E7 peptide and magnesium oxide-functionalized coaxial fibre membranes enhance the recruitment of bone marrow mesenchymal stem cells and promote bone regeneration

Background

The repair of bone defects remains a significant clinical challenge. Although magnesium (Mg)-based biomimetic scaffolds are widely utilized for bone defect repair, the release of Mg2+ ions often leads to an alkaline microenvironment, thereby adversely affecting bone regeneration. Regenerative medicine strategies that leverage the recruitment of endogenous bone marrow mesenchymal stem cells (BMSCs) offer a novel approach to treating bone defects.

Methods

In this study, we employed poly(L-lactic acid) (PLLA) and polyethylene glycol (PEG) as shell materials and nanomagnesium oxide (nMgO) combined with gelatin (G) as core materials to fabricate coaxial fibre membranes with a "core‒shell" structure via coaxial electrospinning technology. Additionally, we grafted the BMSC-affinitive peptide E7 (EPLQLKM) onto the fibres to achieve specific recruitment of endogenous BMSCs.

Results

Morphological and structural analyses confirmed the successful formation of the "core‒shell" structure of the fibre membranes. Grafting E7 peptides enhanced the hydrophilicity and mechanical properties of the fibre membranes and maintained pH stability in vitro. In vitro experiments demonstrated that the functionalized fibre membranes significantly promoted BMSC proliferation, migration, and osteogenic differentiation. When implanted into a rat cranial defect model, we observed the formation of new bone tissue and the repair of the bone defect.

Conclusions

E7 peptide-functionalized coaxial fibre membranes effectively facilitated bone defect repair by promoting the recruitment and osteogenic differentiation of BMSCs, demonstrating substantial potential for tissue engineering applications.

Annually, more than two million bone grafting surgeries are performed worldwide to address bone defects, imposing significant physical and economic burdens on patients [1]. Currently, the most commonly employed clinical treatments are autologous bone grafts and allogeneic bone grafts. However, these methods are limited by the scarcity of donor bone tissue, the risk of immune rejection, and potential infections. Consequently, the treatment of bone defects remains a major clinical challenge [2,3,4]. The development of novel synthetic bone repair biomaterials to reconstruct damaged bone tissue is considered one of the most promising approaches for treating bone defects [5].

Bone tissue engineering (BTE) comprises three fundamental components: scaffolds, cells, and bioactive factors. In terms of materials, electrospinning is one of the simplest techniques for preparing micro/nanofibres in tissue engineering. It can produce fibre membranes with different functions by controlling factors such as voltage, receiving distance, temperature, solution concentration, and solution composition [6]. Electrospinning can simulate the structure and function of the extracellular matrix (ECM), which is conducive to cell proliferation, adhesion, migration and differentiation. It is regarded as an important biomaterial carrier in bone defect repair [7,8,9]. PLLA is a commonly used artificial polymer biomaterial in electrospinning and features excellent biocompatibility, degradability and mechanical properties [10, 11]. For example, Lv [12] et al. prepared polylactic acid-hydroxyapatite (PLLA-HA-Hap) fibre scaffolds and used them for the regeneration of rotator cuff tendons. Han [13] et al. prepared polylactic acid/bovine serum albumin/nanohydroxyapatite (PLLA/BSA/nHAp) fibres. Compared with pure PLLA nanofiber pads, the PLLA/BSA/nHAp composite nanofibers stimulated the proliferation of mouse osteoblasts (MC3T3 cells) more effectively. However, the application of PLLA is limited by its relatively low biological activity, hydrophobicity and acidic degradation products [14, 15]. Gelatin is a natural polymer material that has excellent biocompatibility, low immunogenicity, and easy chemical modification. It has been widely applied in fields such as wound healing, drug delivery carriers, and tissue engineering [16]. However, its drawbacks include poor mechanical properties and structural stability, rapid degradation, and complete dissolution in aqueous media, which have led to significant limitations in the application of gelatin/collagen electrospun nanofibers in biomedicine [17, 18]. At the cellular level, BMSCs can differentiate into osteoblasts and promote bone repair; thus, enhancing the local enrichment and osteogenesis of BMSCs is crucial for accelerating bone regeneration [19,20,21]. Chen et al. [22] combined stromal cell-derived factor-1α (SDF-1α) and M2 macrophage-derived exosomes (M2D-Exos) with a hyaluronic acid (HA-HA) hydrogel precursor solution, the developed HA-HA@SDF-1 α/M2D-Exo hydrogel promoted the proliferation and migration of BMSCs and accelerated the repair of bone defects. With respect to bioactive factors, the incorporation of cytokines, bioactive peptides, natural polymers, and nucleic acid-based factors with biomaterials can significantly enhance the bioactivity and osteogenic performance of the materials [23,24,25]. The loading and delivery methods of bioactive factors are critical during material modification [26, 27], as these strategies facilitate precise regulation of the bone regeneration process, thereby improving the efficacy of bone defect repair.

Fracture repair consists of three stages: inflammation, repair and remodelling. In the field of bone repair, the vast majority of studies tend to focus on the latter two stages while ignoring the inflammatory stage of early cell recruitment. During this period, due to the lack of targeting and controllability in the recruitment of BMSCs and the obstruction of factors such as inflammation, the number of BMSCs involved in the process of bone repair often fails to meet the needs of the body [28,29,30,31]. Therefore, the timely and controlled recruitment of endogenous BMSCs and the promotion of their osteogenic differentiation to accelerate bone defect repair have remained focal points and challenges in research [23, 32]. Previous studies [33, 34] have demonstrated that the E7 peptide is a member of the SDF-1 family. It is derived from human bone marrow mesenchymal stem cells (hBMMSCs) and has simple and stable molecular structure, long half-life, high stability, easy synthesis and modification, and high biosafety. It can specifically and efficiently promote the proliferation, adhesion and migration of BMSCs to the targeted site without any species specificity. As essential trace elements in the body, magnesium ions (Mg2+) can upregulate the expression of osteogenic marker genes and proteins such as alkaline phosphatase (ALP), osteocalcin (OCN), and bone morphogenetic protein-2 (BMP-2). Additionally, their degradation products can neutralize acidity, thereby increasing the local pH and creating a favourable microenvironment for osteogenesis [35, 36]. However, the combination of these two bioactive factors within electrospun biomaterials for bone defect repair has rarely been reported.

Coaxial electrospinning is an advanced variant of conventional single-fluid electrospinning. Different from conventional electrospinning, its nozzle is composed of two "concentric circles" with different inner diameters but located on the same axis. Different inner diameters are respectively connected to different electrospinning solutions. By controlling the composition and proportion of the inner and outer layer fluids, the continuous and controlled release of the loaded molecules can be achieved. This distinctive "core-shell" structure can separately load different bioactive components, endowing the material with multi-functional properties and demonstrating significant advantages in the delivery of biological factors/drugs [37, 38]‌. Therefore, In this study, we utilized coaxial electrospinning technology to fabricate coaxial fibre membranes, using gelatin and nMgO as the core materials and PLLA with polyethylene glycol (PEG) as the shell material (the purpose of incorporating PEG was to introduce carboxyl groups (-COOH)). The E7 peptide was grafted via EDC/NHS(N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride)/(N-hydroxysuccinimide)). The core layer (gelatin /nMgO) enables the sustained release of bioactive factors, while the shell layer (PLLA/PEG) provides mechanical support and serves as a grafting platform for E7 polypeptides. We expect that the unique "core-shell" structure of coaxial electrospinning can achieve the controllable release of Mg²⁺ ions and maintain the stability of pH in vitro to avoid the side effects of the alkaline microenvironment caused by the rapid release of Mg²⁺. In addition, after grafting with E7 peptide, the hydrophilicity of the shell is enhanced, endowing it with the ability to recruit BMSCs. Ultimately, under the action of Mg²⁺, it promotes osteogenic differentiation and accelerates the repair of bone defects (Fig. 1). In summary, this study proposes a novel material design for bone defect repair. Previous studies on promoting bone repair through the E7 peptide and nMgO have been reported, but no research has combined these two materials. Therefore, we designed a new type of bone defect repair material. This research not only fills the gap in the related field but also provides new ideas and a theoretical basis for bone tissue engineering.

Preparation of coaxial electrospun fibres

PLLA (MW = 16 kDa, Ji’nan Daigang, Shandong) at 0.256 g and PEG (MW = 3400, Beijing Baidai, Beijing) at 0.064 g were dissolved in 2 mL of hexafluoroisopropanol (HFIP, McLin, Shanghai) to prepare a 16% w/v shell-layer solution. G (Aladdin, China) at 0.1 g and nMgO (Aladdin, China) at 0.02 g were dissolved in 1 mL of HFIP to prepare a core-layer solution with a nMgO concentration of 2% w/v. The two electrospinning solutions were separately placed on a magnetic stirrer (Shanghai Sile, China) and stirred overnight, followed by ultrasonic treatment (40 kHz, Crownboshi, Shenzhen) for 30 min. The two electrospinning solutions were then transferred to 10 mL syringes and connected to corresponding 17/22G coaxial nozzles (Yongkang Leye, Beijing). The coaxial electrospinning parameters were adjusted as specified in Sig. 1 to fabricate coaxial fibre membranes. All electrospun membranes were stored at room temperature for three days to remove residual organic solvents.

The EDC/NHS(Solaibao, China) solution was prepared in phosphate-buffered saline (PBS pH 7.4) at a molar ratio of 2:1. The scaffold was immersed in the solution and reacted at room temperature in the dark for 2 h. Then, E7 polypeptide solution (0.1 mM, Beijing Zhongke Yaguang, Beijing) was added and the reaction continued at room temperature in the dark for 2 h. Finally, the membranes were washed three times with PBS. On the basis of the presence of the E7 peptide and nMgO, the fibre membranes were designated the PLLA/G, PLLA/MgO, PLLA/G@E7, and PLLA/MgO@E7 groups.

Characterization of coaxial electrospun fibres

The fibre membranes were prepared as 1 cm ×ばつ 1 cm pieces and gold-sputtered. The surface morphologies were scanned by scanning electron microscopy (SEM, Hitachi Japan) at different magnifications in random areas. The microstructure was observed via transmission electron microscopy (TEM, Japan Electronics Co. LTD). Hydrophilicity was measured using a water contact angle meter (Oca 20, Dataphysics Co. Ltd. Germany). A 5 μL droplet of deionized water was placed at a height of 5 cm above the sample, and the contact angle was immediately captured via ImageJ software (ImageJ, USA) (n = 3). Fourier transform infrared spectroscopy (FTIR, PerkinElmer, USA) was employed to analyse the fibre membranes. Each group of fibre membranes was prepared as 0.2 cm ×ばつ 0.2 cm pieces and scanned in the wavelength range of 600 cm−1 to 4000 cm−1 (n = 3). The mechanical properties were tested via a universal testing machine (EZ-LX, Shimadzu, Japan). All the samples were prepared in rectangular shapes of 3 cm ×ばつ 1 cm and clamped at both ends with sandpaper, and the length between the clamps was recorded. A load of 50 N was applied at a tensile rate of 1 mm/min, and the stress‒strain curves were recorded (n = 3). The ultimate tensile strength (UTS), elongation at break, and elastic modulus were calculated via Origin software.

In vitro degradation of electrospun fibres

Fibre membranes were prepared as 18 mm diameter circles and placed into a 12-well plate, with 3 mL of PBS added to each well. The fiber membrane was fixed to the bottom of the orifice plate with sterile stainless steel mesh to prevent floating displacement during PBS incubation. The samples were incubated at 37 °C, and the soaking solutions were collected at 2, 4, 8, 12, 24, 48, 72, 120, 168, 216, 288, and 336 h, with the same volume of fresh PBS added each time. Finally, the pH of the soaking solutions was measured via a pH meter (n = 3). For degradation studies, the fibre membranes were weighed before incubation W0 and then soaked in 3 mL of PBS at 37 °C on a constant-temperature shaker, with the medium replaced every three days. After 1, 3, 7, 14, 21, and 28 days, the membranes were removed, dried, and Wt was weighed (n = 3). The remaining mass percentage was calculated as follows:

E7 peptide release assay

The in vitro release profile of the FITC-labelled E7 peptide (EPLQLKM-FITC, Beijing Zhongke Yaguang, Beijing) was determined. A 0.1 mM E7 peptide solution was prepared and serially diluted. The absorbance was measured at 485/538 nm to construct a standard curve. Each group of fibre membranes was prepared as 18 mm diameter circles, grafted with the E7 peptide, and washed three times with PBS, and the soaking and washing solutions were collected for absorbance measurement. The grafting efficiency was calculated on the basis of the standard curve (n = 3). The fibre membranes were then placed in test tubes with 2 mL of PBS and incubated at 37 °C in the dark. The fiber membrane is fixed to the bottom of the orifice plate with sterile stainless steel mesh to prevent floating displacement during PBS incubation. At 1, 3, 7, 14, 21, and 28 days, the soaking solutions were collected and replaced with an equal volume of fresh PBS. The absorbance was measured at 485/538 nm to determine the release profile (n = 3).

Cells and materials

Rat BMSCs were purchased from Cyagen Biosciences (Cyagen, China), and P3-P5 generation cells were cultured at 37 °C and 5% CO₂ in α-MEM (HyClone, Wuhan) supplemented with 10% FBS (Newzerum, New Zealand) and 1% penicillin-streptomycin (Thermo, China).

Prior to BMSC seeding, all the fibre membranes were sterilized by soaking in 75% ethanol for 2 h, followed by ultraviolet (UV) irradiation for 1 h.

Biocompatibility of electrospun fibres

Fibre membranes were prepared as 16 mm diameter circles and fixed in 24-well plates. The plates were seeded with BMSCs at a density of 3 ×ばつ 104 cells/mL per well. For live/dead staining, a Calceiin-AM/PI kit was used. Each well received 300 μL of the staining solution and was incubated at 37 °C for 15 min in the dark. A fluorescence microscope (Olympus, Japan) was used to observe and photograph the samples at excitation wavelengths of 490 nm and 560 nm (n = 3). For the CCK-8 assay, 300 μL of medium containing 10% CCK-8 reagent (Dojindo, Japan) was added to each well and incubated at 37°C for 2 h in the dark. Subsequently, 100 μL of the solution was transferred to a 96-well plate, and the absorbance was measured at 450 nm (n = 3).

In vitro transwell assay

Fibre membranes were prepared as 16 mm diameter circles and fixed in 24-well plates. The ability of the fibre membranes to recruit BMSCs was evaluated via Transwell experiments. P3 BMSCs that grew well were serum starved for 6 h in serum-free medium. The cells were digested and centrifuged and then resuspended at a density of 2 ×ばつ 105 cells/mL. Two hundred microliters of the cell suspension was added to the upper chamber of the Transwell, and 700 μL of complete medium was added to the lower chamber. The cells were incubated at 37 °C in a 5% CO2 incubator for 12–24 h. After incubation, the cells were fixed with 4% paraformaldehyde (Thermo, China) for 15 min, washed three times, and stained with 10% crystal violet (Solaibao, Beijing) for 10 min. Nonmigrated cells in the upper chamber were removed, and the migrated cells were mounted on slides using neutral resin and observed under a microscope (Olympus, Japan) (n = 3).

Osteogenic differentiation and staining of BMSCs

Fibre membranes were prepared as 16 mm diameter circles and fixed in 24-well plates. P3 BMSCs in logarithmic growth phase were seeded at a density of 5 ×ばつ 104 cells/mL in 24-well plates. After 24 h of incubation, the medium was replaced with osteogenic induction medium (complete medium containing 100 nM dexamethasone, 10 mM β-glycerophosphate disodium, and 50 μg/mL ascorbic acid). The osteogenic induction medium was changed every two days. After 7 and 14 days of incubation, the cells were fixed with 4% paraformaldehyde for 30 min and stained with a BCIP/NBT alkaline phosphatase staining kit (Beyotime, Shanghai) according to the manufacturer’s instructions. Each well received 300 μL of the staining solution and was incubated at 37 °C for 30 min in the dark, followed by three washes with PBS. The stained cells were observed under a stereomicroscope (OLYMPUS, Japan). The cells cultured under the same conditions were lysed with 1% Triton X-100, the lysate was obtained after 30 min, and the supernatant was collected via high-speed frozen centrifugation(12,000 rpm, 15 min, 4°C). ALP activity was measured via an ALP assay kit (Beyotime, Shanghai), and the absorbance was measured at 405 nm. The total protein content was determined via a BCA assay kit (Beyotime, Shanghai), and the ALP activity was normalized to the total protein content as described in the kit instructions (n = 3).

Alizarin Red S (ARS) staining was performed to assess mineral deposition during osteogenic differentiation. After 14 and 21 days of coculture under osteogenic conditions, the samples were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and washed three times. ARS staining solution (OriCell, China) was added and incubated at 37 °C for 2 h, and images were collected under a stereomicroscope (OLYMPUS, Japan). The solution was subsequently dissolved in cetylpyridinium chloride (CPC, 10%, Sigma Diagnostics, USA), and the absorbance was measured at 562 nm (n = 3).

Establishment of the SD rat cranial defect model

Eight-week-old male SD rats weighing 250–300 g were used to establish cranial defect models. The rats were anaesthetized via the intraperitoneal injection of 2% sodium pentobarbital. The scalp was shaved, and the surgical area was disinfected with povidone-iodine. After the cranium was adequately exposed, a 5 mm diameter full-thickness defect was created via a trephine drill. Before implanting the fiber membrane, we cut the fiber membrane into 5 mm diameter circles and disinfected and sterilized them. The rat skull defect model also has a 5 mm skull defect. Therefore, the fibrous membrane can be directly placed at the skull defect site to make its surface flush with the outer surface of the skull. Stable fixation is achieved by relying on the surrounding soft tissues and periosteum, without the need for additional reinforcement measures. Finally, we carefully sutured the soft tissue to the skin layer by layer. Postoperatively, the rats received intraperitoneal injections of carprofen (5 mg/kg, Merck, Germany) for three days to alleviate postoperative pain. All the rats were housed in an SPF animal room with free access to food and water during the study period.

Tissue sectioning and Immunofluorescence staining

Fibre membranes from the PLLA/G, PLLA/MgO, PLLA/G@E7, and PLLA/MgO@E7 groups were implanted into the cranial defects of SD rats, with six rats per group. At 3 and 7 days, the rats were euthanized, and the cranial bones were fixed in 4% paraformaldehyde for one week, followed by decalcification in EDTA (Thermo, China) for three weeks. Then, the paraffin was sliced, dewaxed and rehydrated, and 10% donkey serum (BSA; Servicebio, China) was added, after which the samples were blocked for 30 min. The samples were incubated overnight with CD90 (derived from mice, green fluorescence) and CD29 (derived from rabbits, red fluorescence) monoclonal dilutions (1:300) (Abcam, Beijing) at 4 °C. Following incubation, the sections were treated with Cy3-conjugated goat anti-mouse secondary antibodies (1:300) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (1:400) (Servicebio, China) at room temperature for 1 h. After washing with PBS, the nuclei were stained with DAPI (Servicebio, Wuhan) for 10 min in the dark. The slides were fixed with glycerin and viewed under a fluorescence microscope.

Micro-CT scanning of bone defective tissue

Fibre membranes from the PLLA/G, PLLA/MgO, PLLA/G@E7, and PLLA/MgO@E7 groups were implanted into cranial defects of SD rats, with an additional group serving as a blank control without fibre membrane implantation. At 4 and 8 weeks, the rats were euthanized, and their cranial bones were fixed in 4% paraformaldehyde for one week. Microcomputed tomography (micro-CT, NEMO Micro-CT, Pingseng Healthcare Inc., Kunshan, Jiangsu, China) was used to scan and analyse the femurs of the rats. The scanning parameters included a layer thickness of 30 μm, a voltage of 90 kV, and a current of 500 mA. Three-dimensional images were reconstructed via Avatar software. The bone volume/total volume (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were quantified via Origin software.

Haematoxylin and Eosin (HE) and Masson staining

Cranial bones were decalcified in EDTA solution for six weeks, with weekly changes in the decalcifying solution. Following decalcification, the samples underwent gradient dehydration and were embedded in paraffin. Sections were cut along the axial plane at a thickness of 10 μm. Deparaffinization and rehydration were performed through successive immersions in xylene I (20 min), xylene II (20 min), absolute ethanol I (5 min), 75% ethanol (5 min), and distilled water (5 min). HE staining and Masson’s trichrome staining were conducted according to standard protocols. The stained sections were observed under a microscope.

Statistical analysis

The data were analysed via Origin 2021 (OriginLab, Massachusetts, USA). All experimental data are presented as the mean ± standard deviation (SD). Univariate analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for analysis. A p value of < 0.05 was considered to indicate statistical significance. "ns" denotes no statistically significant difference.

Physical characterization of coaxial fibres loaded with nMgO and the E7 peptide

SEM revealed that the surfaces of the fibre membranes across all four groups were smooth, with uniform diameters and random orientations (Fig. 2A), indicating that grafting the E7 peptide did not alter the morphology of the fibre membranes. In the two groups containing nMgO, occasional bead-like protrusions were observed, likely resulting from the aggregation of nMgO into particulate matter (red arrow in Fig. 2A). TEM confirmed the distinctive "core‒shell" structure of the coaxial electrospun fibres, where the light gray region comprised the PLLA and PEG shell layers, and the black region consisted of G or nMgO, which formed the core layer. The contact angles of the four fibre membrane groups were as follows (Fig. 2B): the PLLA/G group (116.23 ± 2.81°), the PLLA/MgO group (102.97 ± 8.60°), the PLLA/G@E7 group (90.13 ± 10.00°), and the PLLA/MgO@E7 group (49.77 ± 8.89°). Compared with the PLLA/G group, the PLLA/MgO group exhibited lower hydrophilicity, but the difference was not statistically significant (p > 0.05) because the nMgO was wrapped in the inner layer rather than exposed on the surface. However, there were significant differences in hydrophilicity before and after the grafting of the E7 peptide (p = 0.017, p < 0.001), demonstrating that the E7 peptide enhances the hydrophilicity of the fibre membranes.

FTIR analysis of the four fibre membrane groups and their respective components is shown in Fig. 2C. The characteristic peaks of amide I and amide II of gelatin (light yellow) were at 1620 cm^-1 and 1517 cm^-1, respectively. The peak at 1750 cm^-1 corresponds to the typical asymmetric stretching of the ester carbonyl group in PLLA (light green), whereas the absorption peaks at 2995 cm^-1 and 2945 cm^-1 are attributed to asymmetric and symmetric C–H stretching vibrations (light tan), respectively. The absorption peak between 1452 cm^-1 and 1382 cm^-1 corresponds to the symmetric and asymmetric bending of C-H in PLLA (light orange). Weak amide I and amide II peaks were observed at 1620 cm^-1 and 1517 cm^-1 in the PLLA/G@E7 and PLLA/MgO@E7 groups, which may have resulted from the release of gelatin following crosslinking in aqueous solution. The characteristic peaks of each component were observable in the fibre membranes without the formation or significant shift of new absorption peaks, indicating that no chemical reactions occurred between the components.

Mechanical testing revealed that all four fibre membranes initially followed Hooke’s law, exhibiting elastic deformation during the early stages of stretching, followed by plastic deformation upon reaching the yield point (Fig. 2D). Ultimately, the materials fractured, leading to a linear decrease in stress. There were significant differences in the ultimate tensile strength (UTS) and elongation at break of the fibre membranes before and after the grafting of the E7 peptide (Fig. 2E, F) (p < 0.001), indicating that the E7 peptide increased the UTS of the fibre membranes but reduced their extensibility. The elongation at break of the PLLA/MgO and PLLA/MgO@E7 groups was lower than that of the PLLA/G and PLLA/G@E7 groups (p < 0.001) (Fig. 2F), suggesting that nMgO reduces the extensibility of the material. The Young’s moduli of the four fibre membrane groups were as follows (Fig. 2G): the PLLA/G group, 44.23 ± 0.02 MPa; the PLLA/MgO group, 34.44 0.03 MPa; the PLLA/G@E7 group, 106.97 ± 0.03 MPa; and the PLLA/MgO@E7 group, 65.245 ± 0.03 MPa. Compared with the groups without nMgO, the two groups containing nMgO presented a lower Young’s modulus (p = 0.017). The E7 peptide level after grafting was greater than that before grafting (p = 0.011, p < 0.001). The results revealed that the addition of nMgO reduced the Young’s modulus of the fibrous membrane, whereas the addition of the E7 peptide had the opposite effect (p < 0.001).

In vitro degradation of coaxial fibres

Figure 2H shows the pH changes in the four groups of fibre membranes in vitro. Within the first 6 h, the pH of all four groups increased, likely due to the rapid release of gelatin or Mg²⁺ from the fibre edges. In particular, the pH of the PLLA/MgO group was approximately 8.3. The two groups without nMgO presented a mildly acidic pH, which decreased from 7.3 to approximately 7.0 and subsequently stabilized at approximately 6.9. In contrast, the groups containing nMgO maintained a more stable pH, decreasing from 7.5 to approximately 7.2 within the first 9 days and then further stabilizing at approximately 7.1. These results indicate that the fabricated coaxial fibre membranes effectively maintain pH stability in vitro.

In vitro release of the E7 peptide

The remaining mass of the fibre membranes decreased progressively over time (Fig. 2I). Within the first week, all the membranes exhibited rapid weight loss, with the PLLA/MgO@E7 group showing the most significant decrease (approximately 30%). The degradation rate of the fibre membranes subsequently slowed. After 28 days, the degradation rates of the four materials were approximately 27%, 36%, 30%, and 40%, respectively. The degradation rates of the fibre membranes containing nMgO were higher than those of the two groups without nMgO, which was attributed to the rapid release of nMgO. After E7 was grafted, the degradation rate increased. We speculated that this might be due to the loss of some of the peptides during soaking in the aqueous solution when the E7 peptides were grafted. Figure 2J shows that the amount of E7 peptide grafted onto the two fibre membrane groups was not significantly different (p > 0.05). Both E7-grafted fibre membrane groups released approximately 55% of the E7 peptide within the first 7 days, followed by a gradual stabilization of release up to approximately 90% (Fig. 2K).

Coaxial fibres exhibit good biocompatibility

Biocompatibility is a crucial characteristic of ideal biomaterials. Live/dead staining was used to evaluate the survival of BMSCs on the fibrous membranes (Fig. 3A). On day 1, the BMSCs were scattered on the fibrous membranes of all four groups, with no or few dead cells observed. As the culture time increased, the number of proliferating BMSCs on the fibrous membranes of all four groups gradually increased, almost covering the entire field of view. Especially on the seventh day, the number of cells in the PLLA/MgO@E7 group was the highest. The results of the CCK-8 assay were consistent with those of the live/dead staining (Fig. 3C). On the first day, there was no statistically significant difference in absorbance among the four groups (p > 0.05). On the fourth day, the absorbances of the PLLA/G@E7 and PLLA/MgO@E7 groups were greater than those of the PLLA/G and PLLA/MgO groups, respectively (p < 0.001, p = 0.002). On the seventh day, the differences among the groups further increased. The absorbances of the PLLA/MgO and PLLA/MgO@E7 groups were greater than those of the PLLA/G group (p < 0.001) and the PLLA/G@E7 group (p = 0.017), respectively. The two groups grafted with E7 had greater absorbances than the two groups without grafting did (p < 0.001), with the PLLA/MgO@E7 group having the highest absorbance. In conclusion, nMgO and E7 can promote the proliferation of BMSCs on fibrous membranes.

Coaxial fibres recruit bMSCs in vitro via a transwell assay

A Transwell assay was employed to investigate the ability of the fibre membranes to recruit BMSCs in vitro (Fig. 3B, D). Over time, the number of BMSCs migrating to the lower chamber of the Transwell increased. At 12 h, the PLLA/G group and PLLA/MgO group presented only a few migrated cells, with no significant difference compared with the blank control group (p > 0.05). At 24 h, the number of BMSCs recruited by all the fibre membrane groups increased, and it was greater in the E7-grafted groups than in the nongrafted groups (p < 0.001). These results demonstrate that the E7 peptide can promote the recruitment of BMSCs in vitro.

Coaxial fibres promote the osteogenic differentiation of BMSCs

Osteogenic differentiation of BMSCs was assessed by quantifying alkaline phosphatase (ALP) activity and calcium nodule deposition. ALP activity is typically regarded as an early biochemical marker of osteogenic differentiation, whereas calcium nodules signify late-stage differentiation. After coculture with BMSCs, all the groups promoted osteogenic differentiation, and over time, the ability of each group to induce osteogenic differentiation gradually increased (purple in Fig. 4A). At both 7 and 14 days, the PLLA/MgO@E7 group exhibited significantly deeper staining than the other three groups did. Figure 4C presents the semiquantitative analysis of ALP activity, which revealed that Compared with the other three groups, the PLLA/MgO@E7 group presented markedly greater ALP activity at both time points (p < 0.05). Additionally, there was no significant difference between the PLLA/MgO and PLLA/G@E7 groups at 14 days (p > 0.05).

Mineral deposition, another key indicator of osteogenic efficiency, was evaluated via alizarin red S (ARS) staining to observe the formation of mineralized nodules. After 14 and 21 days of coculture with BMSCs, the PLLA/MgO@E7 group produced more calcium nodules than the other groups did (Fig. 4B). At 14 and 21 days, semiquantitative analysis revealed no significant difference between the PLLA/MgO and PLLA/G@E7 groups (p > 0.05), yet both were significantly lower than those of the PLLA/MgO@E7 group, which was consistent with the staining results (Fig. 4D). Collectively, these findings indicate that the combined use of nMgO and the E7 peptide optimally enhances the osteogenic differentiation of BMSCs, whereas the individual use of nMgO or the E7 peptide does not significantly affect the osteogenic capacity (p > 0.05).

Coaxial fibres recruit BMSCs in vivo

To evaluate the recruitment of endogenous BMSCs to the cranial defect area in mice, fluorescent labelling of the BMSC surface markers CD29 (green) and CD90 (red) was performed (Fig. 5A, B). At 3 days postimplantation, there was no statistically significant difference in the average fluorescence intensity between the PLLA/G and PLLA/MgO groups (p > 0.05). However, the average fluorescence intensities of the PLLA/MgO@E7 and PLLA/G@E7 groups were greater than those of the other two groups, although the difference was not statistically significant (p > 0.05) (Fig. 5C). At 7 days, the PLLA/MgO@E7 and PLLA/G@E7 groups presented stronger fluorescence, and the PLLA/MgO@E7 group presented greater compared with the PLLA/G@E7 group (p = 0.002) (Fig. 5D). These results indicate that both the nMgO and E7 peptides promote the migration of BMSCs to the targeted site in vivo.

Coaxial fibres enhance cranial bone defect repair in rats

A critical-sized (5 mm) cranial defect model was established in rats, with the four groups of fibre membranes implanted into the cranial defects and an additional group serving as a blank control without fibre membrane implantation to evaluate in vivo bone regeneration. In the experimental groups, the growth of new bone tissue increased over time, and the PLLA/MgO@E7 group presented significantly more new bone formation than the other four groups did (Fig. 6A). The blank control group exhibited no or only minimal new bone ingrowth, confirming that critical-sized bone defects in rats do not naturally heal under physiological conditions. Transverse bone ingrowth was observed in all the experimental groups, especially in the PLLA/MgO@E7 group. New bone almost continuously crossed the defect site and closely integrated with the surrounding normal bone tissue by the eighth week. Quantitative analysis of new bone formation was conducted via bone volume/total volume (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) (Fig. 6B-F). At 4 and 8 weeks, the PLLA/MgO@E7 group presented higher BV/TV and BMD values than the other groups did, whereas no significant differences were observed between the PLLA/MgO and PLLA/G@E7 groups (p > 0.05). Quantitative analysis of Tb.N, Tb.Th, and Tb.Sp revealed that the PLLA/MgO@E7 group had the greatest number of new trabeculae, the greatest thickness, and the shortest spacing. In summary, the PLLA/MgO@E7 group demonstrated the most effective promotion of cranial bone defect repair in rats.

HE and Masson staining

At the fourth week, in the experimental group, incomplete degradation of the fibrous membrane and no obvious inflammation or necrosis were observed (Fig. 7A). The blank control group only showed ingrowth of fibrous connective tissue without new bone formation. In the PLLA/G and PLLA/MgO groups, only a small amount of new bone was observed at the defect margins, with weakly stained fibrous tissue filling the central area and noticeable gaps between the scaffold and host bone. In contrast, the PLLA/G@E7 and PLLA/MgO@E7 groups presented new bone tightly connected to the original tissue. In particular, in the PLLA/MgO@E7 group, more extensive new bone formation was observed, spreading from the defect margins towards the center. At the eighth week, the blank control group still only presented fibrous connective tissue ingrowth, whereas the experimental groups presented more new bone formation and minimal residual undegraded fibre membranes. The PLLA/MgO@E7 group presented thicker and more mature new bone that nearly spanned the entire defect area. The results of Masson’s trichrome staining were consistent with those of HE staining (Fig. 7B). The blank control group presented no new bone formation. The PLLA/G and PLLA/MgO groups displayed varying degrees of blue staining, indicating that the newly formed bone was composed of sparsely distributed collagen fibres and that there was a minor infiltration of inflammatory cells on the fibre membranes. In contrast, the PLLA/G@E7 and PLLA/MgO@E7 groups exhibited deeper and more extensive blue staining, confirming the formation of thicker and more mature new bone. Additionally, varying amounts of new blood vessel formation, which is crucial for functional bone regeneration, were observed.

Electrospinning is one of the easiest ways to prepare micro/nanofiber scaffolds, and it is also one of the easiest methods to load bioactive factors/drugs into micro/nanofiber scaffolds [39]. Compared with other bionic biomaterials, such as hydrogels, electrospinning has unique advantages in simulating the structure and function of ECMs [40]. In addition, as carriers for transportation, conventional electrospinning often results in the rapid release of drugs or biological factors due to its simple structure. This initial and rapid release is often explosive, which may cause irreversible damage to surrounding cells [41,42,43].

Coaxial electrospinning is an advanced variant of conventional electrospinning. Its unique "core-shell" structure can achieve sustained and controlled release of biological factors according to the relative thickness of the inner and outer layers, which is one of the easiest ways to prepare multifunctional scaffolders [38, 44,45,46]. In this study, we utilized coaxial electrospinning technology to use nMgO and G as the core layer, PLLA and PEG as the shell layer, and the E7 peptide was grafted through EDC/NHS. The PLLA in the shell degrades into lactic acid monomers, increasing the local acidity. However, the release and hydrolysis of MgO in the inner shell to produce OH^- can neutralize acidic products. The grafted E7 peptide can achieve in situ recruitment of BMSCs and promote their osteogenic differentiation through Mg²⁺. We hope to maintain the stability of PH in vitro through the unique structure of coaxial electrospinning and accelerate the repair of defects by in situ recruitment of BMSCs and promotion of their osteodifferentiation.

Mg has the potential to promote bone integration and angiogenesis. Therefore, the applications of modern magnesium-based biomaterials can be divided into two main fields: vascular applications and orthopedic applications [47]. In the field of osteogenesis, Mg²⁺ can promote osteogenesis through multiple signalling pathways, such as the MAPK/ERK, Wnt/β-catenin, and TRPM7/PI3K pathways [48,49,50]. Mg²⁺ has been reported to be strictly concentration dependent in regulating bone formation. An appropriate concentration of Mg²⁺ can promote the proliferation and osteogenic differentiation of BMSCs. However, the rapid release of Mg²⁺ leads to an excessively high concentration of Mg²⁺, which results in an alkaline microenvironment and instead inhibits the proliferation and osteogenic differentiation of BMSCs [51,52,53]. Studies have shown that a Mg²⁺ concentration gradient of 6–10 mM may be favourable for the proliferation and osteogenic differentiation of BMSCs, whereas a concentration greater than 10 mM inhibits the proliferation of BMSCs. This is attributed to the fact that a local high concentration of Mg²⁺ hinders the mineralization of the bone matrix and disrupts the bone regeneration process [54,55,56,57]. For example, Wang [58] et al. reported that 6 mM and 10 mM Mg²⁺ could enhance cell osteogenesis, and they attributed this effect to at least the activation of the PI3K/Akt signalling pathway; however, a high concentration (18 mM Mg²⁺) has an inhibitory effect on the biological behavior of osteoblasts. Liu [59] et al. prepared fibre membranes containing different concentrations of nMgO (nMgO-0, 0.5, 1, 1.5, 2). They reported that with increasing nMgO content, the osteogenic effect of the membrane significantly increased (nMgO-0, 0.5, 1, 1.5), whereas there was no significant difference between the nMgO-2 membrane and the nMgO-0 membrane. These findings indicate that a blended fibre membrane containing 1.5% or less nMgO is beneficial for the osteogenic differentiation of BMSCs. Similarly, Zhang [60] et al. reported a similar phenomenon. They reported that when the magnesium content in calcium phosphate ceramics exceeded 10%, it inhibited the osteogenic differentiation of stem cells.

Consequently, we prepared fibre membranes with varying concentrations of nMgO (0%, 0.5%, 1%, 1.5%, 2%, and 2.5%) and evaluated their cytotoxicity via CCK-8 and live/dead staining assays (Sig 2). Our results indicated that all the fibre membrane groups promoted BMSC proliferation at all the time points. Especially on the 7th day, the number of proliferating cells in the PLLA/MgO-2 group was the greatest, and the absorbance was the greatest. Therefore, we selected a 2% nMgO concentration for subsequent experiments. Additionally, we observed that the PLLA/MgO-2.5% group had a lower absorbance than the PLLA/MgO-2% group but was still higher than that of the PLLA/MgO-0% group. These findings indicate that the scaffold still promoted BMSC proliferation as the nMgO concentration continued to increase. We speculate that this might be because the coaxial fibre membrane can effectively control the rapid release of Mg²⁺.

This study successfully fabricated nMgO coaxial electrospun fibres grafted with the E7 peptide and systematically evaluated their potential for repairing extensive bone defects. SEM observations revealed uniformly smooth and randomly oriented fibres across all four groups. The "core‒shell" structure of coaxial fibers has been verified through direct observation of shell/core delamination using TEM(Fig. 2A). Although cross-sectional SEM/EDS analysis was not performed due to limitations in sample preparation techniques, the FTIR spectrum(Fig. 2C) and pH stability data(Fig. 2H) further confirm the structural design. This structure providing a solid foundation for subsequent biological functions [61]. The hydrophilicity of biomaterials is a crucial physicochemical property that directly influences their biological performance [62]. We found that the addition of nMgO alone did not significantly alter the hydrophilicity of the fibre membranes, likely because the nMgO was encapsulated within the inner layer. In contrast, the grafting of the E7 peptide significantly improved the contact angle, increasing hydrophilicity. This improvement is attributed to the presence of numerous hydrophilic groups in the E7 peptide, such as amino groups (-NH2), which facilitate better cell adhesion and spreading on the fibre membranes, thereby promoting cell proliferation and migration [63]. The mechanical properties of the scaffold are vital for bone tissue repair and largely determine the behavior of osteoclasts and osteoblasts [64]. The addition of nMgO reduced the Young’s modulus and extensibility of the fibre membranes, but the overall mechanical properties remained within suitable ranges, as confirmed by our study [65, 66]. This reduction may be due to the tendency of the nMgO to aggregate into clumps (red arrows in Fig. 2A) [52], forming stress concentration points that diminish the overall mechanical performance. Grafting the E7 peptide increased the Young’s modulus and ultimate tensile strength of the fibre membranes but significantly reduced their extensibility. This may be because the biomimetic material undergoes crosslinking in an aqueous solution. And in aqueous solution, H₂O molecules decompose hydrogen and electrostatic bonds or other chemical bonds that fix collagen fibers together, which may be the reason for the decrease in the elongation at break of the material [67]. In addition, gelatin will absorb water and expand in aqueous solution; The Mg (OH)₂ generated by the degradation of nMgO will cause volume expansion, while the outer shell PLLA restricts the expansion of both. This pressure will force the two phases to adhere more tightly, thereby forming a "pressure welding" effect, which improves the material’s stress transmission efficiency and resistance to deformation. 2During the crosslinking process, the better hydrophilic properties of EDC/NHS absorb more water molecules as plasticizers for collagen, while water molecules are not removed during air drying, resulting in more freedom of movement for fibers and allowing for greater reorientation during stretching, ultimately affecting the Young’s modulus and tensile strength of the material [68, 69]. In vitro degradation experiments demonstrated that fibre membranes containing nMgO exhibited accelerated degradation rates, facilitating the gradual replacement of the material by new bone tissue during bone regeneration [70]. For pure or mixed electrospinning, the initial rapid release of Mg²⁺ can cause the pH of the microenvironment to rise to approximately 8–10 [71]. In this study, the rapid increase in pH in the PLLA/MgO group in the first 4 h may have been caused by the rapid release of the cutting edge when the fibrous membrane was cut. While the pH of the PLLA/MgO@E7 group did not increase rapidly because of the grafting of the E7 peptide, the fibrous membrane of this group was soaked in solution for a period of time. During the following period, the pH was maintained at approximately 7.5 and then gradually decreased to approximately 7.1. This finding shows that the stability of the pH value of the coaxial fibre membrane in vitro can avoid, to a certain extent, the adverse effects of the alkaline environment caused by the release of Mg²⁺ on bone regeneration and provide a favourable microenvironment for bone repair. In addition, our research also revealed that a fibrous membrane can achieve the sustained release of the E7 peptide, thereby promoting the long-term recruitment of BMSCs. Studies have shown that the recruitment of the E7 peptide may be related to the stroma cell-derived factor-1/G protein-coupled receptor C-X-C motif chemokine receptor 4 (SDF-1/CXCR4) signal transduction pathway [72], which plays a crucial role in the migration and homing of mesenchymal stem cells to damaged tissues [73]. When stent implantation is defective, the release of the E7 peptide promotes the upregulation of SDF-1 and CXCR4 gene expression in MSCs. SDF-1 and CXCR4 are upstream switches of many migration pathways. Their combination activates a variety of downstream signalling pathways, including extracellular signal-regulated kinase(ERK) [74], PI3K/Akt [75, 76] and p38 [77], which in turn promotes MSC migration, proliferation, differentiation, survival and apoptosis.

Both the E7 peptide and Mg²⁺ promoted BMSC proliferation [78, 79], as evidenced by our results. The PLLA/MgO@E7 group showed more pronounced BMSC proliferation, indicating that the nMgO and E7 peptides synergistically enhance BMSC proliferation (Fig. 3A, C). Previous studies [80, 81] have demonstrated that the E7 peptide can promote the migration of BMSCs to targeted sites both in vitro and in vivo, which our study confirmed (Fig. 3B, D). Additionally, in the Transwell assay, the groups containing nMgO had greater numbers of migrating cells than did those without nMgO, suggesting that Mg²⁺ also facilitates BMSC migration to some extent [36, 59]. However, the PLLA/G@E7 group exhibited greater cell recruitment than the PLLA/MgO group did (p < 0.001), indicating that, compared with Mg²⁺, the E7 peptide alone has a superior ability to recruit BMSCs. ALP and ARS staining are early and late indicators of osteogenic differentiation, respectively. Compared with the other three groups, the PLLA/MgO@E7 group demonstrated superior osteogenic capabilities at all time points (Fig. 4), indicating that the synergistic effect of Mg²⁺ and the E7 peptide enhances the osteogenic differentiation of BMSCs.

In the in vivo rat skull defect model, compared with the blank group, more new bone formation could be observed in the other four groups. The formation of new bone in the PLLA/MgO group was greater than that in the PLLA/G group, indicating that Mg²⁺ has osteogenic ability, which is consistent with previous reports [82]. Through Micro-CT quantitative indexes (BV/TV, BMD), we observed that the PLLA/MgO@E7 group had the most significant bone repair effect. Furthermore, the histological staining results further confirmed that in the PLLA/MgO@E7 group, not only more new bone formation was observed, but also mature bone tissue with vascularization was observed and closely bound to the host bone tissue at the margin. In addition, although qRT-PCR, Western blot and immunohistochemical staining of osteogenic markers (such as OCN, BMP-2) were not performed in this study, the existing Micro-CT, histological and in vitro experimental data have provided multi-level evidence support for the bone promoting ability of the PLLA/MgO@E7 scaffold, among which ALP/ARS is recognized as a reliable indicator of osteogenic differentiation. Previous studies have reported: Mg²⁺ promotes osteogenic gene expression by activating PI3K/Akt and Wnt/β-catenin pathways [83, 84], while E7 polypeptide enhances BMSCs recruitment through the SDF-1/CXCR4 axis [39]. Therefore, we speculate that under the combined effect of the two, fibrous membrane exhibits a good role in promoting bone repair, and further demonstrate the potential of functionalized fibre membranes in practical bone defect repair applications.

We successfully fabricated coaxial electrospun fibre membranes with a "core-shell" structure and grafted the E7 peptide using EDC/NHS to study their effects on BMSC osteogenic differentiation. Through characterization, we found that the hydrophilicity of the coaxial fibre membrane was significantly improved and that it could maintain the stability of the pH in vitro. In vitro studies demonstrated that the fibre membranes promoted the in situ recruitment of BMSCs and exhibited excellent biocompatibility and osteogenic differentiation capabilities. In vivo studies have shown that fibrous membranes can better promote the repair of bone defects. This novel material design offers a new approach and methodology for treating bone defects, showing promising application prospects and advancing the feasibility of its clinical application. However, previous studies have shown that the E7 peptide is involved in the SDF-1/CXCR4 axis signalling pathway, but its downstream signalling pathway has not yet been clearly identified. In addition, in this study, we did not research the osteogenic mechanism of the fibrous membrane, which is a shortcoming. However, whether Mg²⁺ and the E7 peptide promote BMSC migration via the same signal target is unclear. Future studies should include immunohistochemical/immunofluorescence staining of osteogenic markers (such as OCN, Runx2) and research on the E7 peptide signaling pathway to further analyze the molecular mechanism of functionalized fibrous membrane promoting bone regeneration.

The datasets used during the current study are available from the corresponding author on reasonable request.

ALP:

Alkaline phosphatase

ARS:

Alizarin Red S

BMSCs:

Bone marrow mesenchymal stem cells

BTE:

Bone tissue engineering

BMP-2:

Bone morphogenetic protein-2

BV/TV:

Bone volume/total volume

BMD:

Bone mineral density

BSA:

Bovine serum albumin

CXCR4:

G protein-coupled receptor C-X-C motif chemokine receptor 4

E7:

EPLQLKM

ECM:

Extracellular matrix

EDC:

N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

ERK:

Extracellular signal-regulated kinase

G:

Gelatin

HA-HAp:

Hydroxyapatite

HAHA-HA:

Hyaluronic acid

hBMMSCs:

Human bone marrow mesenchymal stem cells

HFIP:

Hexafluoroisopropanol

HE:

Haematoxylin and eosin

Mg:

Magnesium

M2D-Exos:

M2 macrophage-derived exosomes

nMgO:

Nanomagnesium oxide

nHAp:

Nanohydroxyapatite

NHS:

N-hydroxysuccinimide

OCN:

Osteocalcin

PLLA:

Poly(L-lactic acid)

PEG:

Polyethylene glycol

SDF-1α:

Stromal cell-derived factor-1α

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscopy

Tb.N:

Trabecular number

Tb.Th:

Trabecular thickness

Tb.Sp:

Trabecular separation

UTS:

The ultimate tensile strength

We thank Professor Di Lu and Teacher Jiazhi Guo of the Scientific and Technological Achievements Incubation Center of Kunming Medical University for providing the venue and equipment and Chengyong Li and other senior brothers for their help.

This study was supported by The National Natural Science Foundation of China (Grant number: 82460428, 82460421, 82160417, 2025JJ81034), the 76th Batch of the China Postdoctoral Science Foundation General Funding-Regional Special Support Program (Grant number: 2024MD763983), Yunnan Provincial Department of Science and Technology-Kunming Medical University Joint Special Fund for Basic Research General Program (Grant number: 202501AY070001-166), Project Contract of Science and Technology Plan of Yunnan Provincial Department of Science and Technology(Grant number: 202402AD080006), Yunnan Health Training Project of High Level talents (Grant number: H-2024026), Youth Project of Yunnan Basic Research Programme of Yunnan Provincial Department of Science and Technology (Grant number: 202401AU070046), Teachers’ Project of Scientific Research Fund of Yunnan Provincial Department of Education- Special Project on Basic Research for Young Talents (Grant number: 2024J0180), The fifth batch of "535" young academic backbone training subjects of the First Affiliated Hospital of Kunming Medical University (Grant number: 2025535Q08), 2024 Yunnan Province "Colorful Cloud Postdoctoral Program" Innovation Project. Yunnan Provincial Education Department Scientific Research Fund(Grant number: 2024Y249, 2025Y0373).Clinical Medical Technology Innovation Guidance Project of Hunan Province(Grant number: 2021SK51717).Kunming Medical University Graduate Innovation Fund Project(Grant number: 2025B038).

1. Shengyu Long and Wentong Wang contributed equally to this work.

Authors and Affiliations

1. Orthopedics, Qujing First People’s Hospital, Qujing, Yunnan, 655000, China

   Shengyu Long, Yunrong Xu & Fei He

2. Trauma Center, the First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, 650032, China

   Wentong Wang, Zhihua Wang, Hao Duan, Ping Yuan, Denghui Li & Wan Zhang

3. Orthopedics Department, the First Affiliated Hospital of KunmingMedical University, Kunming, Yunnan, 650032, China

   Yongcheng Chen & Weizhou Wang

Contributions

Sy. L. and Wt.W. contributed equally to this work. Sy. L. and Wt. W. conducted the experiment and wrote the paper; Yc. C., F. H. and Wz. W. conceived the project and designed the experiment; Zh. W., H. D., P. Y., Yr. X., Dh. L., W. Z. sorted the findings. The final paper was read and approved by all the authors.

Corresponding authors

Correspondence to Weizhou Wang or Fei He.

Ethics approval and consent to participate

All Sprague-Dawley (SD) rats used in this study were obtained from the Animal Experimentation Department of Kunming Medical University and were housed at the center. The study protocol was approved by the Animal Protection and Use Committee of Kunming Medical University (KMMU2020244) and conducted in accordance with the guidelines of the Animal Ethics Review Committee of Kunming Medical University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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