A strategy of 3D-printed Ta4C3 MXenes nanohydroxyapatite/methacryloylated silk fibroin scaffolds for postsurgical management of osteosarcoma

Abstract

The postoperative repair of bone tumors remains a significant clinical challenge requiring urgent solutions, and this holds particularly true for regenerating large bone defects, completely removing residual tumor cells, and effectively preventing and controlling postoperative infections. The efficacy of traditional single treatments is limited, as they generally target only one of these challenges. To overcome this dilemma, this study designed a multifunctional composite nanoscaffold by applying Ta4C3 MXenes nanosheets to the surface of a 3D printed nano-hydroxyapatite (nHA)/methacrylated silk fibroin (MASF) composite scaffold through in situ anchoring. 3D printing technology fabricated a porous hierarchical architecture matching the target bone defect, which provides a suitable three-dimensional microenvironment for bone cell adhesion, proliferation, and differentiation. The main inorganic component of natural bone, nHA, was included to promote bone tissue mineralization. Additionally, with good biocompatibility and degradability, MASF acts as the organic matrix and facilitates the stable loading of the Ta4C3 MXenes nanosheets. To be more specific, the introduction of Ta4C3 MXenes nanosheets endows the scaffold with excellent photothermal conversion capabilities, enabling the generation of precise and controllable local high temperatures upon near-infrared light irradiation. This property allows it to efficiently kill residual tumor cells through thermal ablation while also exhibiting excellent broad-spectrum antibacterial activity through its thermal effects. This implantable material integrates bone defect repair, tumor clearance, and anti-infection functions, thereby effectively solving the limitations of traditional single therapies. It also provides new ideas for the clinical treatment of tumor-related bone defects and exhibits great application prospects.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13036-026-00730-0.

Introduction

Osteosarcoma (OS) is the predominant primary malignant tumor in orthopedic practice, exhibiting a slightly higher incidence among males compared to females [1, 2]. It is characterized by its high degree of malignancy, elevated mortality rate, and unfavorable prognosis. While osteosarcoma prevalence peaks in adolescents and the elderly, it is more frequently observed among children and adolescents [3, 4]. Between 1988 and 2012, the global incidence rate of osteosarcoma remained relatively stable at 1 to 3 cases per million people annually [5]. However, starting from the 21st century onwards, there has been a threefold increase in osteosarcoma incidence among individuals aged between 0 and 24 years. Despite this rise in incidence rates, survival rates for osteosarcoma have shown stability over recent decades; however metastatic bone tumors exhibit even lower survival rates leading to an unfavorable prognosis [6]. The management of osteosarcoma primarily involves surgical intervention and adjuvant chemotherapy. The five-year survival rate following solely surgical intervention stands at a mere 25%, which only reaches up to 44% with the addition of adjuvant chemotherapy [7]. Various treatment modalities for bone tumors including molecular targeted therapy, immunotherapy, and gene therapy are associated with distinct limitations [8–10]. Adjuvant chemotherapy commonly entails concurrent administration of various chemotherapy agents but faces challenges such as drug resistance development along with multiple adverse effects that may harm overall patient health. Surgical intervention alone proves insufficient in completely eradicating cancer cells while potentially resulting in significant deficits at the tumor site [11]. It is evident that within the realm of surgical practice ongoing research and innovation are necessary to develop techniques addressing bone defects effectively while targeting cancer cells.

The irregular and multiple nature of bone tumors frequently leads to substantial bone defects at single or multiple sites following surgical excision. These areas of bone defect typically exhibit diverse shapes and distinct characteristics, presenting challenges and implications for subsequent treatment interventions post excision [12]. The repair of tumor-associated bone defects primarily entails utilizing techniques including bone grafting [13], tumor bone inactivation [14], and the implantation of artificial prosthesis [15]. Artificial implants represent a frequently used approach for addressing extensive bone defects due to their favorable biocompatibility, mechanical characteristics, and wide-ranging clinical utility [16–18]. Nonetheless, the mismatch between implants and the irregular surgical defects associated with bone tumors poses constraints on their usage [19, 20]. Exploring personalized customization of implanted materials featuring superior biological and mechanical attributes emerges as an optimal strategy for treating tumor-related bone defects, given its potential anti-tumor effects that play a crucial role in mitigating tumor recurrence and metastasis.

In addition, these materials have good properties, including broad-spectrum ROS scavenging capabilities and piezoelectric effects [21–23]. Tantalum carbide (Ta₄C₃), as a component of MXene, shows excellent biocompatibility and near-infrared thermal properties, with a photo-thermal conversion efficiency of up to 44.7% [24]. Ta₄C₃ MXene was selected as the photothermal component because of its strong near-infrared responsiveness, high photothermal conversion efficiency, favorable hydrophilicity, and good compatibility with the scaffold matrix. In addition, its reported biocompatibility and ROS-scavenging capability make it particularly suitable for this scaffold system. It is widely used for intravenous injection for tumor tissue ablation. In a study conducted by Dai, aluminum carbide was surface functionalized through redox reactions to produce customized MXene composite nanosheets for use in multi-wave imaging-guided photothermal tumor ablation. These composite nanosheets exhibit enhanced photothermal conversion capabilities, thereby improving contrast in photoacoustic imaging and significantly inhibiting tumor growth through photothermal therapy [25]. Research on MXene materials for tumor treatment has focused on intravenous administration, followed by local light exposure at the tumor site to convert light energy into heat energy to ablate tumor tissue [26]. Recently, increasing efforts have focused on integrating MXene nanosheets with 3D-printing technologies to fabricate composite scaffolds for tissue engineering and related biomedical applications [27–29]. However, research into the integration of these materials with 3D printing technology is still in its early stages.

To reduce Ta₄C₃ MXene aggregation driven by van der Waals interactions, we modified Ta₄C₃ MXene with PVP. In this design, 3D printing was used to fabricate a porous hierarchical scaffold architecture that matches the target bone defect and provides a suitable three-dimensional microenvironment for bone regeneration. To reduce aggregation of Ta₄C₃ MXene nanosheets caused by van der Waals interactions, Ta₄C₃ MXene was treated with PVP to improve its aqueous dispersibility and hydrophilicity. The PVP-treated Ta₄C₃ MXene with favorable photothermal properties was then anchored in situ into the nano-hydroxyapatite/methacrylated silk fibroin scaffold fabricated by photocurable 3D printing [30]. MASF served as the organic matrix of the scaffold and facilitated the stable integration of Ta₄C₃ MXene nanosheets within the composite system. Subsequently, we developed a custom 3D printed composite nanoscaffold composed of Ta₄C₃ MXenes/ nHA/MASF for the treatment of bone defects caused by osteosarcoma or infection. Importantly, the significance of this design lies not in a simple combination of existing components, but in the development of an integrated scaffold platform for the clinically challenging scenario of postoperative bone tumor defects, particularly those with irregular shapes. By combining defect-matched 3D printing with stable MXene anchoring and the coordinated realization of bone regeneration, photothermal tumor ablation, and antibacterial activity, this scaffold provides both practical conformity to irregular bone defects and a distinct “three-in-one” therapeutic advantage. This scaffold not only promotes effective bone regeneration, but also exhibits a local photothermal mechanism that can effectively eliminate tumor cells and bacteria, thereby reducing the risk of tumor recurrence and infection [30] (Fig. 1). In addition, incorporating silk fibroin into scaffolds enhances biocompatibility while reducing any potential toxicity associated with Ta₄C₃ MXenes. Therefore, this composite scaffold offers many advantages and holds great promise in addressing lytic bone defects caused by bone tumors or infections, as well as other potential future applications.

Fig. 1.

Synthesis of 3D printed PVP-MXene/nHA/MASF composite scaffolds and their functions as bone-promoting, antibacterial and anti-tumor

Materials and methods

Synthesis of MHS

According to our previous research, we synthesized a 3D-Printed NanoHydroxyapatite/Methylacrylylated Silk Fibroin Scaffold (HS) in a ratio of 1:0.5. Methacrylated silk fibroin (SFMA) was purchased from Engineering For Life (EFL), and nano-hydroxyapatite (nHA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tetrapropylammonium hydroxide (TPAOH), and hydrofluoric acid (HF) were obtained from Sigma-Aldrich. Ta₄AlC₃ MAX phase was purchased from MXene Nano Tech. To prepare the printing precursor, 1 g SFMA and 0.5 g nHA were mixed with 5 mL of 0.25% (w/v) LAP solution, which was prepared by dissolving 0.05 g LAP in 20 mL PBS, followed by shaking at room temperature for 1 h. The precursor solution was sterilized by filtration before printing. Scaffolds were fabricated using a BP8600 light-curing 3D printer (EFL) with a layer height of 100 μm, a light intensity of 10 mW/cm², and an exposure time of 15 s per layer. After printing, the scaffolds were washed three times in PBS, with each washing step lasting 2 days. The HA/SF scaffold was first fabricated by light-curing 3D printing. After printing and washing, PVP was dissolved and mixed with Ta₄C₃ MXene to form the PVP-MXene dispersion. The printed HS scaffolds were then immersed in the PVP-MXene dispersion for surface modification.The printed scaffold was a porous cylindrical construct with a thickness of 1 mm. For surface modification, 10 mg PVP was dissolved in 4 mL deionized water and mixed with Ta₄C₃ MXene to form a PVP-MXene dispersion. HS scaffolds were then immersed in PVP-MXene dispersions with Ta₄C₃ concentrations of 0.5, 1.0, and 1.5 mg/mL at room temperature for 12 h, followed by shaking treatment to obtain 0.5MHS, 1.0MHS, and 1.5MHS, respectively. This modification mainly formed a surface-associated photothermal functional layer rather than a fully penetrative loading throughout the construct.

Characterization

Morphological images, energy-dispersive X-ray spectroscopy (EDS) elemental Line analysis, and elemental mapping were acquired using a scanning electron microscope (SEM, Hitachi Regulus8100). Transmission electron microscopy (TEM) images were captured with a transmission electron microscope (TEM, FEI Talos F200X). The elemental banding energy was examined through X-ray photoelectron spectroscopy (XPS, Thermo Kalpha). Fourier Transform Infrared Spectroscopy (FTIR, Thermo Scientific Nicolet) analysis was conducted to analyze functional groups and the structure of the scaffold. For SEM morphological observation, the samples were mounted on conductive adhesive tape, sputter-coated with a thin layer of gold, and examined under vacuum conditions. For EDX elemental mapping and line-scan analysis, separately prepared unsputtered samples were mounted on conductive adhesive tape and analyzed under vacuum conditions to avoid interference from metallic coating. EDX mapping and line-scanning were used to evaluate elemental distribution. For FT-IR analysis, the spectra were collected in ATR mode over the range of 400–4000 cm⁻¹.

Photothermal effect

The scaffolds (HS, 0.5MHS, 1.0MHS, and 1.5MHS) were first equilibrated at the same initial temperature (room temperature, 19 °C) before irradiation. They were then individually exposed to an 808 nm near-infrared laser at 1.0 W/cm² using a near-infrared device (FuZhe, FU808AD3000-F100). Thermal images were recorded every 1 min using a thermal imager (HIKMICRO, TPK10). The highest temperature at the scaffold core at each time point was extracted from the thermal images and plotted against irradiation time to generate the temperature–time curves shown in Fig. 3B.

Fig. 3.

Photothermal effect and structural inspection of MHS scaffolds. (A) The thermal images illustrate scaffolds with varying concentrations exposed to 808 nm near-infrared light for 5 min. (B) Temperature change curves after illumination of different brackets within 10 min. (C) X-ray photoelectron spectrometry of 1.0 MHS. (D) FTIR of 1.0 MHS

Staining of living/dead cells

SD rat bone marrow mesenchymal stem cells were cultured at a density of 1 × 10^5 cells/ml and plated in wells. Three control groups were established: a blank group, an HS group, and an MHS group. The corresponding HS and MHS scaffolds were introduced in each group, followed by a 24-hour incubation at 37 °C in a 5% CO₂ cell culture incubator. Post-incubation, the culture medium and scaffolds were removed, and the wells were rinsed with PBS buffer. Subsequently, the 10× Assay Buffer was diluted to 1× with pure water for washing the cells twice for a duration of 2 min each. Following this, 1 ml of 1× Assay Buffer was added to each well along with 2 µl of Calcein-AM, and the plate was gently agitated and kept in the dark at 37 °C for 30 min. Next, 5 µl of PI was added to each well, and the plate was incubated in darkness at room temperature for 5 min. After staining, the excess staining solution was aspirated, and the wells were examined under dark conditions using an inverted fluorescent microscope to differentiate between viable (green) and non-viable (red) cells. To evaluate the in vitro photothermal response of 143B cells under NIR irradiation, the cells were divided into three groups: N + NIR, HS + NIR, and MHS + NIR. All groups were exposed to an 808 nm laser at 1.0 W/cm² for 5 min, followed by live/dead staining.

CCK-8 cell experiment

The BMSCs were seeded in 96-well plates at a density of 1 × 10^5 cells/ml, distributed across four experimental groups: CCK-8 reagent group, blank group, HS group, and MHS group. Three sets of 96-well plates were cultured for 1, 3, and 5 days at 37 °C in a 5% CO₂ atmosphere, with medium replenished every 48 h. Following the incubation period, 20 µl of CCK-8 reagent was introduced into each well of the respective groups, along with 200 µl of complete medium. Subsequently, the plates underwent a 2-hour incubation in a cell culture incubator, resulting in a color change to orange-yellow. Post-incubation, the plates were extracted, and the absorbance at 450 nm was quantified utilizing an ELISA reader (Varioskan, LUX).

Alizarin red staining

For osteogenic differentiation-related staining assays, bone marrow mesenchymal stem cells were seeded onto the corresponding group scaffolds at a density of 1 × 10^5 cells per scaffold and cultured in osteogenic induction medium for 2 and 3 weeks, respectively. Following this period, the well plate was processed for alizarin red staining. The osteogenic induction medium was aspirated, and the cells were fixed with a 10% paraformaldehyde solution for 30 min. Subsequently, the cells were rinsed twice with PBS buffer to remove excess paraformaldehyde before being treated with alizarin red S staining solution. The staining solution was left to incubate at room temperature for 10–20 min, then any remaining alizarin red S solution was washed off with PBS. Finally, the well plate was left to air-dry at room temperature before images were captured using a fluorescence microscope.

Alkaline phosphatase staining

Cells were grouped and cultured utilizing a technique akin to the Alizarin red staining method. Staining was performed at 3 and 7 days, respectively. The cells were then fixed in a 4% paraformaldehyde solution for approximately 15 min before preparing a 10 ml staining working solution. Subsequently, 1.5 ml of the working solution was added to each well, followed by incubation in darkness at room temperature. After a 12-hour staining period to achieve optimal results, the staining process was concluded by removing the staining solution and proceeding with photography.

Supplementary PCR analysis of osteogenic-related genes

Bone marrow mesenchymal stem cells were cultured under osteogenic induction conditions, and after 14 days, total RNA was extracted using the TRIzol method. Complementary DNA (cDNA) was synthesized from the extracted RNA, and real-time PCR was performed using a SYBR Green qPCR reagent kit (Servicebio, China) on an ABI Step One Plus Real-Time PCR system. Relative expression levels of Col1a1, BMP2, OPN, and OCN were normalized to GAPDH and calculated using the 2^−ΔΔCt method, with the N group used as the calibrator and set to 1. The primer sequences used for qPCR analysis are provided in Supplementary Table S1. This analysis was included as a supplementary molecular-level assessment of osteogenic-related gene expression.

Construction of rat skull bone defect model

The female SD rats were selected to establish a cranium bone defect model. The rats, approximately 4 weeks old, were divided into three groups: control group (N), HS group, and MHS group, each consisting of 10 rats. The rats were anesthetized through intraperitoneal injection of 0.4mL/100 g of 10% chloral hydrate, followed by disinfection and covering with a sterile drape. A scalpel was used to make a vertical incision in the skin, bluntly separating the tissue layers to identify the anatomical location of the frontal bone and cranium. Subsequently, a 0.5 cm diameter hollow drill was used to create a hole. Once the drill bit penetrated slightly, eyepiece forceps were utilized to extract the circular bone defect from the cranium. A 3D-printed scaffold was then implanted based on the groupings, ensuring secure placement and proper alignment of the edges, followed by sequential rinsing and suturing. Post-surgery, the dressing was changed after three days under sterile conditions at a temperature of (25 ± 2)°C in the animal facility.

Micro-CT of the skull

After successfully inducing a cranial bone defect, the rats were fed according to standard protocols for four and eight weeks, respectively. Five rats from each group were randomly selected at designated time intervals for tissue sampling, with specimens being preserved in polyformaldehyde solution. Subsequently, the specimens underwent Micro-CT scanning using the Skyscan1276 system from Bruker in Germany for quantitative and visual analysis of new bone formation within the defect area. The original images of the bone defect were then subject to selective analysis and reconstruction through NRecon (V1.7.4.2, Bruker, Germany). Parameters such as bone volume (BV), tissue volume (TV), bone volume fraction (BV/TV), trabecular spacing (Tb.Sp), and trabecular number (Tb.N) were computed using CT Analyser (1.20.3.0, Bruker, Germany) to assess the extent of new bone formation in the tissue.

Pathological staining of cranial bone tissue

The three sets of rat skull specimens were removed from the formalin solution, decalcified, embedded in paraffin, and sectioned. Thereafter, the sections underwent HE, Masson, and Goldner staining processes. Masson staining primarily targets collagen fibers for distinguishing muscle fibers, whereas Goldner staining can differentiate mineralized bone in green color from non-mineralized bone in orange-red hue.

Construction of osteosarcoma model in nude mice

Eighteen 4-week BALB/c nude mice were selected and randomly assigned to one of six groups: N, N + NIR, HS, HS + NIR, MHS, and MHS + NIR groups. Tumor cells 143B were injected into the well-vascularized armpit region of the mice. When the tumor volume in each group reached approximately 1000 mm³, mice in the HS, HS + NIR, MHS, and MHS + NIR groups underwent anesthesia, and their tumor sites were surgically opened for the implantation of corresponding scaffolds, followed by layered suturing. The following day after scaffold implantation, mice in the N + NIR, HS + NIR, and MHS + NIR groups were exposed to 808 nm NIR at a power of 1.0 W/cm² for 10 min daily. After two weeks, the mice were euthanized, and both tumors and internal organs (heart, liver, spleen, lungs, and kidneys) were harvested.

Tumor indicator monitoring

After subcutaneous tumor formation, the nude mice were routinely fed, and to dynamically detect the nutritional status and tumor condition of the nude mice, we measured the changes in body weight and tumor volume of each group of nude mice every three days, and the tumor volume was calculated as V = 1/2*long axis*short axis.

Pathologic staining of osteosarcoma

To verify whether the implanted scaffolds and external factors such as light and heat irradiation possessed a tissue-toxic effect on nude mice, we collected the hearts, livers, spleens, lungs, and kidneys of the nude mice and subjected these specimens to HE staining to visualize the condition of each organ. Moreover, subcutaneous tumors were stained with HE, Tunel, and Ki67.

Coated Plate Method of Labeling

A 500 µL aliquot of the recovered bacterial suspension was transferred into a sterile culture tube, and the volume was adjusted to 10 mL with LB broth. After the suspension became visibly turbid, the optical density at 600 nm (OD600) was measured using a UV spectrophotometer. The bacterial suspension was then serially diluted from 10 − 1 to 10 − 8. For each dilution, 100 µL of suspension was spread evenly onto LB agar plates. The OD600 value of each dilution was also recorded. The plates were incubated at 37 °C for 2–3 days until countable, well-separated colonies were formed. The colonies were then counted, and the corresponding CFU and OD600 values were used to establish the standard curve.

Flatbed coating experiment

Staphylococcus aureus and Escherichia coli suspensions were adjusted to 1 × 10⁶ CFU/mL. For each bacterial strain, the corresponding scaffolds were added to the bacterial suspensions according to the experimental grouping, and the indicated groups were exposed to near-infrared irradiation. Subsequently, 100 µL of each bacterial suspension was spread onto LB agar plates. After incubation at 37 ℃ for 24 h, the plates were photographed, and the colonies were counted.

Bacterial culture and morphological experiments

After treatment as described above, the bacterial samples were collected, fixed with 2.5% (v/v) glutaraldehyde, and dehydrated using a graded ethanol series of 20%, 40%, 60%, 80%, and 100% for 10 min at each concentration. After dehydration, the samples were observed using scanning electron microscopy to evaluate changes in bacterial morphology.

Live/dead staining assay of bacteria

Bacteria were treated as described above. Samples from each group were transferred into blank wells and washed twice with PBS, using 300 µL each time. After removal of PBS, Calcein-PI cell viability/cytotoxicity staining solution was added to each well. The samples were incubated for 30 min and then imaged using a fluorescence microscope to distinguish live and dead bacteria.

Statistical analyses

Data were statistically analyzed using SPSS V21.0 (IBM, Armonk, IL, USA). All quantitative data are presented as the mean ± standard deviation (SD), and error bars in all quantitative graphs represent SD unless otherwise indicated. GraphPad Prism 8.0.2, ImageJ V1.8.0, and Thermo Avantage V7.0 were used for data analysis and graph generation. A P-value of < 0.05 was considered statistically significant. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results

Morphological characteristics of the scaffolds

SEM examination of the multilayer Ta₄C₃ MXene powder revealed a multilayered lamellar morphology (Fig. 2A). Energy-dispersive X-ray spectroscopy (EDS) further demonstrates the elemental composition of the material, with carbon (C) represented in red, aluminum (Al) in green, and tantalum (Ta) in blue (Fig. 2B). An atomic semi-quantitative analysis indicates that the atomic percentages of C, Al, and Ta are 65.97%, 0.62%, and 33.41%, respectively, highlighting Ta₄C₃ as the predominant constituent. The EDS spectrum and line-scan analysis further confirmed the elemental distribution of Ta₄C₃ MXene, as shown in Figure S6. These results indicated that tantalum was the dominant metallic element in the prepared Ta₄C₃ MXene. Transmission electron microscopy (TEM) was employed to examine the morphology of both single-layer (Fig. 2C) and multi-layer (Fig. 2D) Ta₄C₃ materials. The observations in Fig. S1 and Fig. S2 suggest that PVP treatment affected the hydrophilic behavior of Ta₄C₃ MXene, which may facilitate its dispersion and scaffold incorporation. Because these phenomena may also be influenced by swelling-related effects, the related interpretation was kept cautious. Additional UV–Vis characterization of the PVP-treated MXene is provided in the Supplementary Materials (Fig. S10).

Fig. 2.

Morphological analysis of Ta₄C₃ material. (A, B) SEM images and elemental mapping (C, Al, Ta) of multilayer Ta₄C₃. (C, D) TEM images of single and multilayered Ta₄C₃

The photothermal response of different scaffolds was evaluated under near-infrared irradiation using a thermal imaging system. The results show that the temperature of the HS scaffold remains stable over a 5-minute period. In contrast, the temperatures of the other three groups with varying concentrations of MHS scaffolds gradually rise upon exposure to infrared light, with a faster and higher temperature increase observed as the concentration of MHS scaffold increases. These findings are illustrated in Fig. 3A. During each minute irradiation cycle, the temperatures of various scaffolds were recorded and illustrated using a line graph (Fig. 3B). The HS scaffold showed only a slight temperature change, whereas the MHS scaffolds exhibited a gradual temperature increase and reached a plateau after approximately 5 min. After comprehensive comparison in the preliminary experiments, 1.0MHS was selected for subsequent investigations because it showed the most favorable overall performance, including better structural stability, good biocompatibility, stable printability, and a more suitable microenvironment for cell attachment and growth. XPS analysis of the selected 1.0MHS scaffold confirmed the presence of Ta-related signals, supporting the attachment of Ta₄C₃ MXene to the scaffold after immersion modification (Fig. 3C). By utilizing Fourier transform infrared spectroscopy for the detection of organic components within the scaffold, both functional group qualitative analysis and molecular structure analysis can be conducted. Peaks within the 1600–1700 cm^− 1 range (C = O stretching) correspond to amide I, while those within the 1500–1600 cm^− 1 range (N-H bending) are indicative of amide II. Amide III (C-N stretching) is identified near 1235 cm^− 1, while amide V is represented by the peak near 568 cm^− 1, and amide A (hydrogen bonding) is observed near 3274 cm^− 1. The presence of the phosphate group (PO₄³⁻) is indicated by the peak near 1023 cm^− 1, reflecting PO bending and confirming the existence of hydroxyapatite (Fig. 3D). The secondary β-sheet structure of the fibroin protein is discernible in the peaks surrounding amide I, amide II, and amide III, suggesting that the scaffold’s β-sheet folding and the configuration of its associated amide groups remain unchanged.

Biocompatibility of scaffolds

We used control group, HS group and MHS group in in vitro experiments to initially study the effect of scaffolds on cell proliferation and vitality. Cell viability was assessed using live/dead cell staining techniques to clarify the impact of scaffolds on cell proliferation and survival. After rat BMSCs were co-cultured with blank control, HS scaffold and MHS scaffold for one day, the cells were stained using a live/dead cell dual staining kit. Fluorescence microscopy showed abundant Calcein-AM-positive viable cells and only a small number of PI-positive dead cells in all groups (Fig. 4A). Statistical analysis showed that there were no significant differences between the three groups, indicating that the manufactured scaffolds had minimal effect on normal cell proliferation and did not show significant cytotoxic effects (Fig. 4B).

Fig. 4.

Biocompatibility of MHS scaffolds. (A) Fluorescence images and (B) quantitative graphs of Live/Dead staining BMSCs co-cultured with different scaffolds. (Scale bar = 200 μm), (C) Quantitative graphs of CCK-8 experiments in co-culture for 1, 3, and 5 days. Data in B and C are presented as mean ± SD

In addition, we also conducted CCK-8 experiments to enhance mutual verification. Rat BMSCs were cultured in three groups for 1, 3 and 5 days. Subsequently, an enzyme-linked immunosorbent assay was used to measure OD450 values, reflecting color intensity and number of viable cells. The results showed that there were no significant differences between the groups at the three time points (P > 0.05) (Fig. 4C). The OD450 values increased over time in all groups, indicating continued cell proliferation during the culture period. Overall, the 3D-printed Ta₄C₃ MXene/nHA/MASF scaffold showed good biocompatibility and did not significantly affect BMSC viability or proliferation (P > 0.05).

To assess the biotoxicity of the scaffolds-promoted anti-tumor process, we preserved the hearts, livers, spleens, lungs and kidneys of euthanized mice. Subsequently, the organs are embedded in wax, sectioned, and stained to generate corresponding pathological sections. The slices were examined at a magnification of x100 (Fig. S3). The results of the study showed that there were no significant differences between the N, HS and MHS groups, all showing typical organ structures. This suggests that scaffold implantation will not produce biological toxicity in mice, which is consistent with previous reports showing that MXene-integrated 3D scaffolds did not induce significant pathological abnormalities in major organs in vivo [31]. In addition, comparing infrared radiation between groups exposed to infrared radiation and those not exposed to infrared radiation did not produce any significant differences.

The scaffold’s ability to promote osteogenic differentiation

Under consistent grouping conditions, we conducted an in vitro osteogenic differentiation experiment to evaluate the osteogenic capacity of the fabricated scaffolds. The Alizarin Red S staining solution combines with calcium salts to create orange-red complexes, effectively marking the calcium salts produced by osteoblasts and providing an indirect measure of their osteogenic potential. The stained samples were examined visually and through microscopy to analyze the color, size, number, and density of calcium nodules, thus determining the osteogenic efficacy. The findings revealed a substantial presence of calcium nodules at 14 and 21 days, with increasing sizes and densities observed in the N, HS, and MHS groups (Fig. 5B). The number of calcium nodules formed through the differentiation of mesenchymal stem cells from the bone marrow was higher in the HS and MHS groups compared to the N control group. Furthermore, at 21 days, there was a significantly greater quantity of calcium nodules than at 14 days (P < 0.05). No significant statistical variance was observed in the production of mineralized matrix by cells between the HS and MHS groups over the entire process (P > 0.05). Alkaline phosphatase staining at 3 and 7 days enhanced the osteogenic effects of osteoblasts, with minimal coloration observed at 3 days, but with a relatively larger area of staining in the HS and MHS groups, particularly evident by day 7 (Fig. 5A). This observation is consistent with previous reports showing that MXene-containing scaffolds can enhance ALP activity and early osteogenic differentiation of stem cells or osteoblast-related cells in vitro [32]. The expression of osteogenic-related genes provides a supplementary molecular-level observation of bone formation. In the present study, this analysis was included as supportive evidence only and was not used as the sole basis for evaluating the osteogenic performance of the scaffolds. In this study, we examined the expression levels of osteogenic genes at 14 days by selecting representative osteogenesis-related genes and performing qPCR analysis. Specifically, we focused on the Alpha 1 type I collagen gene (Col1a1), bone morphogenetic protein-2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) as they represent genes associated with different stages of osteogenic differentiation (Col1a1 and BMP2 in the early stage, and OPN and OCN in the secondary stage). The results showed an overall increase in the expression of OCN, OPN, Col1a1, and BMP2 in both the HS and MHS groups compared with the control N group (P < 0.05). Interestingly, no statistically significant differences were observed in gene expression between the HS and MHS groups (Fig. 5C and F).These PCR findings were therefore interpreted with caution and considered in conjunction with the staining-based and in vivo osteogenic results.

Fig. 5.

The scaffold promotes osteogenic differentiation in vitro. (A) Alkaline phosphatase staining at 3 and 7 days. (B) The Alizarin Red S staining at 14 and 21 days. (C-F) Relative mRNA expression levels of osteogenic genes at 14 days (Col1a1, BMP2, OPN, and OCN), normalized to GAPDH using the 2^−ΔΔCt method, with the N group as the calibrator and set to 1. Data in C–F are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Microcomputed tomography analysis

The in vitro experiments indicated that the scaffolds promoted osteogenic differentiation. Subsequent animal experiments were conducted using SD rats to induce cranial bone defects. After cranial defect creation and scaffold implantation, no postoperative infection was observed, and all animals survived. Tissue samples were then collected for Micro-CT scanning and reconstruction of the cranial defect area to assess new bone formation (Fig. 6A). In this protocol, the corresponding treatment group received NIR irradiation daily during the experimental period. Micro-CT reconstruction showed new bone formation in all groups, mainly extending inward from the defect margins and surrounding bone tissue (Fig. 6B). The bone mass in the control group at 4 weeks was generally lower compared to that at 8 weeks. Interestingly, even in the control group without scaffolds, some new bone formation was observed. In contrast, the HS group exhibited inward bone growth from four weeks onwards, aligning closely with the structure of the printed scaffold and showing noticeable scattered bone formation in areas with evident defects. This suggests that our printed scaffolds not only fill and support the defective areas but also enhance tissue and cell adhesion, consequently expediting bone repair. In addition, the scaffold was suturable under the present experimental conditions, further supporting its practical applicability for surgical handling. Particularly noteworthy, the MHS group displayed significant new bone formation in imaging studies, alongside evident scaffold degradation. These findings highlight the advantageous osteogenic impact and degradability of the MHS group. Moreover, the degraded inorganic calcium and proteins from the scaffold can serve as nutrients, fostering in-situ bone formation.

Fig. 6.

Analysis of in vivo bone repair by Micro-CT. (A) Schematic diagram of scaffold promoting osteogenesis in vivo. (B) Micro-CT scanning of the cranial bone model. (C-E) Bone volume ratio (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N) at 4 and 8 weeks in each group. Data in C–E are presented as mean ± SD

We conducted an in-depth analysis using CTAn software to evaluate the bone volume fraction (BV/TV), trabecular separation (Tb.Sp), and trabecular number (Tb.N). The results from bone histomorphometry indicated a notable increase in the bone volume fraction and trabecular number at 8 weeks compared to the 4-week time point (Fig. 6C-E). Moreover, there were statistically significant differences (P < 0.05) observed between the control group and both the HS and MHS groups. The trabecular separation (Tb.Sp) demonstrated a gradual decrease over time, with the scaffold group exhibiting reduced trabecular separation compared to the control group (P < 0.05). This outcome emphasizes the osteogenic effect observed and serves to validate the micro-CT images of the reconstruction process.

Cranial osteogenic staining

To further investigate the in vivo osteogenic impact of the scaffold, we conducted histological staining using HE, Masson’s trichrome, and Goldner’s trichrome on rat skull specimens collected at 4 and 8 weeks post-implantation. This approach facilitated a visual assessment of the osteogenic response in the vicinity of the scaffold. HE staining selectively targets cell nuclei and cytoplasm, revealing a scarcity of cells at the defect site in the control group. In contrast, following scaffold implantation, the scaffold’s surface provided crucial physical support, fostering enhanced cell adhesion and promoting osteogenic activity (Fig. 7A). The Masson staining technique revealed that collagen fibers and cartilage were stained blue, whereas cytoplasm and muscles exhibited a red stain. Notably, the blank group displayed minimal blue staining, predominantly highlighting red-stained cell structures. In contrast, the scaffold group exhibited a prominent blue hue, indicative of its significant osteogenic effect (Fig. 7B). Goldner staining distinguished mineralized bone in green and non-mineralized bone in orange-red. Furthermore, the blank group illustrated a scarcity of mineralized bone, while the scaffold group demonstrated the formation of dense mineralized bone around the scaffold, accompanied by numerous non-mineralized bones at both ends and within the scaffold, suggesting a positive trend toward new bone formation, particularly evident at the early 4-week stage (Fig. 7C). Importantly, a comparison between the HS and MHS of the same group at different time points revealed a partial degradation of the scaffold at 8 weeks, resulting in the formation of beneficial pores for cell attachment and fostering long-term bone formation.

Fig. 7.

Pathological staining after skull implantation of the scaffold. (A) Images of (A) H&E, (B) masson trichrome and (C) Goldner staining of skull on postoperative week 4 and week 8

Anti-tumor effect of the scaffold

To evaluate the in vitro photothermal cytotoxicity of the scaffold, live/dead staining was performed on 143B cells under NIR irradiation. Compared with the N + NIR and HS + NIR groups, the MHS + NIR group showed reduced Calcein-AM-positive live-cell signals and increased PI-positive dead-cell signals, as further supported by quantitative analysis(Figure S15).

Excised tumor tissues from each group were photographed and measured (Fig. 8A and B). The MHS + NIR group showed markedly reduced tumor size and tumor weight, whereas the other groups showed no obvious inhibitory effect on tumor growth (Fig. 8C). In addition, mice in each group were affected by tumors and lost their weight to varying degrees, with mice in the MHS + NIR group being the least affected (Fig. S4). Based on the above results, it can be seen that the composite scaffold we developed has effective photothermal tumor killing properties, but no significant differences were detected between the other groups. These findings are in agreement with previous reports that MXene-integrated scaffolds can achieve effective NIR-triggered photothermal tumor ablation, while groups without combined MXene/NIR treatment typically show only modest antitumor effects [33].

Fig. 8.

Representative images and pathological staining of tumor tissues. (A)Schematic diagram of scaffold anti-tumor in vivo. (B) Objective image of tumor tissues. (C) Tumor weight of each group. (D) H&E, Ki-67, and TUNEL staining of tumor tissues in each group. (E) Quantitative analysis of Ki-67 expression in each group. (F) Quantitative analysis of TUNEL-positive expression in each group. Data in C, E, and F are presented as mean ± SD

To further verify the antitumor effect, we performed subsequent histopathological staining analysis on all groups, including HE staining, TUNEL staining, and Ki-67 staining (Fig. 8D and F). Specifically, TUNEL staining is used to identify cell death, and Ki-67 staining can assess the proliferative activity of tumor cells. Through a comprehensive analysis of these three staining results, it can be seen that under the effect of photothermal therapy, the MHS + NIR group not only greatly promoted the apoptosis of tumor cells, but also significantly inhibited the proliferation of tumor cells. These pathological evidence further confirms the reliability of the anti-tumor efficacy of the composite scaffold.

Antibacterial effect of scaffolds

To evaluate the antibacterial effect of HS group, MHS group, HS + NIR group, and MHS + NIR group, we used representative gram-positive cocci (S. aureus) and representative gram-negative bacillus (E. coli) to conduct antibacterial experiments, and used standard plate counting method to intervene in each group. It was found that there was no significant difference in the number of colonies between the blank group and the HS group with or without near-infrared light group, while the bacterial colonies in the MHS group decreased significantly after near-infrared light irradiation. This showed that light irradiation of the scaffolds in the MHS group produced a significant antibacterial effect (Fig. 9A), and colonies were quantified and plotted into image bars (Fig. 9D and F). In addition, bacterial morphology after scaffold treatment was observed by scanning electron microscopy. The bacteria in the control and HS groups maintained relatively intact morphology, and NIR irradiation alone did not induce obvious structural damage. After MHS + NIR treatment, E. coli exhibited apparent morphological damage, including surface deformation and membrane disruption, whereas S. aureus showed relatively mild cell-wall alterations. This difference was consistent with the residual colonies observed for S. aureus in Fig. 9A. These morphological observations supported the antibacterial effect of MHS + NIR treatment, with more pronounced structural disruption observed in E. coli than in S. aureus (Fig. 9B). The viable status of bacterial biofilms was then assessed by fluorescence microscope scanning to determine the treatment effect of different groups. The results can be seen that except the MHS + NIR group, all the other groups showed excited green fluorescence, indicating that the bacteria survived well in these groups, while the MHS + NIR group stimulated significant red fluorescence, indicating that a large number of bacteria died in this group (Fig. 9C, S5).

Fig. 9.

The antibacterial effects properties of different scaffolds in vitro. (A) Macroscopic view plates and (D, E) Quantitative graphs of solid medium inoculated with S. aureus and E. coli after immersion with scaffolds(B) SEM image of S. aureus and E. coli after immersion with scaffolds. (C)Fluorescence images of Live/Dead staining for co-cultured S. aureus with different scaffolds. Data in D and E are presented as mean ± SD

Discussion

3D printing technologies, including 3D bioprinting, have garnered significant interest in the fields of pharmaceuticals, tissue engineering, and healthcare [31–35]. Continuous advances in these technologies have enabled the fabrication of complex structures with diverse physicochemical properties and functions. Within this field, cell-laden bioprinting generally involves the incorporation of cells into printable matrices, which introduces challenges related to cell viability and biological function during fabrication [36–38]. In the present study, the scaffold was fabricated using a cell-free photocurable 3D-printing process; no cells were encapsulated or loaded during printing, and cells were introduced only in subsequent biological evaluations.

This study is focused on the development of a 3D printed composite nanoscaffold with potent anti-tumor and bone-regenerating capabilities for addressing tumor-induced bone defects, while exploring its anti-infective effects. Compared with many previously reported 3D-printed bone scaffolds that mainly emphasize structural reconstruction, and with MXene-based systems that often focus on a single bioactive function, the present scaffold was designed as a more integrated platform. In our study, the role of 3D printing was not limited to scaffold fabrication itself, but extended to constructing a defect-matched porous architecture for local bone regeneration, while the incorporation of Ta₄C₃ MXene further introduced photothermal tumor ablation and antibacterial capability. Therefore, the main advance of this system lies not in a simple material combination, but in the integration of reconstruction, tumor control, and infection prevention within one implantable scaffold, which is particularly relevant for the clinically complex setting of postoperative bone tumor defects. Rather than representing a simple extension of existing MXene-based scaffolds, the present design was intended to address the clinically complex setting of irregular bone defects after bone tumor surgery by integrating defect-conforming reconstruction with the synergistic functions of bone regeneration, photothermal tumor ablation, and antibacterial protection in a single platform. The scaffold is designed to effectively fill and repair these defects while also enabling the selective destruction of cells and bacteria through controlled exposure to infrared light, thereby effectively preventing the recurrence of malignancies. Different research selected different concentration gradients, but all through reasonably comparable experiments [39–42]. We employed light-cured 3D printing technology to fabricate foundational nano-hydroxyapatite/methyl methacrylate silk protein scaffolds, onto which Ta₄C₃ MXenes material was incorporated to facilitate photothermal therapy and realize the intended design. The selection of Ta₄C₃ MXene was based not only on its photothermal performance, but also on its favorable hydrophilicity, biocompatibility, ROS-scavenging capability, and compatibility with the scaffold matrix. These features make it a suitable photothermal component for the present scaffold system, compared with other widely studied nanomaterials such as graphene derivatives and MoS₂. Various 3D-printed Ta₄C₃ MXenes/nHA/MASF composite nano scaffolds underwent examination for their photothermal properties. The photothermal temperature of the 1.5MHS scaffold rose to approximately 60 °C. Wang emphasized the significance of temperature and humidity in influencing the secondary structure and characterization of silk fibroin biomaterials [43]. Due to concerns regarding potential structural damage to silk fibroin and normal tissues at elevated temperatures, the 1.0MHS scaffold was chosen as the ultimate experimental substrate. Performing physical characterization testing on the composite bracket involved utilizing FTIR to identify the organic components of the bracket’s silk protein, revealing that the spatial structure remains intact and chemical bonds are undamaged. Subsequently, an additional analysis was conducted using an XPS to identify the different elements within the bracket, thereby validating the stable attachment of the Ta₄C₃ MXenes material to the bracket. Yin employed a comparable immersion technique to incorporate MXene materials onto the scaffold, demonstrating the efficacy of this approach in fabricating the intended composite scaffold.

The study findings indicate that after co-cultivating composite scaffolds with rat bone marrow mesenchymal stem cells for 1, 3, and 5 days, CCK-8 experiments were conducted. Subsequently, cell viability assays were performed after 1 day of co-culture, revealing that neither the HS group nor the MHS group had a significant impact on cell proliferation or induced cell death compared to the control group. These results suggest that the scaffolds exhibit no apparent biotoxicity, demonstrate favorable biocompatibility, and are suitable for cellular-level applications. Enhancing bone formation is a crucial characteristic of biological scaffolds as it stimulates the proliferation and differentiation of bone marrow mesenchymal stem cells into osteoblasts, ultimately leading to bone structure development [44–46]. Alizarin red staining can confirm that the scaffold group actively promotes calcium salt deposition and calcium nodule formation in contrast to the blank group. Alkaline phosphatase is an enzyme widely present in bone tissue and cells; its content reflects bone activity [47, 48]. Alkaline phosphatase staining indicates a notable increase in enzyme levels within the scaffold group. These findings suggest a beneficial influence of the scaffold group on mineral deposition by bone marrow stromal cells and extracellular matrix mineralization. Furthermore, to further validate our findings at the genetic level, we assessed expression levels of well-known early and late osteogenic genes using PCR technology. Importantly, there was a substantial upregulation in expression of bone-related genes observed in the scaffold group. The MHS scaffold was surgically implanted into a rat skull bone defect model. After 4 and 8 weeks post-implantation period elapsed without any animal mortality indicating non-toxic nature of brackets used hereafter referred as “bracket”. Subsequently retrieved brackets from each experimental groups at designated time points. Notably, the bracket structure exhibited a remarkable ability to enhance cellular migration and adhesion, leading to significant bone formation both surrounding and within the bracket. This observation highlights its potential in promoting bone development. Remarkably, at the 8-week mark, noticeable degradation of the implanted brackets in the MHS group was observed alongside bone accumulation and maturation within the degraded region. This phenomenon suggests that breakdown products of the bracket could potentially serve as substrates for bone regeneration, thereby augmenting osteogenesis beyond what is achievable in vitro settings. Further analysis of CT scan data revealed a higher bone volume fraction (BV/TV) and trabecular number (Tb.N) compared to the control group, indicating enhanced bone formation. As osteogenesis progressed, there was a progressive decrease in trabecular spacing (Tb.Sp). These findings collectively validate the superior osteogenic impact of our developed composite scaffold.

Composites based on MXene are also advancing in their development for oncology applications [49–51]. After incubating 143b tumor cells in vitro for 24 hours, the cells were treated with 1.0 mg mL^{− 1} of Ta₄C₃ MXenes material and compared to a blank control group under controlled near-infrared light exposure. Subsequently, a live/dead cell staining assay was conducted to assess the photothermal cytotoxicity on the tumor cells. The results, observed using fluorescence microscopy, demonstrated a significant reduction in viable tumor cells following material treatment and light exposure, providing evidence of the potent photothermal anti-tumor efficacy of the material. At 4 weeks, subcutaneous tumor models were established in nude mice using these tumor cells. A scaffold was then surgically implanted for phototherapy purposes. The findings revealed a substantial decrease in tumor size within the MHS group exposed to NIR, highlighting the therapeutic effectiveness of photothermal treatment facilitated by the scaffold implantation. Additionally, post-euthanasia extraction of hearts, livers, spleens, lungs, and kidneys from each group’s nude mice was performed for HE staining analysis; no signs of organ damage were observed under electron microscopy examination further indicating non-toxic properties of the scaffolds used. Pathological staining demonstrated that the scaffold exhibited potent cytotoxic effects on tumor tissue. In particular within the MHS + NIR group, there was a noticeable reduction in tumor size accompanied by prominent indications of cellular apoptosis and reduced cell proliferation as determined through histological analysis. Overall findings suggest that this 3D printed Ta₄C₃ MXenes/nHA/MASF composite nanoscaffold exhibits significant anti-tumor effects both in vivo and in vitro settings effectively inhibiting tumor growth.”

Photosensitive antimicrobial properties are a crucial area of investigation in the characteristics of MXene materials, which have the potential to prevent and even treat infections [52–54]. The mechanisms of antimicrobial action encompass disruption of cell membrane integrity [55, 56], induction of oxidative stress [57], and synergistic phototherapeutic effects based on near infrared light [58]. We have preliminarily validated this aspect through in vitro experiments. Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-negative and Gram-positive bacteria, respectively, resulting in a significant reduction in bacterial proliferation after subjecting the composite scaffold to light exposure. Damaging alterations in the bacteria’s cell membranes were observed under electron microscopy, while live-dead cell staining experiments further confirmed the antimicrobial effect of the scaffold.

The combination of MXene with 3D technology has shown promising results in bone regeneration and the treatment of bone tumors [59, 60], as well as in antimicrobial applications [61–63]. Moreover, ongoing research explores MXene’s potential in other medical aspects such as promoting neurogenesis and bone regeneration for repairing bone defects through composite materials [64]. Additionally, studies have identified its antimicrobial and hemostatic activities that can facilitate wound healing and repair wounds [65]. A review of ClinicalTrials.gov indicates that, although no clinical trial directly corresponding to MXene-based multifunctional scaffolds for postsurgical osteosarcoma management has yet been identified, related translational efforts are emerging in the broader field of patient-specific 3D-printed bone grafts and resorbable bone scaffolds. In addition, a registered clinical study involving a multifunctional 3D-printed bone scaffold for multimodal treatment of osteosarcoma and osteomyelitis further suggests that the clinical translation of advanced bone scaffold systems is beginning to develop. Nevertheless, the present work remains at the preclinical validation stage, and the clinical translation pathway, together with the design and implementation of future clinical studies, still requires further systematic investigation. With continued research, the potential applications of MXene-based materials in clinical treatment may be further expanded, ultimately benefiting patients.

Conclusions

In conclusion, the 3D-printed Ta₄C₃ MXenes/nHA/MASF composite nano-scaffolds developed by our research team demonstrated excellent biocompatibility along with strong osteogenic effects in both in vitro and in vivo models. These scaffolds also exhibited remarkable antitumor and antibacterial properties. Such characteristics highlight their potential as an integrated platform for managing irregular bone defects, particularly in postoperative bone tumor settings, through the combination of bone regeneration, photothermal tumor ablation, and antibacterial protection. Future studies are needed to evaluate applicability of the composite scaffold in larger or less confined bone defects. It is also necessary to determine if its structural and functional properties can sufficiently support the remodeling of more complex postoperative bone tumor defects. Systematically optimizing the scaffold architecture, material composition, degradation behavior, and long-term biosafety holds also a crucial step toward improving its performance and enabling future translational application.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Junli proposed the idea and revised the final draft, Liu Yang did the cell experiment and wrote the first draft, Kenan Sun and Shihao Deng were in charge of the animal experiment and bacterial experiment, Shuai Liang and Shaoze Lan did the Scaffolds synthesis, Canming Ye and Ciren Lunzhu conducted part of the cell experiments, while Runguang Li and Runhuai Yang revised the manuscript.

Funding

This study was financially supported by the New Era Education Quality Project of Anhui Provincial Department of Education (No. 2023zyxwjxalk054), the Clinical Research Cultivation Program of the Second Affiliated Hospital of Anhui Medical University (No. 2020LCZD20), the Research Fund of Anhui Institute of Translational Medicine (No. 2022zhyx-C44), the Basic and Clinical Cooperative Research Promotion Plan of Anhui Medical University (No. 2020xkjT040), the Tibet Autonomous Region Natural Science Foundation Grouped Assistance to Tibet Medical Project (No. XZ2023ZR-ZY47(Z)), the Xizang Natural Science Foundation (XZ202601ZR0093), the National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University (No. 2024GMFY01), and the Technology Plan Project of Shannan City (No. SNSBJKJJHXM2023015).

Data availability

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

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Second Hospital of Anhui Medical University, China (NO. YX2022-128).

Consent for publication

Not applicable.

Declaration of AI-assisted language editing

AI-assisted language editing tools were used to improve the wording and sentence structure of parts of the manuscript. All scientific content, data interpretation, and conclusions were developed and verified by the authors.

Competing interests

The authors declare no competing interests.