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Engineered bone-targeting apoptotic vesicles as a minimally invasive nanotherapy for heterotopic ossification

Journal of Nanobiotechnology volume 23, Article number: 348 (2025) Cite this article

Abstract

Heterotopic Ossification (HO), refers to pathological extra skeletal bone formation, and there are currently no reliable methods except surgery to reverse these unexpected calcified tissues. Apoptotic vesicles (ApoEVs) are membrane-bound vesicles released by apoptotic cells, which are involved in metabolism regulation and intercellular communication. Due to its superior trauma-healing ability, the hard palate mucosa is expected to become an essential resource for tissue engineering. This work presents a minimally invasive nanotherapy based on an engineered apoEV. Briefly, apoEVs were extracted from hard palate mucosa and engineered with bone-targeting peptide SDSSD to treat HO. This engineered apoEV not only can achieve directed localization of heterotopic bones but also has the compelling dual function of promoting osteoclastic differentiation while inhibiting osteogenic differentiation. The underlying mechanism involves the activation of Hippo and Notch pathways, as well as the regulation of pyrimidine metabolism. We envision that this engineered apoEV may be a feasible and effective strategy for reversing HO.

Graphical Abstract

Introduction

Heterotopic ossification (HO) is a bone metabolism disorder characterized by abnormal bone formation in extra skeletal tissues [1]. Every year, more than 200,000 patients worldwide who undergo joint replacement surgery suffer from HO [2]. HO can develop as a consequence of dysregulated bone-related signaling pathways. Traumatic stimuli lead to activated and uncontrolled osteogenesis-related metabolic pathways in soft tissues, which eventually results in aberrant bone tissue formation [3]. HO is associated with complications like restricted range of motion and persistent pain, often resulting from burns, tendon injuries, and invasive surgeries. There are currently limited treatment alternatives for HO. Extensive research has focused on the prevention strategies of HO, including the use of nonsteroidal anti-inflammatory drugs, bisphosphonates, etc [4,5,6]. However, no practical approach except surgical intervention is currently available to control the established HO. The efficacy of this therapeutic modality is constrained by operation difficulty and potential complications that may manifest at the surgical site. Considering these clinical requirements, it’s urgent to devise a minimally invasive, reversible, and specific alternative strategy for HO.

Apoptotic vesicles (ApoEVs) are an emerging type of extracellular vesicles (EVs) derived from apoptotic cells [7]. The biomedical applications of apoEVs are growing owing to their desirable properties, including immune compatibility, ethical considerations, and biological barrier-crossing ability [8]. ApoEVs were previously regarded as debris or by-products of apoptosis, but now they have been discovered to inherit substances and information from dying cells that could be transferred, recycled, and reused by other cells in the body [9]. For instance, it has been reported that bone mesenchymal stem cell-derived apoEVs might alleviate alveolar bone destruction [10]. Moreover, compared with other kinds of EVs, the characteristics of comparatively elevated yield, great stability, and undemanding availability endow them with distinguished advantages in clinical treatment [11]. The multipotentiality and plasticity, combined with the accessibility through minimally invasive procedures that avoid aesthetic complications, render oral mucosa-derived mesenchymal stem cells a highly valuable resource for tissue engineering [12, 13]. Prior research conducted by our research team has revealed the bone-regulating potential of cell sheets fabricated with hard palate mucosa-derived mesenchymal stem cells (PMSCs) [14]. Thus, we presume that apoEVs derived from PMSCs might possibly be involved in bone metabolism.

Researchers have demonstrated that the biodistribution profile of apoEVs shows preferential accumulation in organs such as the liver and lungs, indicating a lack of tissue specificity [15,16,17]. Therefore, it’s essential to explore a more precise and specific mode of apoEV delivery to improve the targeted enrichment at relevant sites and minimize toxicity to other tissues [18]. The modification of apoEVs can be achieved through approaches such as genetic engineering, drug loading, and surface modification. Compared with other approaches, surface modification has advantages like low cost, simple procedure, and availability for pre-isolated apoEVs [19]. Targeting peptides are particularly notable among modification materials due to their nontoxicity, tunability, and no intervention in microenvironment homeostasis. A cell-penetrating peptide has been utilized to modify apoEVs for the treatment of ischemic stroke, yielding promising results [20]. A research team successfully conjugated P-selectin binding peptide to apoEVs and confirmed that the targeting ability of apoEVs to injured arteries significantly increased [21]. Taking these inspirations, we propose that the strategic integration of PMSC-apoEVs’ inherent bone regulatory ability with engineering technologies could potentially instigate a paradigm shift in the therapeutic conundrum of HO.

Herein, we highlight the significance of the establishment of a minimally invasive nanotherapy integrating multiple functions (Fig. 1). We illustrate a rapid and efficient one-step method to modify PMSC-apoEVs with bone-targeting peptide (SDSSD), imbuing them with enhanced bone tissue specificity to achieve the precise treatment of HO. Mechanistically, we speculate that engineered apoEVs can not only facilitate osteoclastic differentiation and inhibit osteogenic differentiation via the Hippo and Notch pathways, but also regulate bone homeostasis through pyrimidine metabolism. Collectively, we elucidate a minimally invasive treatment to reverse HO, suggesting a framework for the construction of an apoEV-based delivery and treatment system. Furthermore, this study clarifies that the use of engineered apoEVs can serve as a potent strategy and a safe clinical translational approach for the management of other complicated diseases.

Fig. 1
scheme 1

Schematic illustration of this work. (A) Progression of clinical symptoms of HO. (B) Preparation of engineered apoEVs. (C) Therapeutic mechanism of engineered apoEVs in vivo

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Materials and methods

Cell culture

Six-week-old male C57BL/6J mice were used for the isolation of PMSCs, bone marrow-derived mesenchymal stem cells (BMSCs), and monocyte macrophages (BMMs). To isolate PMSCs, the hard palate mucosa was harvested and then cultured in MEM alpha basic medium (α-MEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 0.272 g/L L-glutamine (Sigma, USA), 1% penicillin (Gibco, USA), and 1% streptomycin (Gibco, USA). The tissues were cultivated at 37℃ in an incubator with 5% CO2 and 95% humidity. BMSCs and BMMs were obtained from the bone marrow of femurs and tibias and cultivated in α-MEM or Dulbecco’s modified eagle medium (DMEM, Gibco, USA) supplemented with 10% FBS, 0.272 g/L L-glutamine, 1% penicillin, and 1% streptomycin. All cells utilized in this study and relevant experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University (Hangzhou, China).

Animals

The Zhejiang University Institutional Animal Care and Use Committee approved each animal test that was conducted in accordance with the China National Institutes of Health’s Guidelines for the Welfare and Use of Lab Animals. Six-week-old C57BL/6J mice (mean weight 20–30 g) were used in this study. All mice were fed with free food and water under specific pathogen-free circumstances (25 °C, humidity: 50–60%, 12 h light/dark cycle).

Isolation of PMSC-apoEVs

When PMSCs reached 85% density, the cells were treated with 0.5 µmol/L staurosporine (STS, Med Chem Express, USA) for 20 h at 37 °C in 5% CO2 for apoptosis induction. Then cell supernatants were collected after 20 h and centrifuged at 400 g for 10 min and 2000 g for 10 min to remove cell debris. Subsequently, the supernatants were centrifuged three times for 30 min at 16,000 g. For in vitro experiments, apoEVs derived from PMSCs were resuspended in phosphate buffered saline (PBS, Servicebio, China) at 20 µg/mL [22]. For in vivo experiments, apoEV pellets were resuspended in PBS at a concentration of 500 µg/ml [23].

ApoEV modification

A bone-targeting peptide sequence as Ser-Asp-Ser-Ser-Asp (SDSSD; Sangon Biotech, China) was designed for modification of apoEVs. ApoEVs at a concentration of 0.5 mg/mL were combined with N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, Macklin, China), N-Hydroxysuccinimide (NHS, Macklin, China), in 4-Morpholineethanesulfonic acid buffered solution (MES, Aladin, China) and rotated for 1 h for the reaction of NHS with the amino on the membrane of the apoEVs. 0.5 µM SDSSD was introduced to the mixture and rotated at 4℃ for the whole night to obtain the bone-targeting apoEVs (BT-apoEVs). BT-apoEVs were then kept at -80℃ for further use.

Characterizations of apoEVs and BT-apoEVs

To ascertain the size distribution and zeta potential of apoEVs and BT-apoEVs, we employed Dynamic Light Scattering (DLS) analysis through the Zetasizer (Zetasizer Nano-ZS, Malvern, UK). To examine the morphology of apoEVs, a Transmission Electron Microscope (TEM, LSM 800 with Airyscan, Zeiss, German) was utilized. The apoEVs were placed onto a copper grid and negatively stained with 2% phosphotungstic acid (Leagene, China). After 1 min staining, they were gently washed with deionized water to remove the excess acid and air-dried overnight.

To characterize the modification of apoEVs, Fourier Transform Infrared Spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were conducted. The freeze-dried sample was mixed with potassium bromide (KBr) powder in the mortar, fully grounded and then pressed into a transparent tablet. The tablet was scanned in an infrared analyzer (Infrared Spectrometer, Nicolet iS20, Thermo Fisher, USA) and infrared spectra were recorded with specific parameters (resolution: 4 cm− 1, wavelength range: 400–4000 cm− 1, scan number: 32). The surface elemental composition of apoEVs and BT-apoEVs was observed using XPS (K-Alpha XPS, Thermo Scientific, USA). An appropriate amount of freeze-dried sample was pressed into a tablet and placed on the sample plate. The spectra were run at 12 kV and 6 mA. For the individual peak regions, a pass energy of 50 eV with a step size of 0.1 eV was employed. Survey spectra were measured at 150 eV pass energy with a step size of 1 eV.

To assess the bone-targeting ability of apoEVs and BT-apoEVs, a hydroxyapatite (HAp) binding-efficiency experiment was performed. In brief, the PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich, USA) was used to label apoEVs and BT-apoEVs. After incubation with HAp (10 mg/mL) at room temperature for 5 h, the mixture was centrifuged to spin down HAp and the apoEVs bound to it. Then the fluorescence intensity of the supernatants was measured using fluorometry. The changes in fluorescence intensity before and after HAp incubation were analyzed to assess their binding ability to HAp.

To determine the biodistribution and bone-targeting effect of apoEVs and BT-apoEVs in vivo, C57BL/6J mice were classified into 3 groups: the PBS group, the apoEV group, and the BT-apoEV group. PKH26 was used to stain apoEVs and BT-apoEVs, and then tail vein injections were conducted at a concentration of 2.5 µg/g. PBS was infused as a control. The mice were sacrificed at the time points of 2, 4, and 6 h after injections [18]. The organs: heart, liver, spleen, lungs, kidneys, and femurs were harvested and scanned with the imaging system (Aniview, Biolight, China) for analysis.

Internalization of apoEVs in vitro

After seeding BMSCs onto confocal dishes, cells were incubated at 37 °C overnight. ApoEVs or BT-apoEVs were treated with the PKH26 Red Fluorescent Cell Linker Kit according to the manufacturer’s procedure. PKH26-labeled apoEVs were then incubated with BMSCs for 5 h at a concentration of 20 µg/ml. After being fixed for 20 min with 4% paraformaldehyde (PFA, Servicebio, China), the nuclei were stained with DAPI (Solarbio, China). A confocal laser scanning microscope (Nikon A1 Ti, Nikon, Japan) was employed to observe the cell morphology of BMSCs with PKH26-apoEVs.

Cytotoxicity assay

The cytotoxicity of apoEVs was examined using a Live/Dead Cell Double Staining Kit (Solarbio, China) and Alamar Blue Cell Viability Reagent (Invitrogen, USA). BMSCs or BMMs were seeded in 24-well plates and treated with PBS, 20 µg/ml apoEVs, and 20 µg/ml BT-apoEVs. After overnight incubation, the cells were stained with the Live/Dead Cell Double Staining Kit, and the cell morphology was observed by a fluorescence microscopy (EVOS M5000, Invitrogen, USA). Alamar Blue Cell Viability Reagent was added to the wells on days 1, 3, and 5 of co-culture, and the cells were incubated at 37 °C for 3 h. The fluorescence intensity of each well was measured at 540 nm excitation and 590 nm emission wavelengths using a microplate reader (Synergy H1, Bio Tek, USA).

Osteogenic differentiation assay

BMSCs were seeded in a 24-well plate. When the cells reached 100% confluence, the medium was replaced with α-MEM supplemented with 2 mM β-glycerophosphate (Sigma, USA), 100 µM L-ascorbic acid (Sigma, USA) and 10 nM dexamethasone (Sigma, USA). The medium was supplemented with PBS, 20 µg/ml apoEVs, or 20 µg/ml BT-apoEVs according to the groups. To evaluate osteogenic differentiation, BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and 1% Alizarin Red S (Solarbio, China) were used to stain the cells at the time point of 1 week and 3 weeks respectively, in accordance with the manufacturer’s instructions.

Osteoclastic differentiation assay

A 24-well plate was used to seed BMMs. The medium applied for the osteoclastic induction was DMEM containing 30 ng/ml M-CSF (R&D Systems, USA) and 50 ng/ml RANKL (R&D Systems, USA). The same volume of PBS, 20 µg/ml apoEVs, or 20 µg/ml BT-apoEVs were added into the medium. The osteoclasts were stained using an Acid Phosphatase Leukocyte Kit (Servicebio, China) on Day 6. Under an optical microscope, the images were captured, and positive multinucleated cells (> 3 nuclei/cell) were defined as osteoclasts (6 random fields for each well).

The induced osteoclasts were rinsed three times with PBS, fixed with 4% PFA for 15 min, and then permeabilized with 0.2% Triton X-100 for 10 min. Next, the cells were treated with Rhodamine Phalloidin (Med Chem Express, USA) for 30 min in a dark environment. The dye was then discarded and cells were washed with PBS. Afterward, the osteoclasts were stained with DAPI for 10 min. Random fields were taken and fluorescence images were captured using a fluorescence microscope.

Quantitative real-time polymerase chain reaction (RT-qPCR)

TRIzol reagent (Invitrogen, USA) was utilized to extract total RNA. The quantification of total RNA was conducted with Nanodrop 2000 (Thermo Fisher Scientific Inc., USA). In accordance with the instructions of the manufacturer, the PrimeScript RT reagent Kit (TaKaRa, Japan) was employed for the reverse transcription (RT) process of RNA. For Real-time PCR, TB Green™ Premix Ex Taq (Takara, Japan) was used to prepare the reaction mix under the manufacturer’s instructions. The reaction was performed in a 384-well optic plate with a qPCR cycler (CFX384 Touch, Bio-rad, USA). The expression of the target genes was shown as 2−△△CT after being normalized to the housekeeping gene GAPDH. The primer sequences used for qPCR analysis are listed in Table S1, Additional File 1.

Western blotting

The lysis of cells was achieved with RIPA lysis buffer (GenStar, China) enhanced with phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). To extract proteins, the cell homogenates were centrifuged at 12,000 g for 10 min at 4 °C. The protein concentration was then determined by the BCA Protein Assay Kit (Beyotime, China). For the gel electrophoresis process, 20 µg of proteins were loaded onto 4–20% SDS-PAGE gels (EpiZyme, China) and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). Subsequently, the membranes were blocked with 5% skim milk followed by overnight incubation with primary antibodies at 4 °C. The membranes were then exposed to corresponding HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were finally identified with Super ECL Plus Chemiluminescence Solution (Applygen, China), and the data were shown using a gel imaging system (Bio-Rad, USA). The following primary antibodies were used in the study: GAPDH (60004-1-lg, Proteintech, USA), Sp7 (ab209484, Abcam, UK), BMP-2 (ab214821, Abcam, UK), Col-1 (ab260043, Abcam, UK), Runx2 (ab92336, Abcam, UK), Alp (DF6225, Affinity Biosciences, USA), TRAP (ab52750, Abcam, UK), NFATc1 (ab25916, Abcam, UK), MMP-9 (ab76003, Abcam, UK), CTSK (PAA267Mu01, Cloud-Clone, USA), c-Fos (CY6596, Abways, China).

RNA-sequencing (RNA-seq)

The total RNA from the cell samples was isolated using TRIzol reagent and then quantified. For the preparation of the RNA samples, three micrograms of RNA were utilized as input material. Sequencing libraries were constructed according to the following steps. First, magnetic beads coupled to poly-T oligos were used to separate mRNA from total RNA. Fragmentation was performed at a high temperature using divalent cations in an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using Super Script II and random oligonucleotides. Second-strand cDNA synthesis was then carried out with DNA Polymerase I and RNase H. After the remaining overhangs were transformed into blunt ends by exonuclease/polymerase activities, the enzymes were eliminated. To be ready for hybridization, Illumina PE adapter oligonucleotides were ligated after the 3’ ends of the DNA fragments had been adenylated. The AMPure XP system (Beckman Coulter, USA) was employed to purify the library fragments in order to screen cDNA fragments of the desired 400–500 bp in length. In a 15-cycle PCR process, DNA fragments with ligated adaptor molecules on both ends were preferentially selected using the Illumina PCR Primer Cocktail. The Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent, USA) was used to quantify the products after they were purified. The sequencing library was subsequently sequenced on NovaSeq 6000 platform (Illumina, USA).

Liquid chromatography-mass spectroscopy (LC-MS)/MS analysis

LC-MS/MS analysis was done with an Easy-nLC HPLC (Thermo Scientific, USA) connected to a Q-Exactive mass spectrometer. At a flow rate of 250 nl/min for 60 min, the labeled peptides were fed into a Thermo Scientific™ EASY-C18 sample loading column (100 μm × 2 cm, 5 μm) and subsequently separated using a Thermo Scientific TM EASY-C18 analytical column (75 μm × 10 μm, 3 μm). The mass spectrometer was run in positive mode. In the MS scan, the automated gain control (AGC) target was 3e6, dynamic exclusion time was 40.0 s, primary mass spectrum resolution was 70,000 at m/z 200, and precursor ion scanning range was 300 to 1,800 m/z. 10 fragment maps (MS2 scan) were collected after each full MS scan. HCD activation type, isolation window of 2 m/z, resolution of 17,500 at 200 m/z, normalized collision energy of 30 eV, and underfill ratio of 0.1% were among the MS/MS parameters.

Burn/tenotomy heterotopic ossification model

The C57BL/6J mice were anesthetized via inhaling isoflurane and their dorsal hair was closely clipped. Afterward, using a 60 °C aluminum block, the dorsal region was heated for 18 s to cause a partial-thickness scald burn lesion. A simultaneous dorsal hindlimb tendon transection was performed under sterile conditions using sterile instruments near the middle of the mice’s Achilles tendon. Subsequently, the wound was closed using a single 5 − 0 Vicryl suture. The mice were divided into three treatment groups (n = 6) at random: the PBS group, the apoEV group, and the BT-apoEV group. These mice were randomized and kept in cages with five mice each, under the conditions of appropriate humidity and temperature levels, with ample food and beverages.

HO treatment in vivo

All materials underwent sterilization by ultraviolet radiation for a duration of 24 h. Each calcified tendon received treatment every seven days with 100 µl PBS (control), apoEVs (50 ug in 100 µl PBS), or BT-apoEVs (50 µg in 100 µl PBS) in situ. To minimize the potential interference from the syringe needle, insulin syringes were employed for the injection of materials into the Achilles tendon in situ. All mice were euthanized 90 days post initial injection [2, 24], and their Achilles tendons were subsequently harvested for histological analysis.

Micro-computed tomography (Micro-CT)

For a period of three months, the Achilles tendon of the mice received weekly injections of 100 µl PBS (control), apoEVs (50 µg in 100 µl PBS), or BT-apoEVs (50 µg in 100 µl PBS). The number of samples in each group was 6. There were four time points for micro-CT scans: 0, 1 month, 2 months, and 3 months [2, 24]. The mice underwent examinations by Skyscan 1276 high-resolution micro-CT (Bruker, Belgium) every month with the following parameters: 50 kV X-ray voltage, 200 µA current, and 8 μm resolution. Data were visualized and analyzed using CTVol and CTAn software (Bruker, Belgium).

Histological analysis

Tissues in each group underwent a 48-hour fixation using 4% PFA. Then excess PFA was washed away with running water. The tissues were decalcified using ethylenediaminetetraacetic acid (EDTA, Servicebio, China) for 21 days. Subsequently, the samples were subjected to dehydration using graded ethanol and were embedded in paraffin. After that, they were sectioned at 5 μm per slice. Hematoxylin and eosin (H&E), Masson, Tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical (IHC) staining were then performed to characterize the harvested tissues. A fluorescence microscope (BZ-X800E, KEYENCE, Japan) was employed to assess images of the stained samples. The number of samples in each group was 6.

Statistics

All data were expressed as mean ± standard deviation (SD). Statistical and graphical analyses were performed using GraphPad Prism 8 (GraphPad Software, USA). To identify differences between groups, one-way ANOVA analysis with Tukey’s test was used to evaluate multiple group comparisons. Independent unpaired two-tailed Student’s t-tests were employed to investigate the significance of two-group comparisons. Values of p < 0.05 indicate statistically significant. All the experiments were done in triplicate.

Results

Preparation and characterization of apoEVs and BT-apoEVs

The study employed the tissue block method to isolate primary cells, visually examining the properties of the PMSCs by microscopy (Fig. S1A, Additional File 1). The hard palate mucosa harvested from mice was cultivated in flasks, revealing spindle-shaped PMSC migration by Day 5 post-cultivation. By Day 9, cell colonies emerged and cellular proliferation accelerated. Apoptosis of PMSCs was then induced for the extraction of apoEVs (Fig. S1B, Additional File 1). Bone-specific labeling of apoEVs is accomplished via surface conjugation of the bone-targeting peptide (SDSSD), implemented by an amidation reaction (Fig. 2A).

Comparison of FTIR between apoEVs and BT-apoEVs identified a distinctive peak at 1543 cm− 1 attributed to the primary amide group of SDSSD, verifying successful SDSSD grafting onto the surface of apoEVs. An increase in characteristic peaks of C = O stretching vibrations post-modification indicated an increase in amide bonds due to amino group-carboxyl group conjugation on apoEVs (Fig. 2B). XPS was utilized to characterize the element composition of apoEVs and BT-apoEVs (Fig. 2C and Fig. S2, Additional File 1). A comprehensive analysis of C1s, O1s, N1s, and S2p elements was provided. Specifically, compared with apoEVs, the S2p content of BT-apoEVs marginally increased from 0.16 to 0.26%, owing to FITC-SDSSD incorporation. Concurrently, the enhanced peaks at ~ 287.2 eV and ~ 530.7 eV associated with carbonyl groups on C1s and O1s high-resolution scans signify successful FITC-SDSSD peptide modification on apoEVs. As shown in Fig. 2D, confocal microscopy images clearly showed that the bone-targeting peptide FITC-SDSSD was successfully attached to apoEVs. Moreover, TEM revealed the spherical shape and size of apoEVs and BT-apoEVs (Fig. 2E). Noticeably, the bone-targeting peptides resulted in a marginal rise in the average hydrodynamic diameter, measuring from 268.1 nm to 397.2 nm (Fig. S3A, B, Additional File 1 and Fig. 2F). The zeta potential was decreased from − 27.7 ± 0.5 mV (apoEVs) to -30.2 ± 0.9 mV (BT-apoEVs) (Fig. 2G), which may be caused by the conjunction with negatively charged SDSSD.

After the preparation procedures, the bone-targeting ability of BT-apoEVs is initially evaluated in vitro. Affinity with hydroxyapatite (HAp), which is the primary component of bones, is the index used to assess the bone-targeting effect of the vesicles. HAp-incubated PKH26-labeled BT-apoEVs partitioned off into solution post incubation, drastically diminishing the supernatant fluorescence (Fig. S4, Additional File 1). Post-incubation fluorescence revealed a high binding efficiency of BT-apoEVs to HAp at 69.3 ± 3.8% compared with apoEVs (22.8 ± 2.1%) (Fig. 2H).

Above all, these findings demonstrate that we successfully fabricated engineered PMSC-apoEVs with an innovative bone-targeting peptide SDSSD without changing their basic morphology and properties. BT-apoEVs exhibit a higher tendency for bone-specific targeting, which is a footstone of our minimally invasive and precise nanotherapy.

Fig. 2
figure 2

Preparation and characterization of BT-apoEVs. (A) Schematic illustration of the preparation of BT-apoEVs. (B) FTIR analysis of apoEVs and BT-apoEVs. (C) XPS survey spectra of apoEVs and BT-apoEVs. (D) ApoEVs and BT-apoEVs observed by a confocal microscopy (Scale bar = 20 μm). (E) Representative TEM images of apoEVs and BT-apoEVs. (Scale bar = 200 nm). (F) Size distribution of apoEVs and BT-apoEVs measured by dynamic light scattering (n = 3). (G) Zeta potential of apoEVs and BT-apoEVs determined by dynamic light scattering (n = 3). (H) Binding affinity of apoEVs and BT-apoEVs with HAp (n = 3). **p < 0.01

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BT-apoEVs promote osteoclastic differentiation in vitro

Regulation of osteoclastic differentiation is a fundamental part of our multifunctional minimally invasive nanotherapy. Given that the specific targeting abilities of BT-apoEVs have been demonstrated, we subsequently assessed the effectiveness of BT-apoEVs for promoting osteoclastic differentiation. First, we examined the process of cellular uptake by incubating PKH26-labeled BT-apoEVs with BMMs. BT-apoEVs can be internalized by BMMs as evidenced by colocalization of fluorescently labeled BT-apoEVs with cells (Fig. 3A). A 24-hour cytotoxicity test was performed to evaluate the biocompatibility of BT-apoEVs (Fig. 3B). The results showed that there was no significant difference in the cell morphology and cell amount after BT-apoEV treatment. A cell viability test was conducted at the time point of 1, 3, and 5 days to further investigate the long-term cytotoxicity (Fig. S5A, Additional File 1). These results indicated that BT-apoEVs had the desired biocompatibility and negligible cytotoxicity with BMMs.

BMMs are osteoclast precursor cells that could be used to mimic the differentiation of osteoclasts in vivo. To ascertain the impact of BT-apoEVs on osteoclastic differentiation, we cultivated BMMs with BT-apoEVs for TRAP staining. The results elucidated that the presence of BT-apoEVs could promote osteoclastogenesis (Fig. 3C and D). Observed under a fluorescence microscope, the immunofluorescence of the F-actin and DAPI further verified that BT-apoEVs evidently enhanced osteoclastic differentiation and formation (Fig. 3E). To measure the mRNA expression of osteoclast markers, qPCR was performed following the incubation of BMMs with BT-apoEVs. As qPCR analysis revealed, BT-apoEVs notably elevate the mRNA expression of TRAP, MMP-9, c-Fos, NFATc1, and CTSK (Fig. 3F). Consistent with qPCR results, Western blotting data demonstrated that BT-apoEVs facilitated the expression of osteoclast-related proteins, such as TRAP, CTSK, NFATc1, and c-Fos (Fig. 3G).

In conclusion, we confirmed that BT-apoEVs can be internalized by BMMs and stimulate osteoclastic differentiation with minimal cytotoxicity. This property endowed BT-apoEVs with the potential to become the critical part of the nanotherapy for HO.

Fig. 3
figure 3

BT-apoEVs promote osteoclastic differentiation in vitro. (A) Fluorescence images of PKH26-labeled apoEVs and BT-apoEVs internalized by BMMs (Scale bar = 20 μm). (B) Live/dead staining of BMMs after treatment (Scale bar = 200 μm). (C) Representative images of TRAP staining in the control, apoEV, and BT-apoEV group (Scale bar = 200 μm). (D) Quantification of TRAP-positive osteoclasts (n = 6). (E) Immunofluorescence images of osteoclast cytoskeleton (Scale bar = 200 μm). (F) In vitro osteoclastic-related gene expression of BMMs measured by qPCR assay (n = 3). (G) Western blotting showed the expression of markers related to osteoclastic differentiation. *p < 0.05, **p < 0.01

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BT-apoEVs inhibit osteogenic differentiation in vitro

Another indispensable function in the multifunctional minimally invasive nanotherapy is controlling osteogenic differentiation. Therefore, we next explored the possible impacts of BT-apoEVs on osteoblasts. The internalization of BT-apoEVs by BMSCs was initially investigated. The fluorescence photographs validated that BT-apoEVs can be taken in by BMSCs (Fig. 4A). The cytotoxicity test and cell viability test collectively implied that BT-apoEVs exhibited the intended biocompatibility and minimal cytotoxicity with BMSCs (Fig. 4B and Fig. S5B, Additional File 1).

The cells used in evaluating osteogenic differentiation are BMSCs, which can be induced into osteoblasts in the presence of dexamethasone, β-glycerophosphate, and L-ascorbic acid. Through the introduction of BT-apoEVs into the culture medium of BMSCs, the influence of BT-apoEVs on osteogenesis was examined. According to the results of staining, the alkaline phosphatase (ALP) accumulation and mineralization were effectively reduced by BT-apoEVs (Fig. 4C). Alizarin Red S staining was also conducted as a traditional and advised experiment of osteogenic differentiation. It was noted that the BT-apoEV group had less mineralized nodule formation following 21 days of induction (Fig. 4D). The qPCR was then employed to detect the expression of osteogenic-related genes. From the results, it can be found that the addition of BT-apoEVs downregulated the expression of genes associated with osteogenic differentiation, including Alpl, Sp7, BMP-2, Runx2, and Col-1 (Fig. 4E). In agreement with the qPCR results, Western blotting displayed that Alp, Sp7, Runx2, Col-1, and BMP-2 proteins were reduced in BMSCs following BT-apoEV treatment (Fig. 4F).

In summary, these findings proved the internalization of BT-apoEVs into BMSCs and suggested that BT-apoEVs significantly inhibit osteogenic differentiation of BMSCs with good biocompatibility. Therefore, BT-apoEVs exhibit the dual function of promoting osteoclastic differentiation while inhibiting osteogenic differentiation, which are expected to realize therapeutic effects in vivo.

Fig. 4
figure 4

BT-apoEVs inhibit osteogenic differentiation in vitro. (A) Fluorescence images showing cellular uptake of PKH26-labeled apoEVs and BT-apoEVs by BMSCs (Scale bar = 20 μm). (B) Live/dead staining of BMSCs treated with PBS, apoEVs, and BT-apoEVs (Scale bar = 200 μm). (C) ALP staining of BMSCs after treatment for 7 days (Scale bar = 200 μm). (D) Alizarin Red S staining of the mineralized matrix after treatment for 21 days (Scale bar = 200 μm). (E) In vitro osteogenic-related gene expression of BMSCs measured by qPCR assay (n = 3). (F) Western blotting analyzed the expression of osteogenic markers. *p < 0.05, **p < 0.01

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BT-apoEVs downregulate osteogenic differentiation through Hippo and Notch pathways

To elucidate the underlying mechanism of PMSC-apoEVs by which PMSC-apoEVs inhibit osteogenesis, RNA-sequencing (RNA-seq) analysis was conducted to identify gene expressions. Broad changes of differentially expressed genes (DEGs) can be found in the BT-apoEV group compared with the control group, according to the heatmap results (Fig. 5A). The bar plot and volcano plot revealed that in BT-apoEV vs. PBS, 1161 genes were upregulated and 991 genes were downregulated (Fig. 5B and C. The distribution of DEGs among the Control, ApoEV, and BT-apoEV groups is visualized via the Venn plot (Fig. S1S6, Additional File 1).

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then performed to detect functions and signaling pathways. Based on the GO annotations, biological processes that are significantly enriched in the BT-apoEV group include negative regulation of cell differentiation, Notch signaling pathway, and negative regulation of osteogenic differentiation, suggesting the negative regulatory effect of BT-apoEVs on osteogenic process. Additionally, there was also positive regulation of other vesicle-related cascades, such as regulation of vesicle-mediated transport and regulation of vesicle fusion. In terms of cellular component and molecular function, DEGs primarily function by binding biomolecules such as proteins and ions, mainly located in the extracellular space. Meanwhile, cellular components including cytoplasm and cytosol were also elevated in GO analysis, which indicates that functional molecules are associated with cytoplasm and cytosol (Fig. 5D). In KEGG analysis, the main enriched pathways were identified, including ECM-receptor interaction, osteoclast differentiation, Hippo signaling pathway, Notch signaling pathway, and signaling pathways regulating pluripotency of stem cells, among which we focused on the Hippo and Notch signaling pathway (Fig. 5E). Further Gene Set Enrichment Analysis (GSEA) analysis determined that the Hippo and Notch pathways were significantly activated after the BT-apoEV treatment (Fig. 5F and G). To verify the potential involvement of the Hippo and Notch signaling pathways in BT-apoEV-mediated effects, we examined the expression levels of pathway-related genes with qPCR. As expected, BT-apoEVs notably reduced the levels of transcription factors YAP (Yap1) and TAZ (Wwtr1) and their mediator Tead2 (Fig. 5H). A significant upregulation of genes associated with the Notch signaling pathways, including Notch1, Hes1, and Hey1, was observed in the BT-apoEV group (Fig. 5I).

Altogether, we improved the underlying molecular mechanism of BT-apoEVs in suppressing osteogenic differentiation. The inhibitory effects of BT-apoEVs may be exerted through Hippo and Notch signaling pathways.

Fig. 5
figure 5

BT-apoEVs downregulate osteogenic differentiation through Hippo and Notch pathways. (A) Heatmap analysis showed enriched expression genes. (B) Bar charts of gene upregulation and downregulation. (C) Volcano plots revealed the downregulation and upregulation of genes of apoEV and BT-apoEV vs. PBS. (D) GO analysis showed enriched genes in the biological process, cellular component, and molecular function. (E) Bubble plot showing KEGG enrichment by mRNAs in pathways. (F) Gene set enrichment analysis of the Hippo pathway. (G) Gene set enrichment analysis of the Notch pathway. (H) Relative mRNA expression of Yap1, Wwtr1, and Tead2 in different groups (n = 3). (I) Relative mRNA expression of Notch1, Hes1, and Hey1 measured by qPCR assay (n = 3). *p < 0.05, **p < 0.01

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BT-apoEVs regulate bone metabolism through pyrimidine metabolism

To gain insights into the relationship between metabolism and BT-apoEV treatment, we performed LC-MS-based metabolomics. The heatmap shown in Fig. 6A displays the cluster analysis of differential metabolites and a clear differential pattern of metabolite expression was observed between groups. Employing partial least squares discriminant analysis (PLS-DA) score plots, we discerned distinct metabolite clusters in the apoEV and BT-apoEV groups compared with the control group (Fig. 6B). We then studied the alternations of metabolomic patterns among three groups based on the differential metabolites. Volcano plots were used to depict fold changes in the levels of annotated metabolites in the apoEV and BT-apoEV groups relative to the control group (Fig. 6C). A total of 285 metabolites were detected, including nucleotides and analogues, organic nitrogen compounds, organic oxygen compounds, etc. (Fig. 6D). Among all metabolite categories, organic acid and its derivatives and organoheterocyclic compounds are the most abundant metabolite types accounting for 25.2% and 19.2%, respectively (Fig. 6E). To comprehend the functional features of the identified altered metabolites, the KEGG analysis was employed for detailed annotation and pathway analysis. KEGG analysis results showed that pathways including pyrimidine metabolism, mineral absorption, biosynthesis of cofactors, protein digestion, etc. were enriched in both apoEV and BT-apoEV groups compared with the control group (Fig. 6F).

Subsequently, we performed an integrated analysis of the RNA-seq and metabolomic data to understand the correlation between the transcript profiles and metabolite profiles. The heatmaps illustrated the correlation between differential genes and metabolites (Fig. 6G). When the results of RNA-seq and metabolomic analysis were collated, 15 key metabolic pathways enriched in both differentially expressed genes and proteins were identified. The analysis revealed that pathways related to pyrimidine metabolism, protein digestion and absorption, mineral absorption, beta-alanine metabolism, and phenylalanine metabolism were involved in the metabolism of both apoEV and BT-apoEV groups (Fig. 6H). Notably, we identified several pyrimidine-related metabolites, such as uracil, uridylic acid, cytosine, cytidine 3’-phosphate, and thymidine 5’-phosphate, as among the altered metabolites. Correspondingly, genes including K01513, K00524, K01511, and K00857 are found to be significantly downregulated after treatment.

Taken together, we further investigated the metabolic mechanisms of BT-apoEVs in regulating bone homeostasis. Our findings implied that BT-apoEVs are possible to prevent osteogenic differentiation by affecting pyrimidine metabolism.

Fig. 6
figure 6

BT-apoEVs regulate bone metabolism through pyrimidine metabolism. (A) Heatmap of metabolites in control, apoEV, and BT-apoEV group. (B) Metabolic profiles were significantly altered in the apoEV and BT-apoEV groups compared with the control group by partial least-squares discriminant analysis. (C) Volcano plot showing metabolites with differential abundance. (D) A Venn diagram visualizing metabolites of the control, apoEV, and BT-apoEV group. (E) Metabolite profiles according to the metabolomic results. (F) KEGG analysis of the identified metabolites. (G) Heatmaps showing the correlation between differentially expressed genes (mRNA) and metabolites. (H) Common pathways identified in the integrated analysis of the RNA-seq and metabolomic data

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BT-apoEVs prevent heterotopic ossification in vivo

In view of the significant function of apoEVs and BT-apoEVs in maintaining bone homeostasis in vitro, we tried to further explore the application of this nanotherapy in an animal disease model. Figure 7A schematically illustrates the development of the HO mouse model and the experiment strategies with BT-apoEVs. To determine the roles of BT-apoEVs, it is essential to ascertain the distribution and metabolism of BT-apoEVs. We started by conducting local injections in the Achilles tendon of mice to generally evaluate the absorption rate of BT-apoEVs in vivo. As the fluorescence images show, the injected BT-apoEVs could be retained for about 4 days and were gradually absorbed by the time point of 7 days (Fig. S7A, Additional File 1). We harvested the heterotopic bones from the injected mice and fluorescence analysis exhibited an elevated average fluorescence intensity in the BT-apoEV group, indicating improved bone-targeting ability (Fig. S7B, Additional File 1). It’s possible that the PKH26-labeled vesicles in the apoEV group are dispersed in other tissues such as muscles and tendons without specificity, thus reducing their concentration in bone tissues. Since we have successfully confirmed that BT-apoEVs targeted bone tissues in vitro, we next studied the in vivo bone-targeting ability of BT-apoEVs with an intravenous administration method. The heart, liver, spleen, lungs, kidneys, and femurs were harvested for fluorescence imaging 2, 4, and 6 h after injections. The fluorescence signals were only detected in the apoEV and BT-apoEV groups, but not in the PBS group. It was discovered that the heart, spleen, and kidneys displayed weak signals in both two groups, while the liver and lungs displayed high fluorescence signals. The distribution of apoEVs may be influenced by the many scavenger receptors of Kupffer cells in the liver, which may bind to ligands on the surface of apoEVs. Additionally, physical capture of lung microcapillaries and the 4.5–5 nm glomerular filtration barrier may also play a role [18, 25,26,27]. Notably, the BT-apoEV group had significant fluorescence signals on the tibia and femur bone, while the apoEV group showed fewer signals (Fig. 7B). These outcomes were in line with the prior work, demonstrating the superior bone-targeting capability of BT-apoEVs.

Given that the precise targeting properties of BT-apoEVs have been elucidated, we then assessed the therapeutic efficacy of BT-apoEVs for alleviating HO in a mouse model. Based on reconstructed three-dimensional micro-CT images, we observed that the heterotopic bone volume was significantly decreased in the BT-apoEV group after in-situ injections. Furthermore, compared with the control group, mice that received injections of BT-apoEVs showed a reduction in the bone mass of heterotopic bones (Fig. 7C). Notably, the BT-apoEV group demonstrated a more remarkable reduction compared with the apoEV group, which may be attributed to their bone-targeting effect. Other structural parameters, including bone volume fraction (BV/TV) and trabecular thickness (Tb. Th), also witnessed an obvious decrease in the BT-apoEV group (Fig. 7D). On the basis of H&E and Masson’s trichrome staining photographs, we further confirmed that large amounts of new bone appeared in the control group, while new bone formation was barely observed in the BT-apoEV group at this time point. Using TRAP staining, it can be seen that the BT-apoEV group saw an increase in the number of osteoclasts (Fig. 7E). The qPCR results of heterotopic bone tissues suggested that the expression of osteogenic-related genes is evidently downregulated (Fig. 7F). The immunohistochemical staining results likewise exhibited a noticeable decrease in the positive area of osteogenic markers SP7, BMP-2, ALP, Col-1, and Runx2 in the Achilles tendon post BT-apoEV treatment (Fig. 7G and H).

Overall, these findings validated that BT-apoEVs could effectively alleviate the disease status of HO and that the SDSSD modification could significantly improve bone-targeting efficacy, which was consistent with the results of in vitro experiments and our speculation.

Fig. 7
figure 7

Injection of BT-apoEVs prevents heterotopic ossification in vivo. (A) Schematic illustration of HO model and experiment timeline. (B) Organ distribution of BT-apoEVs after intravenous injection for 2, 4, 6 h. (C) Representative micro-CT images at HO sites at the 0, 1st, 2nd, 3rd month (n = 6). (D) Quantitative analysis of BV, BV/TV, and Tb. Th of the HO sites (n = 6). (E) H&E staining analysis, Masson’s trichrome staining analysis, and TRAP staining analysis of the HO sites (Scale bar = 50 μm, n = 6). (F) The relative mRNA levels of CTSK, c-Fos, TRAP, Alpl, Sp7, and BMP-2 genes in heterotopic bones (n = 3). (G) Immunohistochemistry staining of heterotopic bones (Scale bar = 50 μm, n = 6). (H) Quantitative analysis of immunohistochemistry (n = 6). *p < 0.05, **p < 0.01

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Discussion

The intricacy of HO presents essential challenges for existing therapeutic approaches as the factors that initiate HO remain elusive. In terms of established HO, surgical excision is a common therapy paradigm in clinical practice often accompanied by complications including contractures, incomplete excision, and scarring [28, 29]. Therefore, it is highly desirable to develop therapies that can improve the life quality of HO patients. Osteoclasts are natural bone-resorbing cells that originate from monocytes/macrophages [30]. Previous research has achieved the treatment of HO through the injection of modified osteoclasts, suggesting that the enhancement of osteoclastogenesis is effective in the management of established HO [2].In this study, we propose a minimally invasive and precise nanotherapy based on bone metabolism regulation. ApoEVs derived from PMSCs were grafted with the bone-targeting peptide SDSSD by adopting a surface modification approach. After the modification, their concentration in the targeted tissues increases, which would reduce the dosage in the clinical use and avoid impacts on other irrelevant tissues. TEM examination demonstrated that the spherical morphology of apoEVs was preserved post-modification. DLS analysis further revealed a slight increase in size and a reduction in zeta potential, attributed to the incorporation of negatively charged bone-targeting peptides. We adopted the local injection method to send engineered apoEVs in this study, as it is one of the most efficient delivery methods. However, predicting which patients will develop HO and where in their bodies is challenging in a clinical setting. Thus, one likely delivery method that can be explored in the future is systemic injection. Engineered PMSC-apoEVs can also be a generation of novel therapeutic options for numerous types of complicated ectopic calcification illnesses.

Currently, cell-free therapies based on extracellular vesicles (EVs) such as apoEVs are considered to be a promising substitute for cell therapies. Studies have shown that EVs from different sources can exert different effects on physiological and pathological cellular processes [31]. According to this, scientists are inspired to employ multiple EVs to deal with challenging clinical problems [32,33,34]. Hard palate mucosa is easily accessible for tissue harvesting and exhibits a notable wound healing ability, which makes PMSCs an appropriate candidate for apoEVs to investigate [14, 35]. In this study, apoEVs were isolated from PMSCs with a differential centrifugation method and characterized. ApoEVs derived from apoptotic PMSCs inherit original components and information from parental cells, which give them the potential to mediate bone metabolism. Fluorescence images showed that apoEVs and BT-apoEVs can be internalized into the cytoplasm after 5 h of exposure. We subsequently evaluated the changes in osteoclastic levels of BMMs by examining the expression of osteoclast-related factors like TRAP, CTSK, NFATc1, and c-Fos. Meanwhile, the levels of osteogenic markers in BMSCs such as BMP-2, Runx2, Alp, Sp7, and Col-1 are assessed to determine the osteogenic effects. Our findings robustly validated that engineered PMSC-apoEVs possess the ability to promote osteoclastic differentiation while inhibiting osteogenic differentiation. Bone homeostasis is a dynamic balance between bone resorption and bone formation, which is mainly controlled by osteoblasts and osteoclasts. Under pathological conditions, the balance between the activity of osteoclasts and osteoblasts breaks, which leads to diseases such as osteosclerosis or osteoporosis [36]. The physiological role of engineered PMSC-apoEVs in intervening bone cell differentiation demonstrates their potential in bone homeostasis regulation. Thus, we can potentially identify novel diagnostic and therapeutic strategies for bone diseases based on engineered PMSC-apoEVs. Obviously, further studies must be conducted to investigate the underlying mechanism and relevant therapeutic targets.

Previous research has determined the biological functions of EVs and utilized them as promising therapeutic agents [37,38,39]. Nevertheless, when used in vivo, obstacles such as limited specificity, short circulation lifetime, and inadequate controls also hinder the further use of EVs [40]. To overcome these shortcomings, researchers have turned to nanotechnologies to modify EVs properly. The genomic edition method for modification seems rather time-consuming and technically challenging. In this context, chemical modification methods exhibit advantages since the modifying substances are connected directly to the membrane. In this study, the fabrication of engineered apoEVs with higher production yield and bone-targeting ability was innovatively explored. We proposed a rapid and effective one-step modification technique that utilizes the amidation reaction between bone-targeting peptide (SDSSD) and amino groups on the apoEV surface to achieve the engineering of apoEVs. According to research findings, our modification will not affect the original functions of PMSC-apoEVs while enhancing the bone-targeting properties. The concentration of BT-apoEVs in bone tissues was substantially higher than that of the bare apoEVs after intravenous injection, underscoring its superior bone-targeting properties. We then investigated the potential role of BT-apoEVs in the progression of HO. Once released into the extracellular milieu, apoEVs can be recognized and metabolized by patrolling phagocytes or neighboring cells such as MSCs, fibroblasts, and macrophages to exert specific effects [41, 42]. ApoEVs also participate in the circulation system to regulate distant cells. Circulating apoEVs have been identified as major mediators of intercellular communication [11]. Endocytosis is the primary pathway of apoEV uptake, followed by fusion with acidified endosomes or lysosomes for cargo release [43]. Phosphatidylserine recognition receptors and integrins play a critical role in the detection and engulfment of apoEVs [44]. To determine the optimal frequency of local injections, we administered BT-apoEVs into the Achilles tendon of mice and monitored their in vivo absorption rate using fluorescence imaging. The results demonstrated that the injected BT-apoEVs were retained for approximately 4 days and were gradually absorbed by the 7-day time point. Based on these findings, a 7-day injection interval was selected for subsequent experiments. The injection dosage was established through reference to prior research [22, 23, 45,46,47,48]. The HO mice underwent a three-month treatment with BT-apoEVs [2, 24], and subsequent analysis of heterotopic bone sections and micro-CT scans revealed that BT-apoEVs evidently interrupted heterotopic bone formation and stimulated bone resorption. Examination of gene expression profiles in heterotopic bone tissues revealed a downregulation of osteogenic-related genes alongside an upregulation of osteoclastic-related genes. As anticipated, BT-apoEVs successfully targeted bone tissues and exerted the dual function of promoting osteoclastic differentiation and inhibiting osteogenic differentiation.

For mechanisms, we detected a close association between suppressed osteogenic differentiation induced by BT-apoEVs and specific signaling pathways. Hippo signaling pathway regulates cell proliferation, differentiation, and apoptosis as well as tissue regeneration in many organs. Core components of the Hippo pathway include upstream kinase cascade transcription and downstream effectors [49]. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors in the Hippo pathway [50]. When the Hippo pathway is activated, mammalian Sterile 20-like kinase 1/2 (MST1/2) combines with Salvador 1 protein (SAV1) for large tumor suppressor 1/2 (LATS1/2) phosphorylation. YAP/TAZ is further phosphorylated and retained in the cytoplasm without entering the nuclei and binding with TEA Domain Transcription Factor (TEAD) to perform its transcriptional activation function. LATS1/2 also facilitates the SCFβ−TrCP E3 ligase-dependent degradation of YAP/TAZ [51]. Consequently, the activation of the Hippo signaling pathway leads to the downregulation of intranuclear levels of YAP/TAZ and their mediator TEAD. Phosphorylated YAP/TAZ can not only suppress Smad phosphorylation to inhibit TGF-β signaling and the transcription of osteogenic-related genes, but also disrupt Wnt signaling by preventing β-catenin nuclear translocation [52]. By inhibiting the transcriptional activity of Runx2, YAP/TAZ regulates osteocalcin expression via the Src/Yes tyrosine kinase pathway [53]. Therefore, the Hippo signaling pathway acts as a negative regulatory pathway to inhibit the differentiation of osteoblast precursor cells in bone metabolism [54]. In line with the bioinformatics results, the levels of YAP, TAZ, and TEAD were decreased in the BT-apoEV group, suggesting that BT-apoEVs exert the inhibiting effect on osteogenesis via the Hippo signaling pathway. The Notch pathway is another important regulatory pathway in bone homeostasis. There are several key transmembrane proteins of the Notch signaling pathway: Notch receptors, Jagged ligands, and Delta-like ligands [55]. The Notch protein is cleaved and Notch intracellular domain (NICD) enters the nucleus for the regulation of downstream gene expression once the Notch pathway is activated. Under physiological conditions, Notch signaling negatively regulates Runx2 transactivation function to control osteogenic differentiation [56, 57]. Notch signaling transduces downstream molecules such as Jagged, Hes, Hey and other genes to mediate osteoclastic differentiation and participate in bone resorption [58, 59]. Previous studies have demonstrated that downstream molecules may influence the Wnt/β-catenin signaling pathway, thereby modulating osteogenesis [60, 61]. The activation of Notch signaling pathway is also involved in a variety of biological processes, triggering downstream secretion of bioactive molecules and affecting the bone microenvironment. Upon comparing key genes associated with the Notch signaling pathway, it was discovered that the expression of genes, such as Notch1, Hes1, and Hey1, was upregulated after treatment with BT-apoEVs. According to our results, we speculate that engineered apoEVs suppress osteogenic differentiation by upregulating Hippo signaling pathway and Notch signaling pathway. These findings indicated that engineered PMSC-apoEVs can be employed as a nanotherapy for facilitating precise management of HO and other calcification diseases.

Metabolomic results showed that multiple enriched pathways are involved in metabolism according to KEGG analysis, including beta-alanine metabolism, mineral absorption, phenylalanine metabolism, protein digestion and absorption, pyrimidine metabolism, and other synthetic pathways. Pyrimidine metabolism, a crucial part of nucleotide metabolism, plays an indispensable role in DNA replication and RNA synthesis [62]. It’s also essential for glycosylation, phospholipid synthesis, cellular biomass, homeostasis, and the regulation of signal transduction [63, 64]. We observed changes in products related to pyrimidine metabolism, such as UMP, uracil, cytidine, 3’-CMP, and dTMP, implying that BT-apoEVs may mediate the regulation of pyrimidine metabolism. Relevant genes such as K01513, K01511, K13421, etc. are found to be downregulated in this pathway. Given that K01511 and K13421 are both genes involved in the process where orotate is converted to UMP, we presumed that BT-apoEVs regulate bone metabolism through this biological process. Furthermore, Yap1, a key transcriptional regulator in the Hippo signaling pathway, has been demonstrated to directly modulate the expression and activity of glutamine synthetase (Glul), thereby influencing pyrimidine metabolism [65]. A potential association between pyrimidine metabolism and the cleavage and activation of the Notch protein has also been established [66, 67]. ApoEVs are vesicles produced in the apoptosis process when the cell membrane wrinkles and encapsulates nucleic acid as well as organelles. Therefore, we speculate that the DNA and RNA inside the apoEVs are released after the internalization by cells, having impacts on pyrimidine metabolism. Regulation of pyrimidine metabolism is hypothesized to be the underlying metabolic mechanism of the inhibitory effects on osteogenic differentiation that BT-apoEVs exert. The potential correlation between these pathways and the effects of BT-apoEVs not only improves the understanding of the roles of BT-apoEVs, but also lays a theoretical foundation for further exploration of the BT-apoEVs’ regulatory functions on bone homeostasis. These findings also suggest a close association between metabolic pathways and the treatment of HO. However, the underlying mechanisms of the functional crosstalk among the Hippo pathway, the Notch pathway and pyrimidine metabolism remain to be dissected in detail.

Overall, our research provides solid evidence for supporting our multifunctional nanotherapy that integrates the regulation of osteogenic differentiation and osteoclastic differentiation, thereby emphasizing the application of BT-apoEVs in the management of HO. From a broader perspective, this straightforward and adaptable engineered apoEV can be the possible treatment of other complex calcification diseases, such as vascular calcification. More promisingly, our research highlights the feasibility and flexibility of employing patient-specific engineered apoEVs in clinical practice without raising ethical controversy or inducing immune rejection. Therefore, this research inspires us to construct multifunctional and x-targeting apoEVs that could be universally implemented for various complicated diseases.

This study still has several limitations. For instance, partial accumulation of BT-apoEVs in the liver and lungs was observed, which may trigger immune responses and other adverse effects. Moreover, our present study is not without the limitations that have been observed in previous EV-based therapeutic strategies, namely, the lack of long-term outcome analysis. Although this study has assessed the therapeutic effects of BT-apoEVs over a three-month period, extending the observation time is valuable for functional evaluation and future clinical translation, considering the chronic nature of HO. In light of these points, we plan to design a more rigorous experimental protocol and extend the observation period in future studies. Looking ahead, this study can further explore innovative modification strategies to enhance the multifunctional properties, such as integrating antimicrobial and sustained-release capabilities, and investigate potential synergistic effects of BT-apoEVs in combination with other therapeutic approaches. Additionally, detailed mechanism studies, including gene knockout or overexpression experiments, are essential to elucidate the underlying biological processes.

Conclusion

In the present study, an innovative bone-targeting delivery system based on engineered PMSC-apoEVs was successfully constructed. This engineered apoEV exhibits multiple functions including bone targeting, osteoclastic differentiation promotion and osteogenic differentiation inhibition. The engineered apoEVs target heterotopic bones under pathological conditions and regulate bone homeostasis through the mechanism of Hippo and Notch pathways, as well as pyrimidine metabolism. Therefore, the findings of this study offer a strong and simple strategy for the customization of apoEV functional attributes, thereby providing a promising minimally invasive and precise nanotherapy for HO with the potential for clinical translation.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (82471005 82201008 82271001 82301018).

Funding

This work was supported by the National Natural Science Foundation of China (grant number: 82471005, 82201008, 82271001, 82301018).

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Author notes

  1. Like Tang, Yuchen Wang and Shihua Mao contributed equally to this work.

Authors and Affiliations

  1. Stomatology Hospital, School of Stomatology, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Engineering Research Center of Oral Biomaterials and Devices of Zhejiang Province, Zhejiang University School of Medicine, Cancer Center of Zhejiang University, Hangzhou, 310000, China

    Like Tang, Yuchen Wang, Shihua Mao, Zhou Yu, Yitong Chen, Wenjin Cai, Kaichen Lai, Guoli Yang & Tingben Huang

  2. Department of Implantology, The Affiliated Stomatology Hospital, Zhejiang University School of Medicine, Hangzhou, 310000, China

    Kaichen Lai, Guoli Yang & Tingben Huang

  3. The Affiliated Stomatology Hospital, Zhejiang University School of Medicine, Hangzhou, 310000, China

    Xiaoqiao Xu

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Contributions

L.T., Y.W., and S.M. designed the experiment, carried out laboratory research and wrote draft of manuscript. Z.Y. and Y.C. performed some experiments and analysed some data. X.X., W.C., and K.L. revised the manuscript. G.Y. and T.H. provided financial support, assisted in designing research, approved the final version and submitted.

Corresponding authors

Correspondence to Guoli Yang or Tingben Huang.

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All animal experiments were approved in accordance with current guidelines for the care of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Zhejiang University (approval ID: ZJU20230285).

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Tang, L., Wang, Y., Mao, S. et al. Engineered bone-targeting apoptotic vesicles as a minimally invasive nanotherapy for heterotopic ossification. J Nanobiotechnol 23, 348 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03431-w

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Journal of Nanobiotechnology

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