yang D, Ying H, Zhu H, Wang J, Xu J, Wang L (2022) Melatonin attenuates the progression of osteoarthritis in rats by inhibiting inflammation and related oxidative stress on the surface of knee cartilage. Orthop Surg 14:2230–2237 10.1111/os.13408 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim KI, Lee WS, Kim JH, Bae JK, Jin W (2022) Safety and efficacy of the intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritic knee: a 5-year follow-up study. Stem Cells Transl Med 11:586–596 10.1093/stcltm/szac024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim KI, Lee MC, Lee JH, Moon YW, Lee WS, Lee HJ, Hwang SC, In Y, Shon OJ, Bae KC, Song SJ, Park KK, Kim JH (2023) Clinical efficacy and safety of the intra-articular injection of autologous adipose-derived mesenchymal stem cells for knee osteoarthritis: a phase iii, randomized, double-blind, placebo-controlled trial. Am J Sports Med 51:2243–2253 10.1177/03635465231179223 [DOI] [PubMed] [Google Scholar]
  21. Lian C, Wu Z, Gao B, Peng Y, Liang A, Xu C, Liu L, Qiu X, Huang J, Zhou H, Cai Y, Su P, Huang D (2016) Melatonin reversed tumor necrosis factor-alpha-inhibited osteogenesis of human mesenchymal stem cells by stabilizing SMAD1 protein. J Pineal Res 61:317–327 10.1111/jpi.12349 [DOI] [PubMed] [Google Scholar]
  22. Liang H, Yan Y, Sun W, Ma X, Su Z, Liu Z, Chen Y, Yu B (2023) Preparation of melatonin-loaded nanoparticles with targeting and sustained release function and their application in osteoarthritis. Int J Mol Sci 24:8740 10.3390/ijms24108740 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liao N, Shi Y, Zhang C, Zheng Y, Wang Y, Zhao B, Zeng Y, Liu X, Liu J (2019) Antioxidants inhibit cell senescence and preserve stemness of adipose tissue-derived stem cells by reducing ROS generation during long-term in vitro expansion. Stem Cell Res Ther 10:306 10.1186/s13287-019-1404-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liao N, Shi Y, Wang Y, Liao F, Zhao B, Zheng Y, Zeng Y, Liu X, Liu J (2020) Antioxidant preconditioning improves therapeutic outcomes of adipose tissue-derived mesenchymal stem cells through enhancing intrahepatic engraftment efficiency in a mouse liver fibrosis model. Stem Cell Res Ther 11:237 10.1186/s13287-020-01763-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liao N, Zhang D, Wu M, Yang H, Liu X, Song J (2021) Enhancing therapeutic effects and in vivo tracking of adipose tissue-derived mesenchymal stem cells for liver injury using bioorthogonal click chemistry. Nanoscale 13:1813–1822 10.1039/D0NR07272A [DOI] [PubMed] [Google Scholar]
  26. Liu Y, Chen XQ, Wang F, Cheng B, Zhou G (2022) Melatonin relieves Th17/CD4-CD8- T cells inflammatory responses via nuclear-receptor dependent manner in peripheral blood of primary Sjögren’s syndrome. Int Immunopharmacol 109:108778 10.1016/j.intimp.2022.108778 [DOI] [PubMed] [Google Scholar]
  27. Liu Y, Wang F, Cheng B, Zhou G (2024) Melatonin improves salivary gland damage and hypofunction in pSS by inhibiting IL-6/STAT3 signaling through its receptor-dependent manner. Mol Immunol 169:10–27 10.1016/j.molimm.2024.02.012 [DOI] [PubMed] [Google Scholar]
  28. Liu-Bryan R, Terkeltaub R (2015) Emerging regulators of the inflammatory process in osteoarthritis. Nat Rev Rheumatol 11:35–44 10.1038/nrrheum.2014.162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Manferdini C, Maumus M, Gabusi E, Piacentini A, Filardo G, Peyrafitte JA, Jorgensen C, Bourin P, Fleury-Cappellesso S, Facchini A, Noël D, Lisignoli G (2013) Adipose-derived mesenchymal stem cells exert antiinflammatory effects on chondrocytes and synoviocytes from osteoarthritis patients through prostaglandin E2. Arthritis Rheum 65:1271–1281 10.1002/art.37908 [DOI] [PubMed] [Google Scholar]
  30. Prakash R, Fauzia E, Siddiqui AJ, Yadav SK, Kumari N, Shams MT, Naeem A, Praharaj PP, Khan MA, Bhutia SK, Janowski M, Boltze J, Raza SS (2022) Oxidative stress-induced autophagy compromises stem cell viability. Stem Cells 40:468–478 10.1093/stmcls/sxac018 [DOI] [PubMed] [Google Scholar]
  31. Qin K, Tang H, Ren Y, Yang D, Li Y, Huang W, Wu Y, Yin Z (2022) Melatonin promotes sirtuin 1 expression and inhibits IRE1α-XBP1S-CHOP to reduce endoplasmic reticulum stress-mediated apoptosis in chondrocytes. Front Pharmacol 13:940629 10.3389/fphar.2022.940629 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Qiu X, Liang T, Wu Z, Zhu Y, Gao W, Gao B, Qiu J, Wang X, Chen T, Deng Z, Li P, Chen Y, Zhou H, Peng Y, Xu C, Su P, Liang A, Huang D (2022) Melatonin reverses tumor necrosis factor-alpha-induced metabolic disturbance of human nucleus pulposus cells via MTNR1B/Gαi2/YAP signaling. Int J Biol Sci 18:2202–2219 10.7150/ijbs.65973 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Richard MJ, Driban JB, McAlindon TE (2023) Pharmaceutical treatment of osteoarthritis. Osteoarthr Cartil 31:458–466 10.1016/j.joca.2022.11.005 [DOI] [PubMed] [Google Scholar]
  34. Sadri B, Hassanzadeh M, Bagherifard A, Mohammadi J, Alikhani M, Moeinabadi-Bidgoli K, Madani H, Diaz-Solano D, Karimi S, Mehrazmay M, Mohammadpour M, Vosough M (2023) Cartilage regeneration and inflammation modulation in knee osteoarthritis following injection of allogeneic adipose-derived mesenchymal stromal cells: a phase II, triple-blinded, placebo controlled, randomized trial. Stem Cell Res Ther 14:162 10.1186/s13287-023-03359-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Waluyo Y, Artika SR, Wahyuni Insani Nanda, Gunawan AMAK, Zainal ATF (2023) Efficacy of prolotherapy for osteoarthritis: a systematic review. J Rehabil Med 55:jrm00372 10.2340/jrm.v55.2572 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang X, Liang T, Qiu J, Qiu X, Gao B, Gao W, Lian C, Chen T, Zhu Y, Liang A, Su P, Peng Y, Huang D (2019) Melatonin reverses the loss of stemness induced by TNF-α in human bone marrow mesenchymal stem cells through upregulation of YAP expression. Stem Cells Int 2019:6568394 10.1155/2019/6568394 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu H, Yin Z, Wang L, Li F, Qiu Y (2017) Honokiol improved chondrogenesis and suppressed inflammation in human umbilical cord derived mesenchymal stem cells via blocking nuclear factor-κB pathway. BMC Cell Biol 18:29 10.1186/s12860-017-0145-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xia TJ, Wang Z, Jin SW, Liu XM, Liu YG, Zhang SS, Pan RL, Jiang N, Liao YH, Yan MZ, Du LD, Chang Q (2023) Melatonin-related dysfunction in chronic restraint stress triggers sleep disorders in mice. Front Pharmacol 14:1210393 10.3389/fphar.2023.1210393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xu MM, Kang JY, Wang QY, Zuo X, Tan YY, Wei YY, Zhang DW, Zhang L, Wu HM, Fei GH (2024) Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis. Respir Res 25:186 10.1186/s12931-024-02815-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yang M, Li L, Chen S, Li S, Wang B, Zhang C, Chen Y, Yang L, Xin H, Chen C, Xu X, Zhang Q, He Y, Ye J (2020) Melatonin protects against apoptosis of megakaryocytic cells via its receptors and the AKT/mitochondrial/caspase pathway. Aging (Albany NY) 12:13633–13646 10.18632/aging.103483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zafari Zangeneh F (2022) Deregulated brain’s central clock management on sleep-wake behavior in women with polycystic ovary syndrome: melatonin & sleep pattern. J Family Reprod Health 16:229–238 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhang Y, He F, Chen Z, Su Q, Yan M, Zhang Q, Tan J, Qian L, Han Y (2019) Melatonin modulates IL-1β-induced extracellular matrix remodeling in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration and inflammation. Aging 11:10499–10512 10.18632/aging.102472 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang Y, Liu T, Yang H, He F, Zhu X (2022) Melatonin: a novel candidate for the treatment of osteoarthritis. Ageing Res Rev 78:101635 10.1016/j.arr.2022.101635 [DOI] [PubMed] [Google Scholar]
  44. Zhao Y, Gao C, Liu H, Liu H, Feng Y, Li Z, Liu H, Wang J, Yang B, Lin Q (2021) Infliximab-based self-healing hydrogel composite scaffold enhances stem cell survival, engraftment, and function in rheumatoid arthritis treatment. Acta Biomater 121:653–664 10.1016/j.actbio.2020.12.005 [DOI] [PubMed] [Google Scholar]
  45. Zhao M, Song X, Chen H, Ma T, Tang J, Wang X, Yu Y, Lv L, Jia L, Gao L (2022) Melatonin prevents chondrocyte matrix degradation in rats with experimentally induced osteoarthritis by inhibiting nuclear factor-κb via SIRT1. Nutrients 14:3966 10.3390/nu14193966 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhou X, Zhang Y, Hou M, Liu H, Yang H, Chen X, Liu T, He F, Zhu X (2022) Melatonin prevents cartilage degradation in early-stage osteoarthritis through activation of mir-146a/NRF2/HO-1 axis. J Bone Miner Res 37:1056–1072 10.1002/jbmr.4527 [DOI] [PubMed] [Google Scholar]

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