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Nano-zinc oxide (nZnO) targets the AMPK-ULK1 pathway to promote bone regeneration

Stem Cell Research & Therapy volume 16, Article number: 206 (2025) Cite this article

Abstract

Background

Nano-zinc oxide (nZnO) has attracted significant attention in bone tissue engineering due to its antibacterial properties, anti-inflammatory effects, biocompatibility, and chemical stability. Although numerous studies have demonstrated the enhancement of osteogenic differentiation by nZnO-modified tissue engineering materials, the underlying mechanisms remain poorly characterized.

Methods

This study aimed to identify the molecular mechanisms how nZnO promoted osteogenic differentiation and bone regeneration using transcriptome analysis, drug intervention, and shRNA knockdown techniques, etc. First, the study evaluated the in vivo effects of gelatin methacryloyl (GelMA) containing nZnO on bone regeneration using a mouse calvarial defect model. The impact of nZnO exposure on the osteogenic differentiation of mesenchymal stem cells (MSCs) was then assessed. The combined treatment of nZnO and MSCs in GelMA for bone regeneration was assessed in the mouse calvarial defect model thereafter.

Results

nZnO induced osteoblastic differentiation to promote bone regeneration. nZnO activated the AMP-dependent protein kinase (AMPK)-ULK1 signals to stimulate autophagosomes formation and facilitate autophagy flow, which was the essential pathway to induce osteogenic differentiation. The combined treatment of MSCs and nZnO significantly enhanced bone regeneration in calvarial defect mice. Conversely, AMPK inhibitor Compound C (C.C) reversed the effects on autophagy flow and osteogenic potentiality induced by nZnO.

Conclusions

These results highlight that nZnO can regulate bone regeneration by activating autophagy through the AMPK/ULK1 signaling pathway, which may provide a novel therapeutic strategy for addressing bone defects using nZnO.

Introduction

The incidence of bone defects has been increasing during the last decades, leading to substantial social and financial burden [1, 2]. Autologous or allogeneic bone transplantation stands as the gold standard treatment for such defects. However, this approach faces limitations due to the scarcity of donor bone and the potential risks of immune system infection and rejection [3]. Bone tissue engineering strategies, employing scaffolds, bioactive molecules, and stem cells, have gathering considerable attention as alternative approaches to promote bone regeneration [4].

Nanomaterials have emerged as highly promising candidates in bone tissue engineering, owing to their unique chemical and physical properties [5]. Nano-zinc oxide (nZnO) is one promising antibacterial inorganic material due to its antibacterial, anti-inflammatory, biocompatible, and chemically stable properties [6,7,8,9,10,11]. Furthermore, nZnO had also been considered as a good tissue engineering material with its anti-cancer, drug delivery and biological imaging activities [12]. It has been found that the incorporation of nZnO into scaffolds enhanced the attachment, diffusion, proliferation, and permeation of cells [13], with good biocompatibility, bone conductivity, and antibacterial properties [14]. However, the mechanism through which nZnO accelerates bone growth and mineralization remains unknown.

Autophagy is shown to play a crucial role of in maintaining bone homeostasis [15,16,17,18]. It serves as an intracellular degradation pathway, facilitating the breakdown and recycling of cytoplasmic components, thereby playing an essential role in homeostasis and response to nutritional deficiencies [19]. The AMP-dependent protein kinase (AMPK)-ULK1 signaling pathway is pivotal in regulating autophagy to manage various cellular stresses [20], with AMPK promoting autophagy by enhancing ULK1 kinase activity [21, 22]. Furthermore, negative pressure therapy could activate autophagy to induce osteogenesis through the AMPK-ULK1 signaling pathway [23]. Whether and how nZnO links autophagy in osteoblasts have not been ever investigated.

This study aims to investigate the impact of nZnO on the osteogenic differentiation and to evaluate the effect of nZnO on autophagy activation, identifying the underlying pathway.

Materials and methods

Cell culture and treatment

Mouse mesenchymal stem cells (MSCs) were procured from the Cyagen Biosciences Inc. (Guangzhou, China). These cells were cultured in α-MEM medium (Gibco, 12571063) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained in a 5% CO2 incubator at 37 °C and subcultured upon reaching 80–90% confluence. Osteogenic differentiation was induced by replacing the medium with cell osteogenic differentiation medium (Cyagen, MUXMT-90021), with medium changes performed every three days.

Nanometer zinc oxide particles (nZnO, 677450) and chloroquine diphosphate salt (CQ; C6628) were procured from Sigma Aldrich (USA). The AMPK inhibitor Compound C (C.C, HY-13418 A), AMPK agonist AICAR (Aic, HY-13417), ULK1 inhibitor SBI-0206965 (SBI, HY-16966), ULK1 agonist BL-918 (BL, HY-124729), and TRP channel blocker SKF-96,365 hydrochloride (SKF, HY-100001) were obtained from MedChemExpress (USA). During the nZnO treatment, the complete medium was replaced with induction medium containing different concentrations of nZnO (0, 1, 5 µg/mL). In the rescue experiment, CQ (10 µM), C.C. (1 µM), Aic (10 µM), SBI (1 µM), BL (10 µM), or SKF (2 µM) was added to the culture medium one hour prior to the nZnO treatment. Each experiment was conducted independently at least three times.

Cell viability

Cell viability was evaluated using the cell counting kit-8 (CCK-8) (Vazyme, A311) across various concentrations of nZnO (0, 0.01, 0.1, 0.5, 1, 5, 10, 50, and 100 µg/mL). Briefly, MSCs (2 × 10^4) were seeded in a 96-well plate with six replicates per treatment condition. After three days of treatment, the medium was replaced with fresh medium containing 10% CCK-8 reagent and incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using the Infinite M200 microplate reader (Tecan, Switzerland). Each experiment was independently replicated at least three times.

Alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining

ALP staining and ARS staining were performed on the 7th and 14th days post cell differentiation induction, respectively. Cells were rinsed twice with PBS, fixed with 4% paraformaldehyde, and then subjected to BCIP/NBT staining solution (Beyotime, C3206) or Alizarin Red S staining solution (Beyotime, C0148S) to ensure complete coverage of the samples. Incubation in the dark at room temperature facilitated color development. Following this, the staining solution was aspirated, and the samples were rinsed thrice with PBS to halt the color reaction. Observations and photomicrographs were captured using an inverted microscope (Nikon, Japan). ImageJ software was utilized for analyzing the ARS staining area. Each experiment was conducted independently with a minimum of three repetitions.

ALP activity assay

ALP activity was evaluated on the 7th day post cell differentiation induction using the alkaline phosphatase activity assay kit (Beyotime, P0321S). Following two washes with PBS, cell lysis was carried out using the provided lysis buffer. Subsequently, the substrate solution was added as per the manufacturer’s instructions. Absorbance at 405 nm was measured using a 96-well plate reader, specifically the Infinite M200 ELISA (Tecan, Switzerland). ALP activity was normalized to protein concentration, with three replicates for each assay.

Cell transfection

The lentiviruses targeting AMPK and ULK1 knockdown were obtained from GeneChem Biotechnology Co., Ltd., with the sequences listed in supplementary materials 1: Table S1. Empty lentiviral vectors served as the negative control (NC). Virus volume was calculated using the formula “Virus Volume = (MOI × Cell Number) / Virus Titer,” diluted in cell culture medium, and supplemented with HitransG A to enhance infection efficiency. After PBS washes, cells were incubated with the virus-containing medium for 24 h, followed by replacement with complete medium. At 90% cell density, selection was initiated using puromycin-containing medium (2 µg/mL).

Autophagy flux detection

The adenovirus expressing Ad-mCherry-GFP-LC3B was procured from Shanghai Beyotime Biotechnology Co., Ltd. It was transfected into MSCs, and after 8 h, the medium was replaced with induction medium, with or without nZnO. After 72 h, cells were rinsed twice with PBS, fixed in 4% paraformaldehyde (Servicebio, G1101) for 30 min at room temperature, and stained with DAPI for 5 min to label the nuclei. Imaging was conducted using a Carl Zeiss AG (Germany) LSM700 laser confocal scanning microscope. Zeiss ZEN software was utilized for image processing. Each experiment was performed with triplicate samples.

Calcium ions (Ca2+) measurement

MSCs were cultured in induction medium with or without nZnO for 72 h. After triple washing with PBS, the cells were treated with 2 µM Fluo-4 AM (Beyotime, S1060) and then incubated at 37 °C for 1 h. Subsequently, intracellular Ca2+ signals were captured using a Carl Zeiss AG (Germany) LSM700 laser confocal scanning microscope. The acquired images were processed using Zeiss ZEN software.

Western blot

Protein extraction and immunoblotting experiments were conducted on the third day following cell differentiation induction. Initially, cells were washed twice with PBS and then lysed with RIPA lysis buffer (Beyotime, P0013C) for 30 min. Protein concentration was determined using the BCA protein concentration assay kit (Beyotime, P0012). SDS-PAGE was utilized for protein separation, followed by transfer onto polyvinylidene fluoride membranes. Immune complexes for proteins such as Osteopontin (OPN), Runt-relaxed transcription factor 2 (RUNX2), SP7, Atg3, Atg5, Atg7, Beclin1, P62, LC3I/II, AMPK, p-Thr172-AMPK, ULK1, p-Ser555-ULK1, Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2), and β-actin were probed using Cell Signaling Technology antibodies at a 1:1000 dilution. Image acquisition was performed using Tanon (China) equipment, and quantification was carried out using ImageProPlus 6.0 software. Each experiment was independently replicated at least three times.

HPLC detection of ATP, ADP, and AMP

Adenosine triphosphate (ATP, S5260), adenosine diphosphate (ADP, S9368), and adenosine monophosphate (AMP, S9366) were procured from Selleck (USA). For calibration, 10 mg of ATP, ADP, and AMP were dissolved in ddH2O to yield a standard stock solution with a concentration of 1 mg/mL. Subsequently, this solution was diluted to various concentrations to establish a standard curve. MSCs, treated with either a control or nZnO for 3 days, were harvested and homogenized (1 × 10^7 cells per sample) using ultrasound. The resulting cell extract underwent centrifugation at 15,000 r/min at 4 °C for 30 min, and the supernatant was filtered through a 0.22 μm filter for detection. HPLC analysis was conducted using an Agilent 1260 Infinity II system equipped with an Ultimate Plus C18 column (Welch, 00208-31043). Each analysis comprised three duplicate samples.

RNA-sequencing

On the third day of inducing osteogenic differentiation, both the control group and the group treated with nZnO (5 µg/mL) prepared mRNA sequencing libraries according to the manufacturer’s instructions. Total RNA was extracted from each cell sample using TRIzol reagent (Invitrogen, 15596018), and its quality was evaluated using gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Enrichment, fragmentation, reverse transcription, library construction, HiSeq X Ten sequencing, and mRNA data analysis were performed by Hangzhou Lianchuan Biotechnology Co., Ltd. (Hangzhou, China). The raw data were converted to Fastq format, and the number of transcripts per sample was calculated using fragments per thousand base transcripts (FPKM) per million mapping fragments. Subsequently, FPKM values were transformed to log2. Gene set enrichment analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), was conducted using differentially expressed genes (DEGs) in Hiplot Pro (https://hiplot.com.cn/). GO analysis was visually represented using a GO enrichment circle plot.

Preparation of hydrogel and cell encapsulation

GelMA (EFL-GM-60) and Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP, EFL-LAP) were procured from Engineering for Life (Suzhou, China). To prepare a 0.25% LAP standard solution, 50 mg of LAP powder was dissolved in 20 mL of PBS and heated in a water bath at 40–50 °C. The solution was then stored in the dark at 4 °C. Subsequently, 1 g of GelMA freeze-drying was dissolved in 20 mL of the LAP standard solution, heated in a water bath at 40–50 °C away from light for 20 min, and sterilized using a 0.22 μm sterile needle filter upon complete dissolution. This process yielded a 5% GelMA solution stored in the dark at -20 °C.

To encapsulate MSCs in the hydrogel, a cell suspension with a concentration of 1 × 10^6 cells/mL was added to the 5% GelMA prepolymer solution preheated at 37 °C. This step was performed with or without nZnO particles and with or without the AMPK inhibitor C.C. To prevent low-temperature gelation, the prepared hydrogel mixture was placed in a 37 °C water bath for subsequent experiments.

Mouse calvarial defect model

Male C57BL/6J mice at 8 weeks of age were used for the experiment. After anesthesia with 3% w/v pentobarbital sodium, circular defects with a diameter of approximately 4 mm were created on both sides of the calvarial, with a distance of 1 mm from the middle of the sagittal suture. To assess the effect of nZnO on calvarial defects, the left side was filled with a mixture of GelMA and MSCs (5 × 10^4 cells/gel) as a control, while GelMA, MSCs, and nZnO (5 µg/mL) were used on the right side. To evaluate the combined transplantation of AMPK inhibitor C.C. with nZnO, each defect was filled with GelMA embedded with nZnO and/or AMPK inhibitor. Mice were euthanized with excessive pentobarbital sodium at the 4th and 8th weeks after surgery, and skull tissue samples were collected for subsequent experiments. All animal procedures were conducted in accordance with the approved protocol by the Animal Protection and Use Committee of Nanjing Medical University (IACUC-2209047). The animal experiment was conducted in accordance with the ARRIVE guidelines 2.0.

Determination of particle size and zeta potential of nZnO

nZnO suspensions were sonicated and then analyzed using the Zetasizer Nano ZS (Malvern Panalytical Ltd., Malvern, UK). Dynamic light scattering (DLS) was employed to determine the size of nanoparticles in water, while their Zeta potential was assessed using electrophoretic light scattering (ELS).

Transmission electron microscopy (TEM)

MSCs treated with either the control or nZnO were fixed at 4 °C for 4 h using a 2.5% glutaraldehyde solution in 0.1 M sodium dihydrogen phosphate (Servicebio, G1102). Subsequently, the cells were exposed to 1% osmium tetroxide at room temperature for 1 h. Dehydration was carried out using a graded ethanol solution, followed by gradual infiltration with EMbed 812 epoxy resin (Ted Pella, 14120). Ultrathin Sects. (60–80 nm) were prepared using an ultra-fine slicing machine (LEICA, Germany) and stained with uranyl acetate and lead citrate. Autophagosomes were visualized using JEM-1010 transmission electron microscopy (JEOL, Japan). Each test included three replicate samples.

Scanning electron microscopy (SEM)

A solution containing nZnO at a concentration of 50 µg/mL was sonicated for 30 min and then deposited onto a silicon wafer. Following drying, the morphology of the nZnO particles was observed using SEM (JEOL, JSM-7900 F, Japan). GelMA prepolymer solution, with or without nZnO particles, was dispensed into a 2 mL centrifuge tube (1 mL solution), followed by curing with a 405 nm light source for 10 to 30 s. Subsequently, the samples were frozen for 5 h at -20 ℃, 2 h at -80 ℃, and then thoroughly dried using a vacuum freeze dryer (Telstar, Spain). The microstructure of the hydrogel was examined via scanning electron microscopy after sectioning. Three replicate samples were utilized for each test.

Micro-CT imaging and analysis

After euthanizing the mice with CO2, the calvariae were dissected from the skin and assessed using the SkyScan 1176 high-resolution micro-CT imaging system (Bruker, USA). Each calvaria was scanned at a resolution of 18 μm. The obtained images were reconstructed using NRecon software (Bruker, USA) and subjected to quantitative analysis utilizing CTAn software (Bruker, USA). Analysis was conducted based on the entire initial circular defect.

Histopathological section

Following micro-CT scanning evaluation, the calvariae were fixed in 4% paraformaldehyde (Servicebio, G1101) for 48 h. Subsequent to decalcification of the bone tissue, paraffin sections with a thickness of 8 microns were prepared. HE staining was performed using the Beyotime HE staining kit (C0105S), and Safranin O-solid green cartilage staining solution trichrome staining was conducted using the Nanjing Senbeijia Modified red O-solid green cartilage staining solution (BP-DL421). Images were captured utilizing a pathological section scanner (3DHISTECH, Hungary), followed by subsequent analysis. ImageJ software was utilized for analyzing the new bone area within the defect tissue.

Statistical analysis

Data were presented as mean ± SEM. The normality of the data was assessed using the Shapiro-Wilk method. For normally distributed data, differences between two or more groups were analyzed using unpaired bilateral Student’s t-tests, analysis of variance (ANOVA), and Dunnett’s multiple comparison test. Statistical significance was determined using SPSS 22.0, with a significance level of P < 0.05 indicating statistical significance.

Results

nZnO promoted bone regeneration in calvarial defects mice

The morphology and size of nZnO particles exhibited a short rod-shaped structure with an average size of 92.24 ± 25.96 nm (Fig. S1A and B). The average surface potential of nZnO particles in water was measured at -16.3 ± 3.21 mV. The electron microscopy image of GelMA hydrogel (Fig. S1C) illustrated an interconnected porous network in the nanocomposite hydrogel, with an average pore diameter of 300 μm. Notably, nZnO particles were uniformly dispersed within the hydrogel network, contributing to the thickening of the porous network structure.

To assess the bone regeneration potential of nZnO, we employed a mouse calvarial defect model. Circular defects, approximately 4 mm in diameter, were created on both sides of the skull, 1 mm away from the midline sagittal suture. These defects were filled with hydrogels with/without nZnO and crosslinked under a 405 nm light source (Fig. 1A). At 4th and 8th week post-surgery, Micro-CT scanning and 3D reconstruction were performed to evaluate bone regeneration levels (Fig. 1B). Quantitative analysis revealed that GelMA treatment combined with nZnO significantly increased the bone volume per unit tissue volume (BV/TV) and bone mineral density (BMD) compared to the control group. After eight weeks of implantation, the GelMA-nZnO group exhibited a BV/TV of 13.92 ± 2.08%, which was 1.6 times higher than the control group’s BV/TV of 8.50 ± 1.67%. Furthermore, the GelMA-nZnO group demonstrated a significant increase in BMD to 201.05 ± 29.68 mg/cm3, compared to the control group’s BMD of 137.65 ± 11.13 mg/cm3, indicating its robust bone repair capability (Fig. 1C).

Fig. 1
figure 1

nZnO promotes bone regeneration in mouse calvarial defects. (A) Schematic illustration of in vivo experiment design. (B) Micro-CT 3D reconstruction of craniums in the control and nZnO groups 4 and 8 weeks after surgery. Scale bars: 2 mm, Magnified Scale bars: 1 mm. (C) Quantitative analysis of micro-CT imaging of defect regions (n = 6 in each group). (D) Histological staining of craniums from the control and nZnO groups, including H&E and Safranin O-Solid Green staining. Scale bars: 1 mm, Magnified Scale bars: 500 μm. (E) Quantitative analysis of the new bone area within the defect tissue in D) histology images (n = 6 in each group). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control

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HE staining and Safranin O-Solid Green staining were performed on calvarial slices to observe the microscopic structure of the regenerated bone (Fig. 1D and E). The control group exhibited a small amount of loose fibrous tissue filling the defect site, with scarcely presence of newly regenerated bone at 4th and 8th week post-surgery. In contrast, the GelMA-nZnO group displayed enhanced new bone formation along the edge of the bone defect. These results indicated that the GelMA-nZnO composite hydrogel promoted greater new bone structures within the defect area compared to the control group. Consequently, the inclusion of nZnO in GelMA significantly improves bone regeneration and stimulates osteogenesis in mice.

nZnO promoted MSCs differentiating into osteoblasts

MSCs viability were not affected at low dose of nZnO (0.01 to 10 µg/mL) (Fig. S2). Therefore, subsequent experiments were conducted using nZnO at concentrations of 1 µg/mL and 5 µg/mL. Treatment with 5 µg/mL nZnO promoted the osteogenic differentiation of MSCs as demonstrated by ALP and ARS staining. The ALP activity in the 5 µg/mL nZnO treatment group was 1.2-fold higher than that in the control group. Moreover, the area of mineralized nodules significantly increased in the 5 µg/mL nZnO treatment group (30.05 ± 2.04%) compared to the control group (18.55 ± 2.31%) (Fig. 2A and B). Significant increases of osteogenic differentiation-related proteins, RUNX2 and OPN, following treatment with 5 µg/mL nZnO also demonstrated nZnO treatment promoted MSCs differentiating into osteoblasts (Fig. 2C and D).

Fig. 2
figure 2

nZnO promotes differentiation of MSCs into osteoblasts. (A) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days under different concentrations of nZnO treatment, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (B) ALP activity assay of MSCs after osteogenic differentiation for 7 days under different concentrations of nZnO treatment (n = 3), and quantitative analysis of the Alizarin Red staining in A (n = 6). (C) Western blot analysis of osteogenic proteins (RUNX2 and OPN) in MSCs under different concentrations of nZnO after osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig. S5. (D) Relative protein expression determined in C (n = 3). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control

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nZnO induced autophagy during osteogenic differentiation

To elucidate the molecular events activated during osteogenic differentiation induced by nZnO, transcriptomic analysis was performed in MSCs after exposed to osteogenic differentiation induction medium (with / without 5 µg/mL of nZnO) for 3 days. Among the 570 differentially expressed genes, 194 genes were up-regulated, while 376 genes were down-regulated (using a statistical threshold of p < 0.05 and fold change > 1.5, Fig. 3A and Supplementary materials 3). GO enrichment analysis of these differentially expressed genes indicated that nZnO treatment affected various biological processes, including cell differentiation, protein phosphorylation, bone mineralization, and osteogenic differentiation (Fig. 3B). Furthermore, KEGG enrichment analysis revealed a significant enrichment of the “phagosome” pathway (Fig. 3C). This was in line with previous findings showing the critical role of autophagy in bone homeostasis [15, 16, 18]. Transmission electron microscopy analysis confirmed an increased number of autophagosomes in nZnO-treated cells compared to the control group (Fig. 3D). Fluorescence results indicated an increased number of red spots (represent autolysosome) in MSCs treated with nZnO, providing further evidence of autophagy activation in response to nZnO treatment during osteogenic differentiation (Fig. 3E). Additionally, dose-dependent up-regulation of autophagy markers (Atg3, Atg5, Atg7, Beclin1, LC3-I/II) upon nZnO treatment were observed, along with a decrease of P62, especially at 5 µg/mL of nZnO (Fig. 3F).

Fig. 3
figure 3

nZnO induces autophagy during osteogenic differentiation of MSCs. (A) Volcano map of the RNA-seq data of MSCs in the control versus nZnO (5 µg/mL) groups after osteogenic differentiation for 72 h calculated by DESeq2 (n = 3 in each). (B) GOCircle plot of GO enrichment analysis of the DEGs. (C) KEGG enrichment analysis of DEGs. (D) Transmission electron microscopy of MSCs under control and nZnO treatment for 72 h. Arrows indicate autophagosomes; Scale bars: 1 μm, Magnified Scale bars: 500 nm. (E) Autophagic flux detection for MSCs transfected with Ad-mCherry-GFP-LC3B under control and nZnO treatment; Scale bars: 40 μm. (F) Western blot analysis of autophagic activation in MSCs under different concentrations of nZnO treatment at osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig.S6. (G) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with autophagy inhibitors chloroquine (CQ, 10 µM) under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (H) ALP activity assay of MSCs cells after osteogenic differentiation for 7 days treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions (n = 6), and quantitative analysis of the Alizarin Red staining in G (n = 6). (I) Western blot analysis of autophagic activation in MSCs cells after osteogenic differentiation for 72 h treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S7. (J) Autophagic flux detection for MSCs transfected with Ad-mCherry-GFP-LC3B treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions; Scale bars: 40 μm. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control

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Then, we examined the protein level of LC3-II, an indicator of autophagy activation, in the presence and absence of autophagy inhibitors. ALP and ARS staining demonstrated that pretreatment with CQ (a reagent that inhibits autophagy by damaging the fusion of autophagosomes and lysosomes) inhibited the osteogenic differentiation effect of nZnO-treated cells, resulting in substantial reduced mineralized nodules and ALP activity (Fig. 3G and H). LC3-II protein was elevated by CQ in MSCs treated with nZnO (Fig. 3I). Fluorescence results revealed an increased number of endogenous yellow spots (represent autophagosome) in MSCs treated with nZnO (Fig. 3J). These findings indicated activation of autophagy in MSCs during differentiation by nZnO.

nZnO activated AMPK-ULK1 autophagy axis to induce osteogenic differentiation

Genome-wide enrichment analysis (GSEA) revealed significant enrichment of differentially expressed genes in the AMPK signaling pathway in cells treated with nZnO (Fig. 4A). AMPK is a regulator to initiate autophagy process through phosphorylation of ULK1 [22,23,24,25]. The phosphorylated AMPK(Thr172) and ULK1(Ser555) proteins significantly increased in cells treated with nZnO compared to the control group (Fig. 4B and C). To ascertain the role of AMPK-ULK1 on autophagic axis activation in nZnO-induced osteogenic differentiation, we pre-treated MSCs with the AMPK inhibitor C.C or the agonist Aic, followed by nZnO treatment. nZnO effectively AMPK(Thr172) phosphorylation, but was abolished by C.C. The AMPK agonist Aic significantly stimulated AMPK phosphorylation, resulting in increased expression of osteogenic markers (RUNX2) and autophagy-related proteins (LC3-I/II) (Fig. 4D). Furthermore, the AMPK inhibitor C.C hindered the osteogenic effects of nZnO, while the AMPK agonist Aic promoted osteogenesis and further enhanced the osteogenic effects induced by nZnO (Fig. 4E and F).

Fig. 4
figure 4

AMPK-ULK1 signaling pathway participates in nZnO induced autophagy and osteogenic differentiation. (A) GSEA analysis demonstrated significant enrichment of AMPK-regulated genes in the nZnO (5 µg/mL) versus control group after osteogenic differentiation for 72 h. (B) Western blot analysis of AMPK and ULK1 activation in MSCs under different concentrations of nZnO for after osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig.S8. (C) Relative protein expression determined in B (n = 3). (D) Western blot analysis of AMPK-ULK1 and autophagic activation in MSCs after osteogenic differentiation for 72 h treated with AMPK inhibitor compound C (C.C, 1 µM) or agonist AICAR (Aic, 10 µM) under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S9. (E) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with AMPK inhibitor C.C or agonist Aic under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (F) ALP activity assay of MSCs after osteogenic differentiation for 7 days treated with AMPK inhibitor C.C or agonist Aic under control and nZnO (5 µg/mL) conditions (n = 3), and quantitative analysis of the Alizarin Red staining in D (n = 6). (G) Western blot analysis of AMPK-ULK1 and autophagic activation in MSCs after osteogenic differentiation for 72 h treated with ULK1 inhibitor SBI-0206965 (SBI, 1 µM) or agonist BL-918 (BL, 10 µM) under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S10. (H) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with ULK1 inhibitor SBI or agonist BL under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (I) ALP activity assay of MSCs after osteogenic differentiation for 7 days treated with ULK1 inhibitor SBI or agonist BL under control and nZnO (5 µg/mL) conditions (n = 3), and quantitative analysis of the Alizarin Red staining in G (n = 6). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control

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To evaluate the dependence of phosphorylation of ULK1 to promote osteogenic effects by nZnO on AMPK activation, we pre-treated MSCs with the ULK1 inhibitor SBI or the agonist BL, followed by nZnO treatment. Induction of ULK1(Ser555) phosphorylation levels by nZnO was not affected by SBI or BL. SBI inhibited the formation of ULK1 (Ser555) phosphorylation and the levels of LC3-II induced by nZnO treatment. In contrast, BL elevated levels of ULK1 phosphorylation and LC3-II production to enhancing the autophagy-promoting effects of nZnO treatment (Fig. 4G). Moreover, SBI inhibited the osteogenic effects of nZnO, while BL further enhanced the osteogenic differentiation induced by nZnO treatment (Fig. 4H and I).

Next, we used knockdown AMPK or ULK1 to further observe the influence on AMPK-ULK1 autophagic axis in nZnO-induced osteogenic differentiation (Fig. S3A and E). AMPK knockdown abolished the enhancement of osteogenic differentiation by nZnO in accompanied by reduced expression of osteogenic-related proteins (RUNX2, SP7, and OPN), decreased calcium deposition, and lower ALP activity (Fig. S3B, C, and D). Similar result was found by ULK1 knockdown (Fig. S3F, G, and H). Furthermore, the phosphorylation levels of ULK1 were reduced by AMPK knockdown (Fig. S3A and E). These findings suggest that AMPK participated in nZnO-induced autophagy and osteogenic differentiation through the activation of ULK1.

nZnO induced osteogenic differentiation was dependent on both Ca2+ and AMP signaling pathways

AMPK signaling pathway can be activated through two distinct pathways: the Ca2+-dependent pathway mediated by CaMKK2 and the AMP-dependent pathway [26, 27]. Following exposure to varying doses of nZnO for 72 h, Ca2+ concentrations increased in a dose-dependent manner (Fig. 5A). When MSCs were stimulated with external Ca2+ (1 mM), nZnO treatment facilitated the release of stored Ca2+ (Fig. 5B). Quantitative analysis of CaMKK2 protein expression revealed that nZnO elevated CaMKK2 protein levels by 2.13-fold (control group: 1.00 ± 0.14, nZnO 5 µg/mL group: 2.13 ± 0.20, P < 0.05, Fig. 5C and D). Increase in nZnO-mediated Ca2+ currents may be associated with TRPC calcium channels [28]. To investigate the role of Ca2+ in the activation of the AMPK signaling pathway by nZnO, cells were treated with the non-selective TRPC inhibitor SKF. SKF treatment decreased cellular CaMKK2 protein expression, and AMPK activation and osteogenic differentiation by nZnO was reserved (Fig. 5E and F).

Fig. 5
figure 5

nZnO induces osteogenic differentiation through Ca2+-dependent pathway. (A) The fluorescence images of Fluo4-AM and DAPI labeled MSCs at different concentrations of nZnO treatment. Scale bars: 40 μm. (B) Representative traces show the transient increase in Ca2+ after treatment with 1 mM Ca2+ to MSCs under different concentrations of nZnO treatment. (C) Western blot analysis of CaMKK2 in MSCs under different concentrations of nZnO treatment after osteogenic differentiation after osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig.S11. (D) Relative protein expression determined in C (n = 3). (E) Western blot analysis of CaMKK2, AMPK activation and RUNX2 in MSCs after osteogenic differentiation for 72 h treated with TRP channel blocker SKF-96,365 hydrochloride (SKF, 2 µM) under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S12. (F) Relative protein expression determined in E (n = 3). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control. #P < 0.05, ##P < 0.01, ###P < 0.001 relative to nZnO

Full size image

AMPK, as a crucial energy sensor, its activity is dependent on the ATP: AMP or ATP: ADP ratio [24]. Significant increase in the AMP: ATP ratio was found in MSCs treated with nZnO compared to the control group (Control: 1.00 ± 0.04; nZnO: 1.54 ± 0.02; P values < 0.001) (Fig. S4 A and B).

Activation of AMPK-ULK1 autophagy axis in mice treated with nZnO

MSCs possess the capacity to differentiate into osteoblasts, actively contributing to the formation and repair of new bone tissue [29]. To explore the translational application of our research findings, we validated the involvement of the AMPK-ULK1 autophagy axis in nZnO-induced osteogenic differentiation in vivo using a mouse calvarial defect model with nZnO alone or combined with MSCs transplantation. After 8-week treatment, enhanced bone regeneration at the defect site was observed using micro-CT scanning in both nZnO alone treatment and nZnO-MSCs combined treatment groups compared to the control group (GelMA only). Notably, the combined therapy of nZnO-MSC showed superior therapeutic effect. Using AMPK inhibitor C.C, the effect could partially be attenuated (Fig. 6A and B). Quantitative analysis indicated that the nZnO-MSCs group achieved the highest BV/TV and BMD values (Fig. 6B). After 8 weeks treatment, the nZnO-MSCs group (20.64 ± 3.08%) exhibited a significantly higher percentage of bone volume per unit tissue volume compared to the MSCs alone group (14.86 ± 1.92%) (P < 0.001). Additionally, the BMD of the nZnO-MSCs group (183.72 ± 8.43 mg/cm3) significantly surpassed that of the MSCs alone group (126.20 ± 9.06 mg/cm3) (P < 0.001). These results underscore the robust bone repair capacity of the nZnO-MSCs combination therapy.

Fig. 6
figure 6

nZnO combined with MSCs transplantation promotes the regeneration of skull defects in vivo. (A) Micro-CT 3D reconstruction of craniums treated by GelMA, GelMA + MSCs, and GelMA + MSCs + C.C (compound C) with or without nZnO (5 µg/mL) 8 weeks after surgery; Doted circles indicate the initial defect regions. Scale bars: 2 mm, Magnified Scale bars:1 mm. (B) Quantitative analysis of micro-CT imaging of defect regions in A (n = 6). (C) Histological analysis of craniums from each group, including H&E and Safranin O-Solid Green staining. Scale bars: 1 mm, Magnified Scale bars: 500 μm. (D) Quantitative analysis of the new bone area within the defect tissue in C) histology images (n = 6 in each group). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control

Full size image

HE and Safranin O-Solid Green staining revealed increased new bone tissue formation within the defect sites in the nZnO-MSCs group (Fig. 6C and D). Consistent with the cellular outcomes, the enhanced osteogenic ability and autophagy induced by nZnO were partially counteracted by the AMPK inhibitor C.C (Fig. 6A-D). Collectively, these findings suggest that nZnO promotes osteogenic differentiation through the AMPK-ULK1 autophagy axis in vivo, thereby enhancing bone regeneration. The combined MSCs transplantation with nZnO added values to more effective treatment of bone defects.

Discussion

Bone defects are commonly present in orthopedics, caused by trauma, osteoporosis, and other bone diseases [30]. With the advancement of nanotechnology, nZnO has become a promising tool in bone tissue engineering, regulating osteogenic differentiation and mineralization [31]. This study explores the potential of nZnO in promoting bone defect healing and elucidates its underlying mechanisms. Our experiments indicate that nZnO initiates autophagy by activating the AMPK-ULK1 signaling pathway, thereby promoting bone regeneration. The activation of the AMPK signaling pathway by nZnO is due to an increase in intracellular Ca2+ levels and AMP: ATP ratio (Fig. 7). In addition, the combined treatment of nZnO and MSCs transplantation in mouse skull defects significantly improved bone regeneration.

Fig. 7
figure 7

A working model of AMPK-ULK1 activating autophagy in nZnO induced bone regeneration. nZnO stimulated the phosphorylation and activation of the AMPK-ULK1 signaling pathway through Ca2+ and AMP-dependent pathways, leading to increased expression of autophagy-related genes. The treatment with nZnO activates autophagic flux, promoting the transcription of osteogenic differentiation genes such as RUNX2 and OPN, promoting bone regeneration

Full size image

In line with our findings, previous studies have indicated that tissue engineering materials modified with nZnO possess the capacity to enhance osteogenic differentiation. It has been found that that nZnO-modified carbon fiber scaffolds can enhance the osteogenic differentiation of mesenchymal stem cells and possess certain anti-infective capabilities. This enhancement was attributed to the physical properties (nano morphology) and chemical properties (gradual release of Zn2+) of the material [31]. Compared with unmodified MEW-PCL, MEB-PCL modified with ZnO nanoparticles exhibits better osteogenic properties. This modification stimulates osteogenic differentiation, bone formation, and mineralization of MC3T3 osteoblasts [8]. Additionally, studies have shown that nZnO-loaded MTC-Ti implants promote cell adhesion, proliferation, and osteogenic activity of BMSCs, leading to remarkable improvements in bone integration and inhibition of bacterial infection [9]. However, the precise mechanism underlying this process remains elusive. Autophagy plays a critical role in bone tissue metabolism, governing cell viability and function, and contributing to bone tissue equilibrium [32,33,34]. Our investigation unveiled that nZnO activates the AMPK-ULK1 signaling pathway, inducing autophagy in osteoblasts and fostering the differentiation of MSCs into osteoblasts. This may be related to the autophagosomes facilitate the transportation of mineral crystals to promote mineralization [15, 35]. It may also be related to the MSCs utilize accumulated autophagosomes to acquire energy during osteogenic differentiation [36].

Although nZnO exhibits significant potential in the treatment of bone defects, its clinical application still faces numerous challenges. Firstly, high concentrations of nZnO may induce cytotoxicity through oxidative stress and mitochondrial damage, leading to cell apoptosis or necrosis [12, 37, 38]. Secondly, the size and morphology of nZnO significantly influence its bioactivity and toxicity; however, current fabrication processes struggle to precisely control these parameters, necessitating optimization of production techniques to achieve scalable manufacturing [39]. Additionally, nZnO tends to aggregate in physiological environments, compromising its dispersibility and bioactivity, which highlights the urgent need to develop surface modification technologies to enhance its stability [40]. In summary, through multidisciplinary collaboration and technological innovation, overcoming challenges related to toxicity, fabrication processes, stability, and the lack of long-term data, nZnO holds promise for playing a critical role in the field of bone repair.

Autophagy regulates cellular energy homeostasis, with the AMPK-ULK1 axis being a key mediator [20, 41, 42]. However, its role in promoting osteogenesis and bone regeneration is less explored. Our research indicates that nZnO treatment increases intracellular Ca2+ concentration, potentially activating AMPK through CaMKK2 [27, 43]. Consistent with our research findings, studies have found that nZnO enhances autophagy by affecting intracellular Ca2+ flow [28]. Moreover, nZnO treatment increases the cell AMP: ATP ratio, potentially activating AMPK and influencing MSCs energy metabolism.

To investigate the therapeutic potential of nZnO in addressing bone defects, we employed a mouse skull defect model in conjunction with MSCs and hydrogel. MSCs represent a subtype of multipotent adult stem cells characterized by their capacity for self-renewal and differentiation into diverse cell types [29]. The transplantation of stem cells assumes a pivotal role in enhancing bone regeneration, primarily through the secretion of paracrine factors aimed at establishing a conducive bone microenvironment at the site of injury [44,45,46]. We used a mouse calvarial defect model with nZnO alone or combined with MSCs transplantation. It was found that the BMD of the nZnO-MSCs group significantly surpassed that of the MSCs alone group. These results bear significant clinical implications and offer potential therapeutic targets for nZnO in bone defect management and related diseases.

Conclusions

In conclusion, our study demonstrates that the combination of nZnO with MSCs transplantation holds promise for treating bone defects. The AMPK-ULK1 signaling pathway plays a crucial role in the activation of autophagy signaling induced by nZnO. We also demonstrate that increased AMPK phosphorylation was linked with increased cellular Ca2+ concentration and elevated AMP: ATP ratio by nZnO. Such effects would be abolished by AMPK inhibitor. These results hold significant clinical implications and provide potential therapeutic targets for the management of bone defects and related diseases using nZnO.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Abbreviations

AMPK :

AMP-dependent protein kinasev

ALP:

Alkaline phosphatase

ARS:

Alizarin red S

BMD:

Bone mineral density

BV/TV:

Bone volume per unit tissue volume

CaMKK2:

Calcium/Calmodulin Dependent Protein Kinase Kinase 2

DLS:

Dynamic light scattering

ELS:

Electrophoretic light scattering

GelMA:

Gelatin methacryloyl

MSCs:

Mesenchymal stem cells

nZnO:

Nano-zinc oxide

OPN:

Osteopontin

RUNX2:

Runt-relaxed transcription factor 2

SEM:

Scanning Electron Microscopy

TEM:

Transmission Electron Microscopy

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Funding

This work was supported by funding from the National Science Foundation of China (U21A20340, 82404302, 82402946); Postgraduate Research & Practice Innovation Program of Jiangsu Province (JX10314110, JX10314114); Natural Science Foundation of Jiangsu Province (BK20240341), and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (24KJB330004).

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Author notes

  1. Xiu Chen, Zhenkun Weng, Hongchao Zhang and Jian Jiao contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Reproductive Medicine and Offspring Health, School of Public Health, Nanjing Medical University, Nanjing, 211166, China

    Xiu Chen, Zhenkun Weng, Jian Jiao, Jingjia Liang, Jin Xu, Qian Liu & Aihua Gu

  2. Jiangsu Environmental Health Risk Assessment Engineering Research Center, Key Laboratory of Modern Toxicology of Ministry of Education, Center for Global Health, Nanjing Medical University, Nanjing, 211166, China

    Xiu Chen, Zhenkun Weng, Jian Jiao, Jingjia Liang, Jin Xu, Qian Liu & Aihua Gu

  3. Changzhou Second People’s Hospital, Changzhou Medical Center, The Affiliated Changzhou Second People’s Hospital of Nanjing Medical University, Nanjing Medical University, Nanjing, 213004, China

    Zhenkun Weng & Dongmei Wang

  4. School of Medicine, Shanghai East Hospital & Institute of Gallstone Disease, Tongji University, Shanghai, Nanjing, 200120, 211166, China

    Hongchao Zhang

  5. Department of Neurosurgery, Children’s Hospital of Nanjing Medical University, Nanjing, 211166, China

    Qing Yan

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Contributions

Conceptualization, D.W. and A.G.; Formal analysis, X.C., Z.W., H.Z. and J.J.; Funding acquisition, Q.L., Q.Y. and A.G.; Methodology, J.L. and J.X.; Project administration, Q.L. and Q.Y.; Writing—original draft, X.C., Z.W. and H.Z.; Writing—review and editing, Q.L. and A.G. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Qian Liu, Qing Yan or Aihua Gu.

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On September 23, 2022, the Animal Protection and Use Committee of Nanjing Medical University approved an animal research protocol titled “Effects of Nano Zinc Oxide on Skeletal Development” (IACUC-2209047).

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The authors declare that they have no competing interests and artificial intelligence is not used in this study.

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Chen, X., Weng, Z., Zhang, H. et al. Nano-zinc oxide (nZnO) targets the AMPK-ULK1 pathway to promote bone regeneration. Stem Cell Res Ther 16, 206 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04322-5

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Stem Cell Research & Therapy

ISSN: 1757-6512