Apelin-13 enhances BMSCs osteogenic differentiation and bone regeneration under hypoxia via upregulation of Ibsp

Abstract

Background

Apelin-13 plays a critical role in promoting angiogenesis and regulating cellular metabolism. However, the short half-life of Apelin-13 in vivo limits its further applications. Stem cell transplantation has been widely applied in tissue regeneration, but the low survival rate of transplanted stem cells in vivo restricts its therapeutic efficacy. This study aims to combine Apelin-13 with stem cell transplantation to enhance the survival rate of transplanted stem cells and promote bone regeneration.

Methods

In this study, BMSCs and HUVEC were pretreated with different concentrations of Apelin-13 to investigate its effects on their proliferation, differentiation capacities under hypoxic conditions (1% O₂). A rat mandibular bone defect model was employed to evaluate the promotive effects of Apelin-13-BMSCs and Apelin-13-HUVEC transplantation on bone regeneration, and RNA sequencing together with a rat closed femoral fracture model was further used to explore the potential mechanisms by which Apelin-13 enhances BMSC proliferation and differentiation.

Results

Apelin-13 enhances the proliferative and differentiation capacities of BMSCs and HUVEC under hypoxic conditions, reduces apoptosis, and exerts these promotive effects in a dose-dependent manner. BMSCs pretreated with Apelin-13 markedly facilitate the repair of rat mandibular bone defects and the healing of femoral fractures. Mechanistically, Apelin-13 promotes proliferation and osteogenic differentiation by upregulating Ibsp expression in BMSCs.

Conclusion

BMSCs pretreated with Apelin-13 exhibited favorable regenerative and differentiation capacities in both in vitro and in vivo experiments, providing a potential strategy to address the challenges of low survival rates and stemness decline following stem cell transplantation in vivo.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-05034-0.

Introduction

Bone regeneration is the physiological basis for treating bone defect–related diseases. Critical-sized bone defects resulting from tumor resection, or femoral fractures in elderly individuals, often exceed the body’s intrinsic regenerative capacity, representing a significant clinical challenge that requires urgent solutions [1, 2]. Bone defects larger than the critical size cannot be completely repaired by the body’s own capacity [3]. Currently, autologous bone grafting is commonly used in clinical practice [4, 5], but it has several drawbacks, including long treatment cycles, complex surgical techniques, high risks of infection and necrosis, and heavy economic burdens [6]. Stem cell transplantation is a widely used technique in regenerative medicine [7], where pluripotent stem cells with self-renewal and differentiation capabilities are transplanted into tissue or organ defects to promote the regeneration and repair of tissue or organ morphology and function [8]. It has been extensively applied in research related to bone [9], myocardial [10], and vascular regeneration [11]. Bone marrow-derived mesenchymal stem cells (BMSCs) are easily accessible stem cells with multipotent differentiation abilities, and they have been applied in the regeneration and repair of various tissues [12], particularly in the treatment of large, refractory bone defects [13], where they show significant advantages. On the other hand, recent studies have revealed that bone regeneration and angiogenesis are closely linked physiological processes. They mutually influence and promote each other, playing a synergistic role in the repair of bone defects. Human umbilical vein endothelial cells (HUVEC) transplantation has also been applied to promote vascular regeneration [14].

However, the stem cells transplanted into the body are exposed to a microenvironment at the bone defect site that is characterized by ischemia, hypoxia, and high levels of inflammatory cytokines. These conditions lead to significant cell senescence, apoptosis, and loss of stemness, which severely impair the regenerative and reparative potential of the transplanted cells [15]. Studies have shown that four days after stem cell transplantation, almost no surviving transplanted cells can be detected at the transplantation site [16, 17]. This suggests that enhancing the adaptability of stem cells to the local microenvironment in vivo is crucial for their role in promoting tissue regeneration. How to enhance the adaptability of transplanted cells has become a key focus in stem cell transplantation research. Combining stem cells with biomaterials for transplantation [18], or improving the nutrient supply [19] and oxygen levels at the transplantation site, has been proven to be effective approaches. However, preconditioning transplanted cells is another more cost-effective and practical method to enhance cell adaptability [20], while avoiding the drawbacks of biomaterial cytotoxicity and immune rejection.

Apelin-13 is an endogenous peptide that binds to the Apelin receptor (APJ), forming the APJ complex, which plays a crucial regulatory role in angiogenesis, energy metabolism, and cell apoptosis [21, 22]. Apelin-13, as a versatile bioactive molecule, has garnered widespread attention for its therapeutic potential in heart failure [23] and vascular regeneration [24]. However, Apelin-13 has a short half-life in vivo [25], requiring frequent administration for cases with long treatment durations, which severely limits its application. Moreover, there is limited research on the application of Apelin-13 in bone regeneration, and the effects of Apelin-13 on bone tissue regeneration and its potential mechanisms remain unclear.

In this study, Apelin-13 was combined with stem cell transplantation to investigate its role in promoting bone regeneration. BMSCs and HUVEC were pretreated with Apelin-13 to examine their proliferative and differentiation capacities under hypoxic and serum-free conditions in vitro. Subsequently, a rat mandibular defect model was established to evaluate the effects of Apelin-13 pretreated cells on mandibular bone regeneration. Furthermore, RNA sequencing identified Ibsp as a potential target of Apelin-13, and its role in enhancing BMSCs osteogenic differentiation under hypoxic conditions was validated through subrenal capsule transplantation and a closed femoral fracture model.

Materials and methods

Cell culture

This experiment was approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University. BMSCs were isolated using the adherent method from three-week-old SD rats followed described previously [26]. Briefly, after humanely euthanizing the rats, the femurs were sterilized in 75% alcohol for 10 min, and then carefully dissected to remove the soft tissue. The marrow cavity was flushed slowly with α-complete medium to collect the bone marrow. The resulting bone marrow suspension was centrifuged, and the pellet was resuspended in complete medium consisting of 90% α-MEM (α-MEM, Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA), and 1% penicillin–streptomycin (Beyotime, China). The cells were then seeded into culture flasks and incubated at 37 °C, 5% CO₂, and 21% O₂ in a cell culture incubator. The medium was changed every two days until the BMSCs reached 80%-90% confluence, at which point they were passaged.

Human umbilical vein endothelial cells(HUVEC, ATCC, USA) were cultured in endothelial cell medium supplemented with 5% FBS(Gibco, USA), 1% penicillin–streptomycin(Beyotime, China), and 1% endothelial cell growth supplement(ECGS, Sigma, USA). The cells were incubated at 37 °C, 5% CO₂, and 21% O₂ in a cell culture incubator. The medium was changed every two days until the HUVEC reached 80%-90% confluence, at which point they were passaged.

Cell counting kit 8 (CCK8)

Well-grown BMSCs and HUVEC were seeded into 96-well plates at a density of 5 × 10³ cells per well. The cells were then treated with medium containing different concentrations of Apelin-13 (MCE, China) (0.01, 0.05, 0.1, 0.15, and 0.2 nM) for 24 h. After treatment, 10 µL of CCK-8 (Beyotime, China) solution was added to each well, and the cells were incubated at 37 °C for 30 min. The absorbance was measured at 450 nm to assess the cytotoxicity of Apelin-13.

Next, we further explored the effect of Apelin-13 on cell proliferation under hypoxic conditions (1% O₂). Following the previously described method, Apelin-13-pretreated BMSCs and HUVEC were cultured in a hypoxic incubator. At days 3, 5, and 7 after Apelin-13 pretreatment, 10 µL of CCK-8 solution was added to the wells, and the cells were incubated at 37 °C for 30 min. Absorbance was measured at 450 nm to determine the effect of Apelin-13 on cell proliferation under hypoxic conditions.

Osteogenic differentiation assay

Well-grown P3 generation BMSCs were seeded into six-well plates at a density of 1 × 10⁵ cells per well. After the cells adhered to the plate, they were pretreated with fresh medium containing Apelin-13 at concentrations of 0.1 nM, 0.15 nM, and 0.2 nM under normoxic conditions for 48 h. Once the cells reached 80%-90% confluence, osteogenic induction was initiated. Osteogenic induction medium was formulated by adding 10^{− 7} mmol/L dexamethasone(Solarbio, China), 50 µg/mL vitamin C(Solarbio, China), and 10 ng/mL β-glycerophosphate(Solarbio, China) to complete medium [27], and the cells were cultured under hypoxic conditions (1% O₂). Alkaline phosphatase (ALP, Beyotime, China) staining was performed on days 3、5 and 7 during osteogenic induction, The alkaline phosphatase (ALP) content in cells was measured using an ALP assay kit (Beyotime, China). The samples were washed three times with PBS buffer, and 400 µL of cell lysis buffer was added to each well. After centrifugation, the supernatant was collected. The subsequent procedures were performed strictly according to the manufacturer’s instructions. The absorbance of each sample well at 405 nm was measured using a microplate reader, and the ALP activity in each well was calculated. Alizarin Red S (ARS, Solarbio, China) staining was performed on days 7、14 and 21 during osteogenic induction, the acquired images were analyzed using ImageJ, and the percentage of the calcium nodule area relative to the total cell area was calculated.

Wound healing assay

HUVEC were seeded into six-well plates at a density of 5 × 10⁴ cells per well. After the cells had fully adhered, they were pretreated with fresh medium containing Apelin-13 at concentrations of 0 nM, 0.1 nM, 0.15 nM, and 0.2 nM under normoxic conditions for 48 h. Subsequently, the cells were starved with serum-free medium for 24 h and cultured under hypoxic conditions. Once the cells reached 80%-90% confluence, a scratch was made along the centerline of each well using a 10 µL pipette tip. After two washes with PBS, serum-free medium was added for further culture. Images were taken at 24 and 48 h post-scratch.

Transwell

HUVEC were pretreated with different concentrations of Apelin-13 and starved according to wound healing assay. After digestion, the cells were prepared into a single-cell suspension at a density of 5 × 10⁵ cells/ml. A 100 µL portion of the single-cell suspension was added to the upper chamber of a 24-well Transwell plate (8 μm, Corning, USA). The lower chamber contained complete medium with 0 nM, 0.1 nM, 0.15 nM, and 0.2 nM Apelin-13. The cells were then cultured under hypoxic conditions. After 8 h, the lower chamber medium was aspirated, and 4% paraformaldehyde was added to the upper chamber to fix the cells for 30 min. After wiping off the non-migrated cells on the upper surface of the chamber with a cotton swab, the cells were stained with 0.1% crystal violet solution for 30 min. Images were captured under a microscope.

Tube formation assay

HUVEC were pretreated with Apelin-13 and starvation as described previously [28]: Specifically, HUVEC were pretreated with culture medium containing 0 nM, 0.1 nM, 0.15 nM, and 0.2 nM Apelin-13 for 48 h. Subsequently, the medium was replaced with serum-free medium, and the cells were cultured under hypoxic conditions for 24 h. Meanwhile, 50 µL of pre-chilled Matrigel (Beyotime, China) was evenly spread into each well of a 96-well plate, ensuring no air bubbles. The plate was then incubated at 37 °C for 30 min to allow the matrigel to solidify. After pretreatment, the HUVEC were digested, resuspended, and seeded into the plate at a density of 5 × 10³ cells per well. The plate was cultured under hypoxic conditions. Images were captured at 2, 4, and 6 h to observe the tube formation by the HUVEC.

TUNEL apoptosis staining

BMSCs and HUVEC, with good growth and 90% confluence, were seeded at 3 × 10⁴ cells per well in a 12-well plate. The cells were pretreated with fresh medium containing 0 nM, 0.1 nM, 0.15 nM, and 0.2 nM Apelin-13 under normal oxygen conditions for 24 h. After that, the medium was replaced with serum-free medium, and the cells were cultured under hypoxic conditions for 7 days. TUNEL staining (Beyotime, China) was performed following the manufacturer’s instructions to assess the apoptosis rate of BMSCs and HUVEC after induction.

Western blot (WB)

Following Apelin-13 pretreatment of BMSCs as described earlier [28], BMSCs were pretreated with culture medium containing 0 nM, 0.1 nM, 0.15 nM, and 0.2 nM Apelin-13 for 48 h, after which osteogenic induction was performed. After 21 days of osteogenesis, the osteogenic medium was removed, and the cells were washed twice with PBS. Subsequently, 100 µL of RIPA (APExBio, USA) buffer and 1 µL of PMSF (SAB, USA) were added to each well of a 6-well plate. Cells were lysed on ice for 30 min, scraped, and collected. After vortexing for 1 min, the samples were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentration was determined using the BCA assay (Beyotime, China), with β-actin as the internal control. The expression levels of PI3K(1ะ1000, CST, USA), AKT(1ะ1000, Abcam, UK), p-AKT(1ะ1000, Abcam, UK), APLNR(1ะ1000, Abcam, UK), BMP2(1ะ1000, CST, USA), and Runx2(1ะ1000, CST, USA) were analyzed in BMSCs. The same pretreatment and protein extraction procedures were applied to HUVEC, with the expression of PI3k(1ะ1000, CST, USA), AKT(1ะ1000, Abcam, UK), p-AKT(1ะ1000, Abcam, UK), and angiogenesis-related factors HIF-1α(1ะ1000, Abcam, UK) and VEGF-A(1ะ1000, Abcam, UK) assessed.

QPCR

BMSCs pretreated with Apelin-13 were harvested on day 14 of osteogenic induction. Total RNA was extracted using TRIzol, and the RNA concentration was measured by absorbance at 260 nm. cDNA was synthesized using a reverse transcription kit (APExBio, USA) according to the manufacturer’s instructions. SYBR Premix Ex Taq (APExBio, USA) was then used to quantify the expression levels of osteogenesis-related genes, including Bmp2, Runx2, and PI3K (Sangon Biotech, Shanghai, China). For HUVEC, total RNA was extracted after 5 days of hypoxic induction following Apelin-13 pretreatment, and the expression levels of HIF-1α, VEGF, and PI3K (Sangon Biotech, Shanghai, China) were measured. β-actin was used as the internal control. Primer sequences refer to Table S1.

RNA sequencing (RNA-seq) analysis

The RNA samples were derived from rat BMSCs that had been pretreated with Apelin-13 or PBS (control) and subsequently cultured under hypoxic conditions for 24 h. Total RNA was extracted using the TRIzol method. RNA purity and concentration were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were prepared with the VAHTS Universal V10 RNA-seq Library Prep Kit (Premixed Version) following the manufacturer’s instructions. RNA sequencing and data analysis were performed by OEbiotech Co., Ltd. (Shanghai, China).

Ibsp^KD BMSCs established via siRNA transfection

A small interfering RNA specifically targeting rat Ibsp (si-Ibsp) was synthesized by Sangon Biotech (Shanghai, China). A scrambled siRNA with no known homology to mammalian genes was used as a negative control (siRNA-NC). The sequences of si-Ibsp are listed in Table S2. Rat BMSCs (P3) were used as recipient cells and seeded at a density of 2 × 10⁵ cells per well in six-well plates. si-Ibsp and negative control siRNA (si-NC) were transfected using a commercial siRNA transfection kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. Briefly, when cell confluence reached approximately 80%, 6 µL of RNATransMate was mixed with 20 pmol of siRNA, and the resulting siRNA/RNATransMate complexes were added to each well. After 24 h of incubation, the resulting Ibsp^KD BMSCs were used for subsequent experiments.

Immunofluorescence staining of BMSCs

BMSCs subjected to the indicated treatments were seeded onto confocal dishes and processed as follows: The culture medium was removed, and cells were washed twice with PBS. Cells were then fixed with 4% paraformaldehyde for 20 min, followed by three washes with PBS. Membrane permeabilization was performed using 0.1% Triton X-100(Beyotime, China) for 30 min, followed by three additional PBS washes. Cells were blocked with 1% BSA (Solarbio, China) for 1 h, then washed three times with PBST. ALP(Abcam, UK)、Runx2(Abcam, UK) and Osterix (Thermo fisher, USA) were diluted according to the manufacturer’s recommended dilution (1:100) and added to the confocal dishes for overnight incubation at 4 °C. After three PBST washes, cells were incubated with secondary antibodies, FITC-Phalloidin (Maokang, China), and DAPI (Solarbio, China) in the dark. Fluorescence was observed using a laser scanning confocal microscope.

Sub-renal capsule transplant surgery

Animal experiments in this study were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 and were approved by the Animal Ethics Committee of Sichuan University (No. WCHSIRB-D-2023-228).

All animal were maintained under a 12-hour light/dark cycle at a controlled temperature of 24 ± 2 °C. Throughout the study, the rats were allowed free movement within their cages and provided ad libitum access to food and water. Prior to the initiation of experimental procedures, all animals underwent 1 week acclimation period. The animals were anesthetized through an intraperitoneal injection combining ketamine (50 mg/kg) and 2% xylazine hydrochloride (5 mg/kg).

The sub-renal capsular assay (SRCA) was employed to assay the osteogenic differentiation capacity of BMSCs pretreated with Apelin-13 in vivo. GFP-BMSCs derived from GFP transgenic rats (Cyagen, Guangzhou, China) were used in this experiment. Third-passage (P3) GFP-BMSCs subjected to the indicated treatments were resuspended in a 1ะ1 mixture of complete culture medium and matrigel (Beyotime, China), and the cell density was adjusted to 1 × 10⁶ cells/mL. The resulting cell suspension was transplanted under the left renal capsule of immunodeficient rats at a volume of 50 µL per rat (5 × 10⁴ cells per rat). Four weeks later, all rats were euthanized by lethal intraperitoneal injection of 1mL sodium pentobarbital (200 mg/mL), and the kidneys containing the grafts were harvested for subsequent immunofluorescence staining and imaging analysis.

Animal model and continuous drug administration

Animal experiments in this study were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 and were approved by the Animal Ethics Committee of Sichuan University (No. WCHSIRB-D-2023-228).

All animal were maintained under a 12-hour light/dark cycle at a controlled temperature of 24 ± 2 °C. Throughout the study, the rats were allowed free movement within their cages and provided ad libitum access to food and water. Prior to the initiation of experimental procedures, all animals underwent 1 week acclimation period. The animals were anesthetized through an intraperitoneal injection combining ketamine (50 mg/kg) and 2% xylazine hydrochloride (5 mg/kg).

The mandible, as a weight-bearing bone, performs complex physiological functions. Mandibular defects are a common type of clinical bone defect with significant clinical relevance. Therefore, in this study, a mandibular defect animal model was established. A total of 24 adult male Sprague–Dawley (SD)(Dossy, Chengdu, China) rats weighing 300–350 g were randomly divided into 4 groups (n = 6 in each group). The surgical procedure was performed by an independent experimenter who was blinded to the group assignments, the surgery order was randomized. A rat mandible defect model was established to investigate the osteogenic and angiogenic effects of Apelin-13-pretreated BMSCs and HUVEC in vivo. Gelatin sponges (GS) were used as scaffolds. In vitro, 100 µL of a single-cell suspension of Apelin-13-pretreated BMSCs (1 × 10⁶/mL) was seeded onto the GS. For the Apelin-13-BMSCs/HUVEC group, Apelin-13-pretreated BMSCs and HUVEC were mixed in equal amounts so that the total number of seeded cells was 1 × 10⁵. The seeded GS was incubated at 37 °C for 2 h to allow cell adhesion. GS without cells and GS seeded with BMSCs not pretreated with Apelin-13 were used as control groups. After anesthetizing the experimental animals, a 1 cm incision was made along the lower edge of the left mandible to expose the bone. A 5 mm circular bone defect was created 1 mm above the lower edge and 1 mm anterior to the posterior notch of the mandible using a ring drill. The GS was then transplanted into the mandibular defect. After achieving complete hemostasis [29]. The incision was sutured, and anti-inflammatory analgesics were administered for 3 days. After 4 weeks, all rats were euthanized by lethal intraperitoneal injection of 1mL sodium pentobarbital (200 mg/mL), and the mandibles were extracted to assess bone defect healing.

A rat closed femoral fracture model was used to evaluate the role of the Apelin-13/Ibsp signaling process in promoting bone regeneration in vivo. A total of 48 rats were randomly divided into 4 groups (n = 12 in each group). The surgical procedure was performed by an independent experimenter who was blinded to the group assignments, the surgery order was randomized. Rats were anesthetized and fixed on the surgical table, and a 1 cm incision was made at the knee joint. A 0.8 mm diameter Kirschner wire was then inserted into the femoral medullary cavity via the patella. Subsequently, following the standard procedure for creating closed femoral fractures, a bone knife was placed at the proximal one-third of the femur, and a 500 g weight was dropped from a height of 20 cm to strike the bone knife vertically, generating a femoral fracture. After surgery, each group of rats received one of the following four types of weekly injections: 200 µl of PBS containing 1 × 10⁶ Apelin/BMSCs, 1 × 10⁶ Apelin/BMSCs-si-NC, or 1 × 10⁶ Apelin/BMSCs-si-Ibsp for 2 or 4 consecutive weeks, while the control group was injected with an equal volume of PBS alone. After 2 and 4 weeks post-surgery, a total of 48 adult rats (n = 12 in each group) were euthanized by lethal intraperitoneal injection of 1mL sodium pentobarbital (200 mg/mL), and the femurs were extracted to assess fracture healing.

All animals received continuous administration of antibiotics and meloxicam for three days postoperatively to prevent infection and relieve pain.

Micro-CT

Four weeks post-surgery, rats were euthanized, and the left mandibles were carefully excised and fixed in 4% paraformaldehyde. Scanning was performed using a SCANO Medical µCT system, and data were analyzed using Skyscan CTAn software. A 2 mm region surrounding the bone defect was selected for analysis. The ratio of bone volume to total volume (BV/TV) within this region was measured to assess new bone formation.

At 2 and 4 weeks after fracture surgery, the rats were sacrificed, and the femurs were carefully harvested in their entirety, followed by processing as previously described for subsequent micro-CT analysis. The following outcome measures were used as reference indicators to evaluate bone healing: BV/TV, Connectivity Density (Conn.D), Trabecular Thickness (Tb.Th), and Trabecular Separation (Tb.Sp).

H&E and Masson staining

After excising the complete bone tissue, it was fixed in 4% paraformaldehyde for 24 h and decalcified with 10% ethylenediaminetetraacetic acid (EDTA) with shaking for 8 weeks. The samples were then processed according to standard procedures for paraffin embedding, serial sectioning (4 μm), and dewaxing. The prepared sections were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome stain.

Statistical analysis

The results were quantified using ImageJ software, and data are presented as the mean ± standard deviation. GraphPad Prism 10.5.0 software was used for analysis and plotting. Statistical analyses were performed using Student’s t-test or one-way ANOVA with Dunnett’s post-hoc test, as appropriate. All statistical tests were two-tailed, and P < 0.05 was considered statistically significant.

Results

Apelin-13 regulates cell number under hypoxic conditions in a dose-dependent manner

First, we characterized the isolated primary BMSCs, as shown in Fig. 1A: P1 cells exhibited spindle-shaped, adherent growth with centrally located nuclei, consistent with the typical morphology of BMSCs. Second, flow cytometry analysis was used to identify characteristic BMSCs markers, and the results (Figure S1) showed that the isolated primary cells highly expressed BMSCs-positive markers such as CD29 and CD90, while CD45 expression was low, meeting the established criteria for BMSCs surface marker identification. Apelin-13 is an endogenous peptide that modulates cellular metabolism and apoptosis. It has been shown to play a significant role in cardiovascular diseases by influencing various physiological and pathological processes, we assessed the Apelin-13 receptor (APLNR) expression in BMSCs and HUVEC under normoxic and hypoxic conditions using Western blotting. The results (Figure S2) showed that both cell types expressed APLNR, and its expression was markedly increased under hypoxia, possibly mediated by hypoxia-inducible factor-1α (HIF-1α), reflecting an adaptive response of the cells to low-oxygen conditions. At fracture or bone defect sites, the formation of hematomas and microcirculatory disturbances create a localized environment characterized by ischemia and hypoxia. This poses a significant challenge to the survival of transplanted stem cells in such conditions. To evaluate the effect of Apelin-13 on cell proliferation, we examined the impact of different concentrations (0, 0.01, 0.05, 0.1, 0.15, and 0.2 nM) of Apelin-13 on the growth of BMSCs and HUVEC under both normoxic and hypoxic conditions(Fig. 1B). Our results showed(Fig. 1C) that under normoxic conditions, different concentrations of Apelin-13 had no significant effect on the number of BMSCs and HUVEC. To further simulate the microenvironment of bone defects in vivo, we investigated the effect of Apelin-13 on cell proliferation under hypoxic conditions. Our results showed (Fig. 1D) that Apelin-13 pretreatment significantly increased the number of BMSCs under hypoxic conditions. Moreover, this effect was dose-dependent within a certain range. When the concentration of Apelin-13 increased from 0 to 0.1 nM, the number of BMSCs significantly increased. At 0.1 nM, the number of BMSCs showed no statistical difference compared to the control group cultured under normoxic conditions. Furthermore, within the concentration range of 0.1–0.2 nM, there was no significant difference between the groups. At the initial measurement after hypoxic culture, no significant differences were observed between the groups. However, over time, the cell count curves for different groups showed clear divergence. Hypoxic culture caused a significant deviation in the BMSCs count curve from that under normoxic conditions, whereas Apelin-13 pretreatment helped restore this deviation. As the concentration of Apelin-13 increased, the cell count curve gradually approached that of the normoxic group. When the concentration reached 0.1 nM, no significant difference in cell count was observed between the hypoxia and normoxia groups. Subsequently, we examined the effect of Apelin-13 pretreatment on HUVEC. Unlike BMSCs, we observed a significant difference in cell numbers between the groups at the first measurement of OD after hypoxic culture. Apelin-13 pretreatment resulted in a marked increase in cell count compared to the normoxia group. Further experiments indicated that Apelin-13 pretreatment could also restore the proliferation curve deviation between the hypoxia group and the normoxia group, but this restorative effect was only observed in the 0.1 nM to 0.2 nM groups. By assessing the effect of Apelin-13 on cell numbers under hypoxic conditions, we observed that Apelin-13 significantly increased the number of viable BMSCs and HUVEC in a dose-dependent manner within a certain concentration range. This promoting effect seemed to plateau when the Apelin-13 concentration reached 0.1 nM. Therefore, we selected the 0.1–0.2 nM concentration gradient for subsequent experiments.

Fig. 1.

Impact of Apelin-13 pretreatment on the number of viable BMSCs and HUVEC. A Morphological images of BMSCs. B Schematic diagram of Apelin-13 pretreatment and hypoxic culture. C Changes in BMSC and HUVEC cell numbers after 24 h of Apelin-13 pretreatment under normoxic conditions, n = 6. D Effect of Apelin-13 pretreatment on BMSC and HUVEC cell numbers under hypoxic conditions. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Apelin-13 inhibits cell apoptosis by upregulating the PI3K/AKT signaling pathway

Previous studies have demonstrated that Apelin-13 pretreatment significantly increases the number of viable BMSCs and HUVEC under hypoxic conditions. However, the exact mechanism underlying this effect remains unclear. Apelin-13 has been shown to effectively enhance cardiac ejection fraction in heart failure patients and reduce cell apoptosis. Based on these findings, we reasonably hypothesize that Apelin-13 exerts its effects by reducing cell apoptosis in hypoxic environments. TUNEL staining of BMSCs cultured in hypoxic, serum-free conditions for 7 days revealed that the TUNEL/DAPI ratio in the control group was approximately 10% (Fig. 2A, B). In contrast, BMSCs pretreated with Apelin-13 demonstrated stronger anti-apoptotic properties, with a TUNEL/DAPI ratio of less than 5%. Moreover, the TUNEL/DAPI ratio gradually decreased with increasing Apelin-13 concentrations. The average apoptosis rate in the 0.15 nM group was lower than that in the 0.1 nM group, but the difference was not statistically significant. However, when the concentration difference increased further, the average apoptosis rate between the 0.1 nM and 0.2 nM groups showed a statistically significant difference. Similar results were observed in the apoptosis assay of HUVEC (Fig. 2A, B). The TUNEL/DAPI ratio in the control group was approximately 25%, while in the three Apelin-13 pretreated groups, the ratio was around 5%. A significant statistical difference was observed only between the 0.1 nM and 0.2 nM groups. Apoptosis assays in both cell types suggest that Apelin-13 may influence the number of viable cells by inhibiting cell apoptosis under hypoxic, serum-free conditions. Apelin-13 has been shown to exert effects in cardiovascular and neurological diseases by activating the PI3K/AKT pathway. Therefore, we became interested in whether Apelin-13 could promote osteogenesis and angiogenesis in bone defects locally by activating the PI3K/AKT pathway in BMSCs and HUVEC. We extracted total RNA and protein from BMSCs and HUVEC pretreated with different concentrations of Apelin-13 under hypoxic, serum-free conditions. The results showed that the mRNA expression levels of PI3K in both BMSCs and HUVEC were significantly increased after Apelin-13 pretreatment compared to the control group (Fig. 2C). WB results showed (Fig. 2D, E) that Apelin-13 pretreatment significantly increased the expression of PI3K in BMSCs under hypoxic, serum-free conditions. Additionally, Apelin-13 also enhanced the expression of downstream signaling molecules AKT and p-AKT. On the other hand, in Apelin-13 pretreated HUVEC (Fig. 2F), we also observed an increase in the expression of PI3K, AKT and p-AKT. Furthermore, as the concentration difference increased, the expression of these proteins between groups showed a noticeable difference. In this part of the experiment, we demonstrated that Apelin-13 pretreatment enables cells to effectively resist apoptosis induced by hypoxic, serum-free culture conditions. The protective effect of Apelin-13 on cells under hypoxic and serum-free conditions may be partially mediated through upregulation of the intracellular PI3K/AKT pathway, enhancing cellular resistance to the external environment. These results provide a theoretical basis for Apelin-13’s ability to reduce apoptosis of cells transplanted into the injury site.

Fig. 2.

Apelin-13 Pretreatment Resists Cell Apoptosis via Upregulating the PI3K/Akt Pathway. A, B TUNEL staining to detect apoptosis activity and quantitative analysis, n = 3, scale bar = 100 μm. Control group BMSCs apoptosis rate: 10%, Apelin-13 pretreated BMSCs apoptosis rate: <5%. Control group HUVEC apoptosis rate: 24%, Apelin-13 pretreated HUVEC apoptosis rate: ~5%. C qPCR analysis of PI3K in BMSCs and HUVEC, n = 3. D–F WB analysis of PI3K, Akt, in BMSCs and HUVEC, n = 3. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Apelin-13 promotes osteogenic differentiation and the formation of mineralized nodules in BMSCs under hypoxic conditions

In the previous experiment, we investigated the effect of Apelin-13 on cell numbers under hypoxic conditions and its underlying mechanism. To further assess the impact of different concentrations of Apelin-13 pretreatment on the osteogenic potential of BMSCs under hypoxic conditions, we evaluated the osteogenic differentiation ability of BMSCs 48 h after Apelin-13 pretreatment, along with the expression of osteogenesis-related genes and proteins. First, ALP staining was performed on BMSCs at days 3, 5, and 7 of osteogenic induction. The results showed (Fig. 3A, C) that at the same time points, Apelin-13 pretreated BMSCs exhibited higher levels of alkaline phosphatase expression. Further analysis revealed that Apelin-13 pretreatment had a sustained effect on BMSCs at different time points, accelerating the osteogenic differentiation process. We further evaluated the effect of different concentrations of Apelin-13 on calcium salt deposition in cells by performing Alizarin Red S (ARS) staining. After Apelin-13 pretreatment, calcium salt content between cells was measured on days 7, 14, and 21 of osteogenic induction. Our results showed (Fig. 3B, D) that on day 7, no significant differences in calcium salt deposition were observed between the groups. This might be due to the relatively short duration, which was insufficient for the cells to produce enough calcium salt deposits. On days 14 and 21, Apelin-13 pretreatment significantly enhanced calcium salt deposition in BMSCs. Moreover, this effect became more pronounced with time, showing a clear cumulative effect. This suggests that Apelin-13’s influence persists over time, providing new insights into how to address its short in vivo half-life. We further investigated the effect of Apelin-13 pretreatment on the osteogenic differentiation of BMSCs under hypoxic conditions at both the gene and protein levels. QPCR results (Fig. 3E) showed that Apelin-13 pretreatment significantly upregulated the expression of osteogenic-related genes Bmp2 and Runx2 in BMSCs compared to the control group. Furthermore, within the different concentrations of Apelin-13, there appeared to be a dose-dependent relationship between the expression of Bmp2 and Runx2 and the Apelin-13 concentration. However, this difference did not reach statistical significance. Further WB analysis confirmed similar results (Fig. 3F, G): Apelin-13 pretreatment enhanced the expression of osteogenic-related proteins in BMSCs under hypoxic conditions. In summary, this section of the experiment validated, at both the cellular and molecular levels, that Apelin-13 pretreatment enhances the osteogenic differentiation ability of BMSCs under hypoxic conditions, and that this effect persists. These results provide a theoretical foundation for the application of Apelin-13 in tissue engineering.

Fig. 3.

The effect of Apelin-13 pretreatment on osteogenic differentiation of BMSCs under in vitro hypoxic conditions. A, C ALP staining and quantitative analysis at days 3, 5, and 7 of osteogenic induction, n = 3; B, D Alizarin Red S (ARS) staining and calcified nodule quantification at days 7, 14, and 21 of osteogenic induction, n = 3, scale bar=200 μm; E qPCR analysis of Runx2 and BMP2 in BMSCs; F, G WB analysis of Runx2 and BMP2 in BMSCs, n = 3. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Apelin-13 enhances the tube formation ability of HUVEC under hypoxic conditions and promotes the expression of genes and proteins related to angiogenesis

To evaluate the effect of Apelin-13 on vascular regeneration at bone defect sites, we cultured Apelin-13 pretreated HUVEC under hypoxic conditions (1% O₂) to investigate the impact of Apelin-13 on HUVEC activity. The results of the wound healing assay and transwell migration assay (Fig. 4A, B) showed that Apelin-13 pretreated HUVEC exhibited stronger collective and individual migration abilities under hypoxic conditions. This enhanced migratory capacity was significantly observed at different time points in the scratch assay, indicating that the effect of Apelin-13 on cell activity is sustained. In both of these assays, no dose-dependent effect was observed between the different concentrations of Apelin-13. We further validated the effect of Apelin-13 preconditioning on HUVEC tube formation ability in vitro using a tube formation assay. The results also showed (Fig. 4C) that HUVEC pretreated with Apelin-13 exhibited stronger tube formation ability under hypoxic conditions. This promotive effect was highly significant at 2, 4, and 8 h, and as time progressed, the differences became even more pronounced. We further explored the molecular mechanisms by which Apelin-13 preconditioning enhances the tube formation ability of HUVEC under hypoxic conditions. We measured the expression levels of angiogenesis-related factors HIF-1α and VEGF-A at both the mRNA and protein levels in HUVEC pretreated with Apelin-13. The qPCR results (Fig. 4D) show that Apelin-13, at all concentrations, significantly increased the expression of HIF-1α in HUVEC. HIF-1α is an upstream signaling molecule of VEGF-A and regulates its expression. The results showed that Apelin-13 pretreatment led to increased expression of VEGF-A in the cells. WB results showed (Fig. 4E, F) that the protein levels of HIF-1α and VEGF-A in cells pretreated with Apelin-13 were significantly increased. This section of the experiment demonstrates that Apelin-13 pretreatment enhances the migration and tube formation ability of vascular endothelial cells in low-oxygen conditions surrounding bone defects, promoting bone regeneration. This effect may be mediated through the HIF-1α/VEGF-A axis.

Fig. 4.

Apelin-13 pretreatment enhances HUVEC migration and tube formation ability under low-oxygen conditions. A Scratch assay assessing the effect of Apelin-13 on HUVEC collective migration, n = 3, scale bar=200 μm; B Transwell assay measuring the effect of Apelin-13 on individual HUVEC migration, n = 3, scale bar=100 μm; C Effect of Apelin-13 on HUVEC tube formation ability under low-oxygen conditions, n = 3, scale bar=200 μm; D qPCR analysis of HIF-1α and VEGF-A expression in HUVEC, n = 3; E WB analysis of HIF-1α and VEGF-A expression in HUVEC, n = 3. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Apelin-13 pretreated stem cells promote the repair and regeneration of critical-sized mandibular bone defects in rats

To investigate whether Apelin-13 pretreated cells can better promote bone regeneration at bone defect sites, we transplanted gelatin sponges(GS) seeded with stem cells into critical-sized mandibular defects in rats. (Fig. 5A). Unpretreated BMSCs and equal volume PBS served as the control group. The standard for critical-sized mandibular bone defects in rats is defined as a circular defect with a diameter of 5 mm, located 1 mm from the lower edge and posterior notch of the mandible. To investigate whether the co-transplantation of BMSCs and HUVEC can synergistically promote bone regeneration by enhancing osteogenesis-angiogenesis coupling, we transplanted GS seeded with Apelin-13-pretreated BMSCs and HUVEC (Apelin-13-BMSCs/HUVEC) into mandibular defects in rats. Four weeks later, the rats’ mandibular bones were carefully removed for micro-CT scanning and histological sectioning. The micro-CT results (Fig. 5B) showed that Apelin-13-BMSCs significantly promoted the repair of mandibular bone defects. The new bone volume at the defect site in the Apelin-13-BMSCs group was significantly higher than that in the BMSCs and control groups. Further analysis of the microstructure of the newly formed bone revealed that the Apelin-13-BMSCs group exhibited denser trabeculae, with a more regular trabecular shape. Subsequently, we analyzed the mandibular bone regeneration in the Apelin-13-BMSCs/HUVEC group and found that the rats exhibited superior regeneration. The BV/TV value was higher than in the other groups, and the difference was statistically significant. This suggests that BMSCs and HUVEC work synergistically in the process of bone regeneration. Given the strong biological connection between osteogenesis and angiogenesis, we further examined the histological features of the newly formed bone through HE and Masson staining (Fig. 5C). Bone tissue within 2 mm of the defect was selected for analysis. HE staining showed (Fig. 5D) that in the regions surrounding the bone defect, both the Apelin-13-BMSCs and Apelin-13-BMSCs/HUVEC groups contained a higher number of mature osteoblasts and clearly defined bone lacunae, indicating a pronounced osteogenic effect in these areas. Interestingly, we observed a higher number of concentrically arranged lamellar bone structures in the mandible tissues of the Apelin-13-BMSCs/HUVEC group. Concentrically arranged lamellar bone structures are characteristic of mature bone and are key structures for the blood supply within bone tissue. This suggests that the Apelin-13-BMSCs/HUVEC group exhibits a higher level of maturity, likely resulting from the synergistic effect of osteogenesis and angiogenesis. Masson staining (Fig. 5E) further confirmed this conclusion: the red-stained areas represent mature bone tissue at the later stages of bone repair, while the blue-stained regions indicate early-stage bone tissue formation during the repair process, the staining results showed that in the control group, most of the bone tissue sections were composed of early-stage regenerating bone tissue, while the BMSCs group exhibited more mature bone tissue. The Apelin-13-BMSCs and Apelin-13-BMSCs/HUVEC groups displayed an even higher proportion of mature bone tissue. The in vivo results confirmed the conclusions from our in vitro experiments, indicating that Apelin-13-pretreated BMSCs exhibit enhanced osteogenic differentiation. Furthermore, the co-transplantation of BMSCs and HUVEC can synergistically promote bone regeneration by facilitating the initiation, mineralization, and maturation of bone regeneration during the biological process.

Fig. 5.

Apelin-13 pretreated BMSCs and HUVEC enhance the regeneration of critical-size mandibular bone defects in rats. A Schematic of rat mandibular critical-size bone defect; B Micro-CT scanning results, 3D reconstruction, and quantitative analysis; C H&E and Masson staining of mandibular defect images after 4 weeks, yellow arrows: empty bone lacunae; blue arrows: bone lacunae; red arrows: concentrically arranged lamellar bone structures, scale bar = 100 μm; D Osteoblast count analysis in mandibular sections; E Quantification of mature bone tissue area. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Apelin-13 promotes the osteogenic differentiation of BMSCs by upregulating Ibsp

To gain deeper insight into the underlying mechanism by which Apelin-13 promotes the osteogenic differentiation of BMSCs, RNA sequencing was performed on BMSCs treated with Apelin-13. A total of 1,322 differentially expressed genes were identified between the two groups, among which 877 were upregulated and 445 were downregulated in the Apelin-13-treated group (Fig. 6A). Subsequent Gene Ontology (GO) enrichment analysis (Fig. 6B) revealed that these genes were predominantly associated with pathways regulating cell migration and adhesion, and were mainly localized within the extracellular matrix. We next focused on the genes upregulated in BMSCs following Apelin-13 treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these upregulated genes (Fig. 6C) revealed enrichment in pathways related to cell proliferation and adhesion, such as the Ras signaling pathway, Rap1 signaling pathway, and cell adhesion molecules, as well as osteogenesis-associated pathways including the Wnt signaling pathway and calcium signaling pathway. Reactome pathway analysis of the upregulated genes (Fig. 6D) further demonstrated that pathways associated with protein glycosylation modifications were most significantly enriched. These findings indicate that Apelin-13 exerts a pronounced positive effect on the proliferation and osteogenic differentiation of BMSCs.

Fig. 6.

Apelin-13 significantly upregulates Ibsp mRNA and protein expression in BMSCs. A Heatmap of all differentially expressed genes; B GO enrichment analysis of all differentially expressed genes; C KEGG enrichment analysis of upregulated differentially expressed genes; D Reactome enrichment analysis of upregulated differentially expressed genes; E Volcano plot of the top 10 upregulated differentially expressed genes; F Western blot analysis of Ibsp and Ocn protein expression, n = 3; G Quantitative analysis of Ibsp and Ocn protein levels. ns: not significant, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Furthermore, we examined the top 10 most highly upregulated genes in the Apelin-13 group (Fig. 6E). Among these genes, Integrin-binding sialoprotein (Ibsp) and Bone gamma-carboxyglutamate protein (Bglap) were found to be closely associated with bone matrix mineralization. The protein encoded by Ibsp, a major structural component of the bone matrix, contains an RGD-binding domain that plays a critical role in guiding hydroxyapatite nucleation and osteoclast fusion. In contrast, the protein encoded by Bglap, also known as osteocalcin (Ocn), is a non-collagenous protein secreted by osteoblasts that regulates bone mineralization and calcium metabolism. To identify the specific target of Apelin-13, total RNA and protein were extracted from hypoxia-cultured BMSCs pretreated with Apelin-13, and the mRNA and protein expression levels of Ibsp and Ocn were examined. QPCR analysis revealed that Apelin-13 significantly upregulated the mRNA expression of both Ibsp and Ocn in BMSCs(Figure S3), which was consistent with the RNA sequencing results. However, subsequent western blot analysis (Fig. 6F, G) showed that while Ibsp protein expression was markedly increased following Apelin-13 treatment, Ocn protein levels remained unchanged. These findings suggest that Apelin-13 exerts its effects primarily through the upregulation of Ibsp.

Maintaining the osteogenic differentiation capacity of BMSCs under hypoxic conditions is essential for bone regeneration following traumatic insults such as fractures. Therefore, we further investigated whether Apelin-13 rescues the impaired osteogenic differentiation capacity of BMSCs under hypoxia through the upregulation of Ibsp. We assessed the expression of osteogenesis-related proteins in BMSCs across different treatment groups using immunofluorescence. The results (Fig. 7A and S4) showed that Apelin-13 pretreatment markedly rescued the inhibitory effect of hypoxia on BMSCs osteogenic capacity. Furthermore, we found that knocking down Ibsp in BMSCs abolished this promotive effect of Apelin-13. Specifically, the expression levels of osteogenesis-related factors in the Apelin-13/BMSCs-si-Ibsp group were significantly lower than those in the Apelin-13/BMSCs and Apelin-13/BMSCs-si-NC groups.

Fig. 7.

Apelin-13 promotes osteogenic differentiation of BMSCs by upregulating Ibsp. A Representative confocal images showing the expression of osteogenic markers Alp, Runx2, and Osterix in BMSCs (scale bar = 50 μm); B Representative gross images (left) and magnified images of the grafts, including bright-field and GFP fluorescence images (right), of GFP-labeled BMSCs transplanted under the renal capsule of nude rats following different treatments; C Representative immunofluorescence images of Runx2 staining in graft sections (scale bar = 100 μm); D Representative digital radiography (DR) images of kidneys containing grafts, with red elliptical regions indicating areas of high-density radiopacity

Next, GFP-labeled BMSCs were mixed with matrigel at a concentration of 1 × 10⁶ cells/mL, and 50 µL of the suspension (5 × 10⁴ cells) was transplanted to the sub-renal capsule of nude mice (Fig. 7B). Four weeks later, high levels of Runx2 expression were observed in the grafts and surrounding renal tissue in the Apelin-13/BMSCs group (Fig. 7C), further supporting the aforementioned findings. Moreover, Digital Radiography (DR) analysis (Fig. 7D) revealed extensive areas of high-density radiopaque signals surrounding the grafts in the Apelin-13/BMSCs group, indicating robust osteogenic activity of BMSCs under the renal capsule and the formation of abundant calcium nodules. Similar high-density radiopaque signals were also observed in the Apelin-13/BMSCs-si-NC group, whereas no such calcification was detected in the kidneys of the control group or the Apelin-13/BMSCs-si-Ibsp group, suggesting insufficient osteogenic capacity of BMSCs in these groups.

In summary, this series of experiments, combining RNA sequencing and molecular biology techniques, identified Ibsp as a downstream target through which Apelin-13 enhances the osteogenic differentiation capacity of BMSCs under hypoxic conditions. Furthermore, subsequent immunofluorescence and renal subcapsular transplantation experiments confirmed that the Apelin-13/Ibsp signaling axis plays a critical role in promoting BMSCs osteogenic differentiation under hypoxia. These findings provide a foundation for further investigation of Apelin-13 in combination with stem cell transplantation for the treatment of bone tissue defects under hypoxic- ischemic conditions in vivo.

The Apelin-13/Ibsp signaling process regulates fracture healing

To further investigate the osteogenic capacity of Apelin-13-pretreated BMSCs under in vivo hypoxic-ischemic conditions, a rat closed femoral fracture model was established. A total of 200 µL of BMSCs suspension (5 × 10⁶ cells/mL) subjected to different pretreatments was slowly injected at multiple sites surrounding the fracture area.

DR and micro-CT analyses (Fig. 8A) demonstrated that at 2 weeks post-surgery, fracture lines remained clearly visible in the control and Apelin-13/BMSCs-si-Ibsp groups, whereas partial callus formation was evident in the Apelin-13/BMSCs and Apelin-13/BMSCs-si-NC groups. Micro-CT 3D reconstructions revealed pronounced defects at the fracture sites in the controland Apelin-13/BMSCs-si-Ibsp groups, with minimal callus formation, while robust callus growth and partial fracture line closure were observed in the Apelin-13/BMSCs and Apelin-13/BMSCs-si-NC groups. By 4 weeks, although limited callus had formed in the control and Apelin-13/BMSCs-si-Ibsp groups, fracture lines were still detectable, suggesting impaired bone regeneration. In contrast, the Apelin-13/BMSCs group showed complete disappearance of the fracture line on DR images, with localized high-density radiopaque areas and abundant callus at both fracture ends on micro-CT reconstructions, indicating active bone repair. The Apelin-13/BMSCs-si-NC group exhibited comparable healing patterns. Parameters such as BV/TV, Conn.D, Tb.Th, and Tb.Sp of the local trabecular structure at the fracture site were analyzed, and the results were consistent with the aforementioned imaging findings(Figure S5).

Fig. 8.

Radiographic and histopathological evaluation of fracture repair at 2 and 4 weeks post-surgery. A Representative DR images and micro-CT 3D reconstructions showing fracture healing; B H&E staining assessing callus morphology and integrity (scale bar = 200 μm); C Masson’s trichrome staining evaluating collagen fiber formation in the fracture region (scale bar = 200 μm)

Histological evaluation revealed marked differences in fracture healing among the groups. At 2 weeks post-surgery (Fig. 8B), extensive infiltration of dark blue inflammatory cells was observed around the fracture lines in the control and Apelin-13/BMSCs-si-Ibsp groups, indicating that the fractures remained in the inflammatory phase. In contrast, the Apelin-13/BMSCs group exhibited the initial formation of thin trabeculae at the fracture site, while the Apelin-13/BMSCs-si-NC group showed evident callus formation bridging both ends of the fracture, suggesting progression into the callus formation and remodeling phase. By 4 weeks post-surgery, fracture healing in the control and Apelin-13/BMSCs-si-Ibsp groups remained limited, with only partial callus formation at the fracture sites. In the Apelin-13/BMSCs and Apelin-13/BMSCs-si-NC groups, newly formed bone at the fracture lines was further mineralized, reflecting robust healing. Specifically, in the Apelin-13/BMSCs-si-NC group, the callus had completed remodeling, forming mineralized trabeculae along the fracture line, whereas the Apelin-13/BMSCs group displayed denser and thicker trabeculae with more organized alignment, characteristic of woven bone.

Masson’s staining was used to assess collagen fibers and new bone formation at the fracture sites. At 2 weeks post-surgery (Fig. 8C), the control and Apelin-13/BMSCs-si-Ibsp groups mainly exhibited abundant myofibers with scattered, disorganized collagen fibers, whereas the Apelin-13/BMSCs-si-NC group showed evident callus formation, and the Apelin-13/BMSCs group displayed orderly aligned mineralized trabeculae. By 4 weeks post-surgery, the proportion of mineralized trabeculae further increased in the Apelin-13/BMSCs and Apelin-13/BMSCs-si-NC groups, resulting in the formation of a considerable amount of woven bone. In contrast, the control and Apelin-13/BMSCs-si-Ibsp groups remained in the callus phase, with abundant chondrocytes within the callus, indicating that mineralization was still incomplete.

Immunohistochemistry and immunofluorescence analyses were performed on samples collected at 4 weeks post-surgery, revealing significant differences among the groups in osteogenic and angiogenic markers as well as cell proliferation. Apelin-13-pretreated BMSCs markedly upregulated osteogenic markers at the fracture sites (Fig. 9A, B, and S6, S7), including ALP, OCN, and Runx2, and also enhanced the expression of the angiogenesis-related marker VEGF-A. Additionally, in vivo cell proliferation was assessed by intraperitoneal injection of BrdU prior to sampling to label S-phase cells undergoing DNA replication. Numerous proliferating cells were observed in the fracture regions of the Apelin-13/BMSCs group. The Apelin-13/BMSCs-si-NC group exhibited similar effects, indicating that siRNA transfection did not significantly impair BMSC function. In contrast, knockdown of Ibsp in BMSCs abolished the therapeutic effect of Apelin-13 pretreatment, demonstrating that the beneficial effects were dependent on Ibsp. Additionally, we examined the expression of type H vessel markers CD31 and Emcn at the fracture site. The results (Figure S8) showed that the Apelin-13/BMSCs group exhibited more pronounced angiogenesis at the fracture site.

Fig. 9.

Evaluation of osteogenic, angiogenic, and cell proliferation-related markers in the fracture region. A Representative immunohistochemical images showing osteogenic markers Alp and Ocn, and BrdU staining indicating cell proliferation (scale bar = 200 μm); B Representative immunofluorescence images of the osteogenic marker Runx2 and the angiogenic marker Vegf in the fracture region (scale bar = 200 μm)

These experiments demonstrate that Apelin-13 pretreatment significantly enhances the resistance of BMSCs to hypoxic-ischemic conditions. Following transplantation into the fracture site, Apelin-13-pretreated BMSCs markedly promoted new bone formation and mineralization, thereby shortening the fracture healing period. This regulatory effect of Apelin-13 on BMSCs is mediated through the upregulation of Ibsp, as knockdown of Ibsp in BMSCs effectively abolished the positive effects of Apelin-13.

Discussion

Bone defects and fracture healing are closely associated with the regenerative and reparative capacity of bone tissue. Stem cell transplantation has been extensively studied for its role in promoting bone regeneration. However, cells cultured in vitro are maintained under high-oxygen conditions (20%), and upon transplantation in vivo, they are suddenly exposed to a low-oxygen environment (1–2%) [30] along with a series of related changes. This abrupt shift creates a harsh microenvironment, making stem cells prone to apoptosis and loss of stemness, thereby limiting the efficacy and further application of stem cell therapy. Our study found that pretreatment with Apelin-13 significantly enhances cell survival and proliferation under hypoxic conditions, better preserves stemness, and exhibits a concentration-dependent effect within a certain range.

Regarding cell apoptosis, previous studies have shown that up to 90% of transplanted cells undergo apoptosis within 6 weeks in vivo [31], which is a major factor limiting the efficacy of stem cell therapy. Similarly, Niknazar et al. [22]reported that Apelin-13 effectively protects cells against hydrogen peroxide–induced apoptosis. A comparable mechanism has also been observed in cardiovascular therapy [32, 33], where Apelin-13 significantly reduces myocardial infarct size by regulating cardiomyocyte metabolism and decreasing apoptosis under ischemic and hypoxic conditions [34, 35]. This mechanism can help explain the protective effect observed in Apelin-13-pretreated cells in our study. We further explored potential molecular regulatory pathways and found that Apelin-13 reduces apoptosis of BMSCs under in vitro hypoxic and serum-free conditions, at least in part, through upregulation of the PI3K/AKT signaling pathway [36, 37]. Collectively, the hypoxic and serum-free in vitro culture conditions in our experiments simulate the microenvironment that transplanted cells encounter at sites of tissue or organ injury in vivo, and the in vitro results reasonably suggest that Apelin-13 exerts its effects by enhancing the adaptive capacity of transplanted stem cells.

After enhancing cell survival and activity, additional strategies are needed to further promote bone regeneration. Considering the critical roles of oxygen and nutrient supply during bone repair [37], we further investigated the contribution of osteogenesis–angiogenesis coupling in bone regeneration [38]. We found that co-transplantation of Apelin-13-pretreated BMSCs with HUVEC exerted a beneficial effect in this context. Blood vessels deliver essential oxygen, nutrients, and growth factors to regenerating tissues, and vascular regeneration is initiated immediately following fracture or bone defect [39]. Previous studies, including those by Miclau et al. [15], have shown that oxygen levels in the fracture site drop significantly in the early stages of fracture, but gradually recover within 24 h, reaching levels comparable to controls by day 3. This suggests that reconstruction of local microcirculation occurs rapidly after tissue injury. Newly formed vessels provide both the initiating signals and necessary nutrients for bone regeneration. Bone tissue repair is a prolonged process, and in regions of bone defects, osteogenesis and angiogenesis are closely coupled both spatially and temporally. Osteogenesis–angiogenesis crosstalk plays a critical synergistic role during bone regeneration. Previous studies have shown that hypoxia-pretreated BMSCs transplanted into mandibular defects in rats exhibit enhanced adaptive capacity and effectively promote bone regeneration [40]. In our experiments, we used Apelin-13 to pretreat the cells, enhancing their resistance to environmental changes. Therefore, Apelin-13-pretreated BMSCs were mixed with HUVEC and transplanted into the mandibular defect region of rats. We observed that rats receiving the combined BMSC–HUVEC transplantation exhibited a significantly greater volume of new bone compared to other control groups, with histological analysis confirming larger areas of newly formed bone tissue. These results indicate that our treatment strategy integrates both osteogenic and angiogenic processes, effectively accelerating the regeneration of critical-sized bone defects. Collectively, our findings provide strong evidence for the beneficial effect of Apelin-13 on mandibular bone regeneration and suggest a novel approach for stem cell pretreatment and transplantation.

Apelin-13, as the endogenous ligand of APJ, requires binding to the APJ receptor to exert its effects, suggesting that the number of APJ receptors on the cell surface may be another factor limiting Apelin-13 activity, This is consistent with our observation that Apelin-13 exhibits dose-dependent effects within a certain concentration range, and aligns with the results reported by Lu et al. in Müller cells [41], and Cai et al. in a cardiac inhibition rat model [42], as well as other in vitro and in vivo studies. In osteogenic and tube formation assays, we also observed another feature of Apelin-13’s effect: a time-dependent cumulative effect. Compared with the control group, the differences in osteogenic or angiogenic capacity between Apelin-13-pretreated cells and untreated cells became increasingly pronounced over time. This demonstrates that by modifying the mode of Apelin-13 application, the duration of its effect on cells can be effectively extended, addressing the limitation of the short half-life of Apelin-13 following direct intravenous injection.

The Apelin-13/APJ complex participates in the regulation of cellular behavior by activating signaling pathways such as PI3K/Akt [42], MAPK/ERK [43], and AMPK [44], playing important roles in cell proliferation and metabolic regulation. RNA sequencing analysis of Apelin-13-pretreated BMSCs indicated that Ibsp is the most likely downstream target. Ibsp is a highly glycosylated and phosphorylated non-collagenous protein containing an RGD sequence, highly expressed in bone and dentin, mediating the adhesion and migration of osteoblasts and endothelial cells [45], and serving as a key initiator of early bone matrix mineralization during new bone formation [46, 47]. Previous studies have demonstrated that Ibsp knockout mice exhibit impaired alveolar bone formation and mineralization, whereas supplementation with exogenous BSP after tooth extraction effectively promotes alveolar socket healing [46, 48]. Our observations are consistent with these findings [49]: Ibsp plays an important role in osteoblast differentiation and bone development and regeneration. In the present study, Apelin-13-pretreated BMSCs maintained high expression of osteogenic markers under hypoxic conditions, This is consistent with the findings of Meng et al. [50], but we further demonstrated that: knockdown of Ibsp significantly abolished the protective effect of Apelin-13. Further subrenal capsule transplantation experiments confirmed that Apelin-13-pretreated BMSCs expressed higher levels of osteogenic markers in vivo and produced more pronounced calcified radiopaque signals, indicating enhanced formation of hydroxyapatite crystals by osteogenic cells transplanted under the renal capsule. Using a closed femoral fracture model, we further validated the functional effects of Apelin-13-pretreated BMSCs at injury sites. Fracture induced by blunt trauma creates a local ischemic and hypoxic microenvironment, where oxygen deprivation, limited blood supply, and elevated inflammatory factors severely compromise the survival of transplanted stem cells. However, Apelin-13-pretreated BMSCs exhibited strong pro-osteogenic activity at the fracture site, significantly accelerating callus formation and mineralization, promoting callus remodeling, and leading to early formation of mature woven bone. In contrast, BMSCs with Ibsp knockdown nearly completely lost this capacity to initiate bone matrix mineralization and accelerate bone turnover.

In summary, we investigated the role of Apelin-13 in enhancing cell survival and activity under hypoxic conditions. In osteogenic experiments, we analyzed the potential and underlying mechanisms of osteogenesis–angiogenesis coupling, and explored the molecular pathways involved. Our findings suggest that Apelin-13 may exert beneficial effects in transplanted cells facing microenvironmental changes, thereby promoting bone regeneration and repair.

This study has several limitations. First, the hypoxic and low-serum culture conditions used in the in vitro experiments cannot fully simulate the local microenvironment at the injury site in vivo. After injury, local tissue acidosis and an inflammatory mediator storm occur, which hinder tissue repair; however, techniques to fully replicate the in vivo microenvironment of injured tissue in vitro are still lacking. Second, although the safety of stem cell transplantation has been widely supported by research, it remains unclear whether cells preconditioned with Apelin-13 may exert adverse effects on systemic tissues and organs.

Conclusion

In this study, we combined Apelin-13 with stem cell transplantation to improve the traditional approach of stem cell therapy. Apelin-13-pretreated stem cells exhibited enhanced proliferation and differentiation in vitro, maintaining biological activity under hypoxic conditions. We further observed that these beneficial effects were positively correlated with Apelin-13 concentration and persisted for a period after pretreatment, even becoming more pronounced over time, indicating a cumulative effect. Using critical-sized mandibular defect and closed femoral fracture models in rats, we validated that Apelin-13-pretreated BMSCs promoted bone defect and fracture healing. Importantly, this promotive effect was mediated through Ibsp. Overall, our study provides a simple, low-side-effect strategy to markedly enhance the efficacy of stem cell transplantation and offers a framework for combining Apelin-13 or other pharmacological agents with stem cell–based therapies.

Author contributions

J. T. and X. Q. helped project design, investigation and manuscript writing. Professor W. J. helped manuscript review and editing. S. W., Z. W. and C. Z. performed project design. M. H., D. F. and S. D. provided data curation.

Funding

This study was supported by the Natural Science Foundation of Sichuan Province (2023NSFSC0564) and National Natural Science Foundation of China (81971319).

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author. RNA-Seq data have been deposited in the NCBI SRA database (PRJNA1378890).

Declarations

Ethics approval and consent to participate

All experimental designs and protocols involving animals were approved by the Ethics Committee of Sichuan University (Title of the approved project: Mechanism of Apelin Activated PI3K/AKT/HIF1-α Pathway in Promoting Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells under Hypoxia. Approval Form ID: WCHSIRB-D-2023-228. Date of IACUC approved: 2023/02/28) and complied with the recommendations of the academy’s animal research guidelines.

Competing interests

The authors declare no competing interests.