Fabrication and Biocompatibility of a 3D-printed Porous Ti-25Ta Alloy Scaffold

Fabrication and Biocompatibility of a 3D-printed Porous Ti-25Ta Alloy Scaffold | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fabrication and Biocompatibility of a 3D-printed Porous Ti-25Ta Alloy Scaffold Di Wu, Yada Li, Wangwei Zhu, Haolin Jiao, Bing Ge, Qinwen Xie, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6134011/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract In this study, a porous Ti-25Ta alloy scaffold was fabricated using 3D printing and compared with pure Ti to evaluate its biocompatibility and osteogenic potential. The scaffolds were fabricated using a selective laser melting (SLM) process, followed by investigations of physical properties, in vitro cytocompatibility, osteogenesis, macrophage polarization, and in vivo bone reconstruction. Pure Ti exhibits the highest wettability, cell proliferation, osteogenic differentiation, mineralized nodule formation, anti-inflammatory capability, and bone reconstruction. The adoption of Ta in the Ti-25Ta alloy significantly increases its wettability, osteogenesis, and anti-inflammatory without a great increase in density. In summary, the Ti-25Ta alloy has a favorable balance of physical properties and biological performance, making it a promising candidate as a bone implant material. 3D printing Ti-25Ta alloy porous scaffold biocompatibility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction In the vast field of contemporary medical materials science, the rapid advancement of medical technology and the increasing refinement of clinical demands have made the selection and in-depth development of bone implant materials one of the core driving forces behind the progress of orthopedic medicine[ 1 , 2 ]. Confronted with the complexities of biological environments and the ever-evolving challenges in healthcare, materials scientists are actively exploring new solutions, striving to preserve the advantages of traditional materials while innovating novel implant materials that better meet human physiological needs. Metals, a historically significant family of materials, remain irreplaceable in bone implant applications due to their outstanding mechanical properties, excellent biocompatibility, and versatile processability[ 3 – 5 ]. In the extensive family of medical metallic materials, stainless steel, cobalt-chromium alloys, titanium alloys, and emerging metals such as tantalum exhibit unique advantages, collectively forming a diverse material system. Stainless steel was widely used in the early stages due to its low cost and ease of processing; however, its limited corrosion resistance restricts its further development in long-term implant applications[ 6 ]. Cobalt-chromium alloys are renowned for their exceptional strength and fatigue resistance, but their biocompatibility still requires continuous optimization to meet higher clinical standards[ 7 – 9 ]. Titanium alloys, particularly Ti-6Al-4V, have become the most widely used metallic materials in clinical applications due to their excellent comprehensive properties, including high specific strength and superior corrosion resistance. Nevertheless, challenges remain, such as the significant difference in elastic modulus between titanium alloys and natural bone, which may cause stress shielding effects, and their biological inertness, which necessitates further research for effective solutions[ 10 ]. Tantalum is currently recognized as the metallic material with the best osteophilic properties. Porous tantalum was primarily used as a bone implant material[ 11 ]. This is because porous tantalum significantly reduces implant weight and elastic modulus[ 12 ], thereby mitigating stress shielding and promoting bone tissue repair and regeneration. Additionally, the micron-scale porous structure guides bone tissue growth into the pores of the tantalum, enabling biological fixation of the implant. Our previous research also revealed that, compared to Ti-6Al-4V, tantalum exhibits superior immunomodulatory properties, promoting macrophage M2 polarization, which facilitates faster bone tissue repair and regeneration[ 13 ]. However, pure tantalum has limitations such as low strength, high density, high cost, and difficulty in machining, which restrict its broader clinical application as a bone implant material. Alloying is one of the most common techniques used to improve the performance of metallic materials[ 14 ]. Titanium alloys are currently among the most extensively studied biomedical metallic materials. Using titanium as the base material and adding an appropriate amount of tantalum to form Ti-Ta alloys is a promising development direction for future biomedical metallic materials[ 15 ]. The studies on titanium-tantalum (Ti-Ta) alloys as biomedical metallic materials are currently limited[ 16 ]. In the early stages, Ti-Ta alloys were typically obtained through casting. Variations in the tantalum (Ta) content significantly affect the phase composition and mechanical properties of the Ti-Ta alloys[ 17 ]. When the Ta content is approximately 30 wt%, Ti-Ta alloys exhibit high strength and low elastic modulus. The advent of additive manufacturing (AM) provides an important technological option for fabricating components with complex structures and has broad application prospects in the aerospace and medical fields. It is particularly suitable for the fabrication of personalized medical implants[ 18 ]. Furthermore, AM has been used for novel alloy materials. Indranath Mitra utilized selective laser melting (SLM) with physically blended powders to fabricate Ti-10Ta and Ti-25Ta alloys and conducted systematic studies on their properties[ 19 ]. The as-printed Ti-Ta alloys demonstrated significantly higher strength than cast Ti-Ta alloys, while the Ti-25Ta alloy exhibited Young's modulus as low as 64 ± 6 GPa. Coupled with the ability of 3D printing to control the porous structure and microstructure of the printed parts, 3D-printed Ti-Ta alloys are considered promising candidates for the next generation of biomedical metallic materials. 3D printing, with its unique advantages, has opened new pathways for the fabrication of titanium-tantalum (Ti-Ta) alloys. Currently, the 3D printing of Ti-Ta alloys is predominantly achieved through the physical powder-mixing method[ 20 ]. This approach offers flexibility and precision in material selection and design[ 21 ]. However, it also faces challenges such as controlling powder uniformity, preventing phase separation during sintering, and ensuring consistency in mechanical properties. Additionally, the resulting alloys often exhibit defects such as unmelted particles and pores. This study aims to address this issue by developing high-performance Ti-25Ta spherical alloy powders using an innovative pre-alloying technique. Subsequently, porous Ti-Ta scaffolds with uniform physical and chemical properties are fabricated using SLM. The potential advantages and feasibility of porous Ti-25Ta alloys in bone repair and regeneration will be validated through a series of in vitro and in vivo experiments. This research not only aspires to provide safer and more effective bone implant solutions for clinical applications but also seeks to offer valuable insights and references for the development of other high-performance biomedical alloy materials, collectively advancing the progress and development of medical materials science. 2 Experimental method 2.1 Material Preparation Spherical powders of pure Ti, and Ti-25Ta alloy were prepared via inductively coupled plasma (ICP) spheroidization. The powders were used to fabricate solid disk samples (Φ10 mm×2 mm) for material characterization via SLM using a 3D printer (Renishaw AM400, UK) under an inert argon atmosphere with an oxygen content below 0.1%. Additionally, porous trabecular-structured disk scaffolds (Φ10 mm×2 mm, Φ30 mm×2 mm) were fabricated for cytological evaluation, and porous cylindrical rod scaffolds (Φ3 mm×10 mm) were produced for in vivo animal experiments. Following heat treatment, the samples were ultrasonically cleaned in anhydrous ethanol and deionized water for 30 minutes to remove residual surface-adhered powders. For ease of reference, the two sample groups—pure Ti and Ti-25Ta alloy—are designated as Ti and Ta25, respectively. 2.2 Material Characterization The surface morphology of powders, solid disc samples, and porous cylindrical scaffolds was observed using a scanning electron microscope (SEM, Hitachi SU3500, Japan). The distribution of powder particle size was analyzed using a laser particle size analyzer (BT-9300SE, China). The elemental composition and distribution on the surfaces of the powders and porous cylindrical scaffolds were analyzed using an energy-dispersive X-ray spectrometer (EDS, Oxford X-Max, UK) attached to the SEM. The elemental composition of the solid samples was determined using an X-ray fluorescence spectrometer (XRF, Panalytical Zetium, Netherlands). The phase composition of the solid samples was analyzed using an X-ray diffractometer (XRD, Rigaku D/MAX 2500, Japan) with Cu Kα radiation (λ = 1.5406 Å). The elemental composition and chemical states on the surface of the solid samples were examined using an X-ray photoelectron spectrometer (XPS, Thermo Scientific ESCALAB 250Xi, USA) with Al Kα radiation. The density of polished solid disc samples (Φ10 mm×2 mm) was measured using a density balance (Ohaus EX-125DZH, USA). Two samples were measured per group, and the mean value and standard deviation were calculated. The hardness (HV0.2) of the solid samples was tested using a Vickers hardness tester (Qness Q10A+, Austria) with a load of 0.2 kg and a dwell time of 10–15 seconds. Three random measurements were performed for each group of samples, and the mean value and standard deviation were calculated. The contact angle and surface energy were measured using a contact angle goniometer (KRÜSS DSA100, Germany). After polishing, the samples were sequentially cleaned with deionized water and anhydrous ethanol and then dried. A droplet (2 µL) of deionized water or dimethyl sulfoxide (DMSO) was dispensed onto the sample surface, and the contact angle was determined from images captured 5 seconds after droplet deposition. Each sample was tested five times with deionized water and DMSO, with the highest and lowest values removed before calculating the mean and standard deviation. Surface energy was calculated using the modified Owens–Wendt method based on three sets of deionized water and DMSO test data input into the software accompanying the contact angle goniometer. 2.3 In Vitro Evaluation 2.3.1 Sample Preparation Two groups of scaffolds were placed in a sterilization box and subjected to moist heat sterilization at 120°C for 20 minutes. After sterilization, the samples were dried using a forced-air drying oven. Once fully dried, they were cooled to room temperature for subsequent use. 2.3.2 Cell Culture The MC3T3-E1 cell line (mouse embryonic pre-osteoblasts) and RAW264.7 cell line (mouse mononuclear macrophage leukemia cells) were used for in vitro experiments. Both cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. MC3T3-E1 cells were cultured in DMEM/F12 complete medium (Hyclone, USA), while RAW264.7 cells were maintained in high-glucose DMEM (DMEM/High glucose, Hyclone, USA). Both culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin antibiotic solution (Hyclone, USA). The cells were incubated at 37°C in a 5% CO₂ humidified atmosphere and were used for in vitro experiments after being passaged twice. 2.3.3 Live/Dead Cell Staining and Cell Proliferation Scaffolds (Φ10 mm×2 mm) from the two experimental groups were placed in a 48-well plate and co-cultured with MC3T3-E1 cells (2×104 cells/mL) in 500 µL of complete culture medium. The plates were incubated at 37°C in a 5% CO2 atmosphere, with the culture medium being refreshed every two days. After 1, 4, and 7 days of culture, the cells were stained using a Live/Dead Cell Staining Kit (Dojindo, Japan) and observed under a fluorescence microscope (Olympus BX53, Japan). Cell proliferation on the scaffold surfaces was assessed using the Cell Counting Kit-8 (CCK-8, Japan). After 1, 4, and 7 days of co-culture in 48-well plates, the culture medium was aspirated, and 500 µL of fresh medium containing 10% CCK-8 reagent was added to each well. Three blank control wells without cells were also prepared. After incubation at 37°C for 2 hours, 100 µL of the supernatant from each well was transferred to a 96-well plate. The optical density (OD) at 450 nm was measured using a microplate reader (BioTek, USA). The mean OD value of the blank control group was calculated, and the final OD value for each sample was determined by subtracting the OD value of the blank control from that of the sample. The results were then subjected to statistical analysis. 2.3.4 Osteoblast Alkaline Phosphatase Immunofluorescence Staining and Extracellular Matrix Mineralization Immunofluorescence (IF) staining was performed to evaluate alkaline phosphatase (ALP) expression in MC3T3-E1 cells cultured on different scaffold surfaces. Two groups of scaffolds (Φ10 mm×2 mm) were placed in 48-well plates and co-cultured with 500 µL of MC3T3-E1 cells (2×104 cells/mL). On the following day, the medium was replaced with an osteogenic induction medium, which consisted of a complete DMEM/F12 medium supplemented with 10 nM dexamethasone, 50 mM ascorbic acid, and 10 mM β-glycerophosphate (Sigma, USA). The osteogenic induction medium was refreshed every two days. On days 7 and 14, the IF staining of ALP was conducted. The cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and blocked with 10% goat serum (Beyotime, China) for 20 minutes. Subsequently, the cells were incubated overnight at 4°C with rabbit anti-ALP primary antibody (1:500, Abcam, UK). After PBS washing, the samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500, Abcam, UK) at room temperature for 60 minutes. After PBS washing, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 10 minutes, and images were captured using a fluorescence microscope. The fluorescent area was quantified using ImageJ software. Extracellular matrix mineralization nodules were assessed using a tetracycline staining kit (Leagene, China). The scaffolds were co-cultured with MC3T3-E1 cells (1×104 cells/mL) for one day, followed by the addition of tetracycline at a concentration of 3% in the culture medium. The culture medium was refreshed every two days, and incubation was continued for 14 days. On day 14, nuclei were stained with DAPI and fluorescence microscopy was used to examine osteoblast mineralization in vitro. The fluorescent area was quantified using ImageJ software. 2.3.5 Macrophage Immunofluorescence Staining Scaffolds (Φ10 mm×2 mm) were placed in 48-well plates and co-cultured with 500 µL of RAW264.7 cells (2×104 cells/mL) for three consecutive days, with high-glucose medium replaced every two days. On day 4, the IF staining was performed. The samples were fixed with 4% PFA for 15 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and blocked with 10% goat serum (Beyotime, China) for 20 minutes. The cells were then incubated overnight at 4°C with rabbit anti-Arginase-1 and rabbit anti-CCR7 polyclonal antibodies (1:500, Sangon, China). The samples were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies (Abcam, UK) at room temperature for 60 minutes. After PBS washing, nuclei were counterstained with DAPI for 10 minutes, and fluorescence microscopy was used to capture images. The fluorescent area was quantified using ImageJ software. 2.3.6 Expression of Inflammation-Related Genes in Macrophages Porous scaffolds (Φ30 mm×2 mm) were placed in 6-well plates and co-cultured with RAW264.7 cells (3×104 cells/mL) to investigate the expression of inflammation-related genes. On day 3, cells were harvested from the co-culture system. Total RNA was extracted using an RNA extraction kit (Accurate Biology, China), and complementary DNA (cDNA) was synthesized using a reverse transcription kit (Takara, Japan). The expression of inflammation-related genes was then quantified by amplifying equal amounts of cDNA. The relative expression levels of IL-10 and ARG-1 genes were analyzed using 2⁻ΔΔCt. 2.4 In Vivo Evaluation 2.4.1 Surgery This study was approved by the Animal Research Institute of Zhongshan Hospital, Dalian University (Approval No. 202404003). Eighteen male New Zealand White rabbits (6 months old, 2.5–3 kg) were randomly divided into two groups. Anesthesia was induced with chlorpromazine hydrochloride at a dose of 0.1 mg/kg. The lateral condyle of the femur was located approximately 1 cm above the knee joint at the lateral femoral platform. A 2–3 cm longitudinal incision was made along the femur, followed by stepwise dissection of the skin, subcutaneous tissue, and muscles until the bone surface was exposed. A bone defect (Φ4 mm×10 mm) was drilled at the right femoral condyle, and a scaffold (Φ3 mm×10 mm) was implanted. The surgical wound was then sutured. Postoperatively, gentamicin was administered for three consecutive days. Euthanasia was performed at 4, and 12 weeks post-surgery using CO2 asphyxiation. 2.4.3 Micro-CT Micro-computed tomography (micro-CT) was employed to scan and analyze the scaffolds in the two groups, using a voltage of 500 kV, a current of 80 mA, and a resolution of 10 µm. Three-dimensional reconstruction of the micro-CT images was performed. A region of interest (ROI) with dimensions Φ5 mm×12 mm surrounding the scaffold was selected to assess the volume fraction of newly formed bone. 2.4.3 Acid Etching Bone specimens were fixed in 4% paraformaldehyde (PFA) for 7 days and dehydrated in a graded ethanol series. The samples were then embedded in polymethyl methacrylate (PMMA). Hard tissue sections were prepared using a microtome, cutting them into thin slices of 80–100 µm. Further grinding and polishing were performed to achieve a final thickness of < 40 µm. For the acid etching process, the hard tissue sections were subjected to wet polishing using 400-, 600-, and 1200-grit sandpapers to refine one surface of each sample. Subsequently, the samples were polished using alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 µm until a mirror-like surface was achieved. The polished samples were then etched with a 9% phosphoric acid solution for 20 seconds. After etching, the specimens were rinsed with deionized water and immersed in sodium hypochlorite solution for 5 minutes. Following bleaching, the samples were rapidly rinsed with deionized water and completely dried. Finally, gold sputter coating was applied before scanning electron microscopy (SEM) imaging to evaluate the tubule-lacunae network. 2.5 Statistical Analysis Data were presented as mean ± standard deviation (SD) and statistically analyzed using GraphPad Prism 8 software. One-way analyses of variance (ANOVA) followed by Tukey’s multiple comparison tests were conducted. A p-value of < 0.05 was considered statistically significant. 3 Results 3.1 Characterizations of the powders and scaffolds The powders were characterized. Figure 1 a shows the SEM images of the powders, where Ti and Ti-25Ta powders exhibit a spherical morphology with a uniform size distribution. Figure 1 b displays the particle size distribution, revealing that all two powders follow a normal distribution with similar particle sizes ranging from approximately 10 µm to 100 µm. The average particle sizes of Ti and Ti-25Ta powders are 36.39 µm and 34.03 µm respectively. The EDS spectra of the powders are shown in Fig. 1 c and Fig. 1 d, indicating that all two powders are composed of Ti and/or Ta, with no detectable impurities. Figure 1 e and Fig. 1 f illustrate the apparent density and tap density of the two powders, respectively. The above results confirm that all two powders meet the requirements for 3D printing. The surface morphology of the porous disk-shaped scaffolds was observed using SEM, and the elemental distribution on the scaffold surface was analyzed using EDS, as shown in Fig. 2 a. The printed porous scaffold exhibits a well-defined structure with uniform pore size and filament diameter, and the Ti and Ta elements are homogeneously distributed. Figure 2 b presents the XRD spectra of the two sample groups, showing that Ti exists as an α-phase pure titanium, while Ta25 is an α-phase titanium alloy. Figure 2 c shows the XPS spectra of Ta25, revealing that the scaffold surface consists of Ta, Ti, and O. The spectra of Ta4f and Ti2p confirm the presence of Ta2O5 and TiO2 oxide layers. The O1s spectrum, attributed to Ta2O5 and TiO2, shows binding energies of 531.87 eV and 530.36 eV, respectively. The O1s peak area ratio of Ta2O5 to TiO2 is 1:4.57, corresponding to an atomic ratio of Ta to Ti of 1:5.71 in the oxide layer. The XRF analysis of Ta25 reveals that the Ta and Ti contents are 24.2 wt% and 75.8 wt%, respectively, corresponding to an atomic ratio of 1:11.84. A comparison of XPS and XRF results suggests that the Ta content in the oxide layer (14.90 at%) is significantly higher than in the alloy (7.79 at%), indicating that Ta is more prone to passivation than Ti. The densities, Vickers hardnesses, contact angles, and surface energies of Ti and Ta25 are summarized in Table 1 . Compared to Ti, Ta25 exhibits an increased density; however, its density remains at only 39% of that of Ta. Ta25 shows significant improvements in hardness over both Ti and Ta. The contact angles of water and DMSO decrease with increasing Ta content, while the surface energy increases, suggesting that Ta exhibits better wettability and higher surface energy than Ti, laying a foundation for the excellent biocompatibility of Ta. Table 1 The densities, Vickers hardnesses, contact angles, and surface energies of the 3D-printed bulks of Ti and Ta25 Sample Mean value Standard deviation Density (g/cm 3 ) Ti 4.51 0.02 Ta25 5.40 0.16 Vickers hardness (HV 0.2 ) Ti 171.7 5.1 Ta25 295.3 5.7 Contact angle of water (degree) Ti 57.5 3.1 Ta25 51.8 2.8 Contact angle of DMSO (degree) Ti 35.1 3.5 Ta25 32.9 3.9 Surface energy (mJ/m 2 ) Ti 43.58 — Ta25 47.85 — In summary, the Ta25 alloy demonstrates excellent printability and improvements in mechanical property and wettability over Ti without significantly increasing the density. 3.2 In Vitro Evaluation 3.2.1 Cell Viability and Proliferation To assess cell growth on the surfaces of two scaffolds, a live/dead cell staining assay was performed (Fig. 3 a). The results demonstrate good biocompatibility across all groups, with minimal dead cells observed. After 7 days, the number of cells on the surface of the Ta25 group was higher than that of the Ti group. Cell viability and proliferation were further evaluated using the CCK-8 assay (Fig. 3 b). At 1, 4, and 7 days, the absorbance of the Ta25 group was higher than that of the Ti group, but the difference between the two groups was not statistically significant at the first day. At 4 and 7 days, the difference between the two groups was statistically significant. These findings suggest that Ta25 has greater cell viability and proliferation than Ti, and incorporating Ta into Ti enhances cellular activity. 3.2.2 ALP Activity and In Vitro Mineralization The osteogenic differentiation of MC3T3-E1 cells in the two sample groups was systematically evaluated using IF staining of ALP (Fig. 3 c) and an in vitro mineralization assay (Fig. 3 d). The results indicate that all groups exhibit a certain level of ALP expression, the ALP expression level of Ta25 group was significantly higher than that of Ti group, especially at 14 days, Ta25 group showed obvious advantages in promoting the differentiation of osteogenic precursor cells. Quantitative analysis (Fig. 3 f, 3 g) further verified this conclusion, and the difference between the Ta25 group and the Ti group was statistically significant, indicating that the addition of Ta metal to Ti metal can effectively enhance the ALP activity of osteoblasts. Tetracycline staining (Fig. 3 e) reveals that after 21 days of culture, MC3T3-E1 cells in both the Ta25 and Ti groups produced mineralized nodules, while the Ta25 group showed the strongest mineralization ability. Quantitative analysis (Fig. 3 h) further verifies this trend, showing statistically significant differences between all pairwise comparisons. These findings suggest that Ta25 alloy has a significant advantage in promoting the differentiation and strengthening of the bone precursor cells, and adding Ta can improve the salinity of osteoblasts in Ti. 3.2.3 Macrophage Immunofluorescence and qRT-PCR Analysis Macrophage polarization was assessed using IF staining for CCR7 and ARG-1 markers. The results showed that the fluorescence signal of the ccr7 immunofluorescence marker (Fig. 4 a) and Ti group was strong in the fluorescence signal of the inflammatory factors in the group of Ti group, which showed that the expression level of the inflammatory factors was higher, and the macrophages showed a typical activation state, and the cytoplastic volume increased and the pseudoponas increased. The fluorescence signals of the inflammatory factors in the Ta25 group of macrophages were weak, indicating that the level of the inflammatory factors was low, the cell morphology of the macrophages was normal, and the cytoplasm was smaller and the pseudoplanes were less. The quantitative analysis results (Fig. 4 c) further confirm this result. In the ARG-1 immunofluorescence marker (Fig. 4 b), the fluorescence signal of the anti-inflammatory factors in the Ti group was weak, indicating that the expression level of anti-inflammatory factors was low. Macrophages may present a typical activation state. The fluorescence signal of anti-inflammatory factors in Ta25 macrophages was significantly enhanced, indicating that the expression level of the anti-inflammatory factor was higher, the formation of the cell morphology of the macrophages was more regular, and the state quantitative analysis of the more static rate was shown in Fig. 4 d, which further confirmed the conclusion. The anti-inflammatory factor of the Ta25 group was significantly stronger than the Ti group, indicating that the Ta25 alloy had a stronger anti-inflammatory effect. The Ta25 alloy may reduce the inflammatory response by regulating the polarization of macrophages and promoting its transformation of anti-inflammatory phenotype (M2). To evaluate gene expression differences between groups at the molecular level, qRT-PCR analysis was performed on macrophage-related genes IL-10 and ARG-1 (Fig. 4 e, 4 f). GAPDH was used as the housekeeping gene for normalization, with gene expression levels in the control group set to 1. Ta25 group IL-10、ARG-1 gene expression was significantly higher than the Ti group. The differences in gene expression between the two groups were statistically significant. This result, further confirmed the anti-inflammatory effect of Ta25 metal from the gene expression level. 3.3 In Vivo Evaluation 3.3.1 Micro-CT Analysis Micro-CT reconstruction of the implants and surrounding bone tissue was performed (Fig. 5 a, 5 b). Compared with Ti scaffold, the repair effect of Ta25 scaffold on bone defects was significantly improved. There was no significant difference in bone defect repair at 4 weeks, and more newly formed bone tissue was observed on the Ta25 scaffold at 12 weeks. In addition, quantitative analysis of bone volume fraction (BV/TV) was performed on the 3D-reconstructed images (Fig. 5 c, 5 d), which further confirmed the enhanced bone regeneration ability of the Ta25 scaffold. 3.3.2 SEM Observation of the Scaffold–Bone Interface To further evaluate the integration of the two scaffolds with bone tissue, acid etching, and SEM were used to observe cellular growth and interactions at the scaffold–bone interface (Fig. 6 ). After 4 weeks of implantation in the lateral femoral condyle of rabbits, all groups exhibited similar material–bone contact. After 12 weeks, a large number of osteocytes were observed around the Ta25 scaffolds, with their quantity significantly exceeding that around the Ti scaffold. The osteocytes formed tight connections with the material surface through canaliculi. Despite a ~ 20 µm gap between the scaffold and the bone, numerous osteocytes are observed within the gap and interconnected via canaliculi that extend toward the material surface, facilitating direct bonding. These findings indicate that bone integration occurs through biological bonding, further confirming the excellent biocompatibility and osseointegration performance of porous Ta25. 4 Discussion This study successfully fabricated porous Ti-25Ta alloy scaffolds using 3D printing. The Ti-25Ta alloy has excellent mechanical properties, good biocompatibility, and promising bone repair capabilities, indicating broad prospects in the orthopedic field. Compared with inert metals such as titanium, tantalum exhibits superior stability, corrosion resistance, and biocompatibility[ 22 ]. Pure tantalum was first introduced into orthopedic implants in 1940. Porous tantalum prepared by chemical vapor deposition (CVD) was approved by the U.S. FDA for artificial acetabular prostheses in 1997[ 23 ]. After nearly 30 years of clinical application, porous tantalum has gained widespread recognition from physicians and patients due to its beneficial clinical performance. In recent years, 3D-printed tantalum implants have emerged as a novel alternative, overcoming the limitations of CVD-fabricated porous tantalum, such as forming difficulties, and significantly expanding the application of tantalum in orthopedic implants[ 24 ]. However, the high density of tantalum remains a major challenge restricting its widespread application. In this study, titanium was alloyed with tantalum to enhance the bioactivity of titanium alloys while mitigating the density issue[ 25 ]. Firstly, compared to pure tantalum, the Ti-25Ta alloy significantly reduces the material density, achieving a lightweight structure. Additionally, the introduction of tantalum considerably enhances the mechanical properties of titanium alloys, with microhardness comparable to that of 3D-printed Ti6Al4V alloys, laying a solid foundation for orthopedic applications. Biocompatibility is a critical criterion for evaluating the success of bone implant materials. In this study, the Ti-25Ta alloy exhibited excellent cytocompatibility. In vitro experiments demonstrate that osteoblast adhesion and proliferation on the alloy surface are superior to those on pure titanium, with enhanced mineralization capacity, indicating that the incorporation of tantalum improves cellular activity. Studies on macrophages revealed that tantalum possesses superior anti-inflammatory properties, and the incorporation of tantalum into the titanium alloy significantly enhances its anti-inflammatory capability. Improved anti-inflammatory effects help suppress immune rejection, thereby promoting osseointegration and bone repair. Therefore, tantalum metal exhibits superior "bioaffinity," which has been confirmed by clinical studies on tantalum[ 26 ]. Particularly in cases involving inflammation or infection, the advantages of tantalum metal are fully demonstrated[ 27 ]. The improvement in cell activity of Ti-25Ta alloy is attributed to its surface oxide layer, which provides a suitable growth environment for cells. This study indicates that tantalum metal possesses higher surface energy and wettability compared to titanium metal, and the introduction of Ta element can enhance the surface energy and wettability of titanium metal. Furthermore, XPS analysis reveals that the content of Ta element in the surface oxide layer of the alloy is significantly higher than that in the alloy itself. This is due to the stronger passivation capability of tantalum metal compared to titanium metal, making it more prone to oxidation[ 28 ]. Thus, even though the atomic percentage of Ta in the alloy is only 7.79 at%, it can significantly improve the cell activity of titanium metal[ 29 , 30 ]. Beyond the intrinsic bioactivity improvements of the Ti-25Ta alloy, the porous scaffold structure provides ample space for cellular infiltration, proliferation, and bone tissue regeneration[ 31 ]. The interconnected pores facilitate the migration of bone marrow and blood vessels to the implantation site, promoting new bone formation[ 32 ]. This enhanced osseointegration underscores the significant potential of porous Ti-25Ta alloys in bone defect repair. Despite the promising biological performance of the 3D-printed Ti-25Ta alloy scaffold, several challenges remain for its clinical translation. Firstly, more tests are required to confirm its mechanical properties and long-term effectiveness. Secondly, optimizing the 3D printing process is needed for better quality and wider application. Finally, the anti-inflammatory effect of the Ti-Ta alloy should be further investigated via animal models of inflammation or infection. 5 Conclusion In this study, porous scaffolds of pure Ti and Ti-25Ta alloy were successfully fabricated using 3D printing. The spherical powders used in the 3D printing show excellent sphericity and purity. The physical properties, biocompatibility, macrophage polarization, and in vivo bone defect repair efficacy of the scaffolds were systematically evaluated. Compared to pure Ti scaffold, the Ti-25Ta scaffold exhibits superior mechanical properties, and enhanced cell adhesion, proliferation, and anti-inflammatory capability. This improvement is due to the incorporation of Ta, which is demonstrated by the highest cell proliferation, osteogenic differentiation, mineralized nodule formation, anti-inflammatory capability, and bone reconstruction of pure Ta scaffold. In summary, Ti-25Ta alloy remains a promising candidate for bone implant applications. Further optimization of its composition and microstructure is required to enhance its osteogenic properties. Declarations Ethics approval and consent to participate The present investigation was conducted in complete adherence to the guidelines outlined in the Guide for the Care of Animals. The research protocol through a rigorous evaluation process and received clearance from the Animal Research Institute of Affiliated Zhongshan hospital of Dalian University, with the assigned approval number 202404003. Consent for publication Not applicable Competing interests The author declares that there was no competing interests Funding This study was funded by the Liao Ning Revitalization Talents Program (project No. XLYC2203102), National Natural Science Foundation of China (project No. 82172398), Distinguished Youth Foundation of Dalian (project No. 2024RJ007). Author Contribution W.D. 、 L.Y.D. and Z.W.W. wrote the article and participated in all experimental projects. J.H.L.、G.B. and X.Q.W participated in the in vitro experiments, X.J.F. participated in the in vivo experiments, W.W.D., C.F. and Y.G.X. participated in the material characterization experiments, and LJL and ZDW provided the main experimental ideas, experimental plan funds and sites. All reviewed the manuscript. 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Investigation of Cytotoxicity, Oxidative Stress, and Inflammatory Responses of Tantalum Nanoparticles in THP-1-Derived Macrophages. Mediators Inflamm. 2020; 2020:3824593. https://doi.org/10.1155/2020/3824593. Seo B, Park HK, Park CS, Kim S, Park K. Manipulating the Cathodic Modification Effect on Corrosion Resistance of High Corrosion-Resistant Titanium Alloy. Materials (Basel). 2023; 16. https://doi.org/10.3390/ma16186217. Dommeti VK, Roy S, Pramanik S, Merdji A, Ouldyerou A, Özcan M. Design and Development of Tantalum and Strontium Ion Doped Hydroxyapatite Composite Coating on Titanium Substrate: Structural and Human Osteoblast-like Cell Viability Studies. Materials (Basel). 2023; 16. https://doi.org/10.3390/ma16041499. Dias Corpa Tardelli J, Lima da Costa Valente M, Theodoro de Oliveira T, Cândido Dos Reis A. Influence of chemical composition on cell viability on titanium surfaces: A systematic review. J Prosthet Dent. 2021; 125:421-425. https://doi.org/10.1016/j.prosdent.2020.02.001. Matuła I, Dercz G, Sowa M, Barylski A, Duda P. Fabrication and Characterization of New Functional Graded Material Based on Ti, Ta, and Zr by Powder Metallurgy Method. Materials (Basel). 2021; 14. https://doi.org/10.3390/ma14216609. Wang X, Ning B, Pei X. Tantalum and its derivatives in orthopedic and dental implants: Osteogenesis and antibacterial properties. Colloids Surf B Biointerfaces. 2021; 208:112055. https://doi.org/10.1016/j.colsurfb.2021.112055. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviews received at journal 22 Mar, 2025 Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor assigned by journal 19 Mar, 2025 Submission checks completed at journal 18 Mar, 2025 First submitted to journal 18 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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10:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6134011/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6134011/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79127353,"identity":"aa4011dd-db08-48e2-b745-42d07f564b7d","added_by":"auto","created_at":"2025-03-24 17:45:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150859,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images (a), particle size distributions (b), EDS spectra (c)(d), apparent densities (e), and tap densities (f) of the powders of Ti and Ta25\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/baa9be313ef7198ab13537d3.png"},{"id":79126762,"identity":"7c2c820f-0388-4f0a-b7b0-788c82d1c3be","added_by":"auto","created_at":"2025-03-24 17:37:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205598,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images and element distribution of the 3D-printed porous scaffolds (a), XRD spectra (b) of the 3D-printed bulks of Ti and Ta25, and XPS spectra (c) of the 3D-printed bulk of Ta25\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/36f07c52de1c9a9df984bae6.png"},{"id":79126764,"identity":"d19fe0d2-cbd1-4f7e-b7e3-278473d136e5","added_by":"auto","created_at":"2025-03-24 17:37:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212847,"visible":true,"origin":"","legend":"\u003cp\u003eLive-dead staining (a), CCK-8 assay (b), ALP IF staining (c, d), tetracycline staining (e) of the MC3T3-E1 cells on the scaffolds. Fluorescence intensities of ALP on 7d (f) and 14d (g), and tetracycline on 21d (h).* p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0,0001\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/7778cd34cb6defc23ba34364.png"},{"id":79126765,"identity":"4fc83bac-6135-40db-a09f-df5bb585eb76","added_by":"auto","created_at":"2025-03-24 17:37:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122406,"visible":true,"origin":"","legend":"\u003cp\u003eIF staining of CCR-7 (a) and ARG-1 (b) of the RAW264.7 cells on the scaffolds. Fluorescence intensities of CCR-7 (c) and ARG-1 (d). Relative expressions of anti-inflammatory-related genes of IL-10 (e) and ARG-1 (f). * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/390ea887a2032979acf863eb.png"},{"id":79126776,"identity":"772ebeba-a6df-4a30-b4df-c2413c62314f","added_by":"auto","created_at":"2025-03-24 17:37:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90500,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-CT images and three-dimensional reconstructions of the rabbit femoral condyles on 4W (a) and 12W (b), and the relative volumes of trabecular bone on 4W (c) and 12W (d). * p\u0026lt;0.05. In (a) and (b), green areas indicate new bone, purple areas indicate implants\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/847aceec8246b09e78278759.png"},{"id":79126767,"identity":"afb7c4da-59c6-4a52-95fc-6c9ae8f97ff3","added_by":"auto","created_at":"2025-03-24 17:37:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137752,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of acid etched scaffold–bone tissue interfaces\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/a1782934451259e504475337.png"},{"id":79129268,"identity":"31646d59-a15d-4181-a166-74dee5e53fad","added_by":"auto","created_at":"2025-03-24 18:09:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1704855,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6134011/v1/e32ff0d0-b4b0-4dc4-a9b3-4d22ff286ca2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fabrication and Biocompatibility of a 3D-printed Porous Ti-25Ta Alloy Scaffold","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eIn the vast field of contemporary medical materials science, the rapid advancement of medical technology and the increasing refinement of clinical demands have made the selection and in-depth development of bone implant materials one of the core driving forces behind the progress of orthopedic medicine[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Confronted with the complexities of biological environments and the ever-evolving challenges in healthcare, materials scientists are actively exploring new solutions, striving to preserve the advantages of traditional materials while innovating novel implant materials that better meet human physiological needs. Metals, a historically significant family of materials, remain irreplaceable in bone implant applications due to their outstanding mechanical properties, excellent biocompatibility, and versatile processability[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the extensive family of medical metallic materials, stainless steel, cobalt-chromium alloys, titanium alloys, and emerging metals such as tantalum exhibit unique advantages, collectively forming a diverse material system. Stainless steel was widely used in the early stages due to its low cost and ease of processing; however, its limited corrosion resistance restricts its further development in long-term implant applications[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cobalt-chromium alloys are renowned for their exceptional strength and fatigue resistance, but their biocompatibility still requires continuous optimization to meet higher clinical standards[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTitanium alloys, particularly Ti-6Al-4V, have become the most widely used metallic materials in clinical applications due to their excellent comprehensive properties, including high specific strength and superior corrosion resistance. Nevertheless, challenges remain, such as the significant difference in elastic modulus between titanium alloys and natural bone, which may cause stress shielding effects, and their biological inertness, which necessitates further research for effective solutions[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTantalum is currently recognized as the metallic material with the best osteophilic properties. Porous tantalum was primarily used as a bone implant material[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This is because porous tantalum significantly reduces implant weight and elastic modulus[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], thereby mitigating stress shielding and promoting bone tissue repair and regeneration. Additionally, the micron-scale porous structure guides bone tissue growth into the pores of the tantalum, enabling biological fixation of the implant. Our previous research also revealed that, compared to Ti-6Al-4V, tantalum exhibits superior immunomodulatory properties, promoting macrophage M2 polarization, which facilitates faster bone tissue repair and regeneration[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, pure tantalum has limitations such as low strength, high density, high cost, and difficulty in machining, which restrict its broader clinical application as a bone implant material.\u003c/p\u003e \u003cp\u003eAlloying is one of the most common techniques used to improve the performance of metallic materials[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Titanium alloys are currently among the most extensively studied biomedical metallic materials. Using titanium as the base material and adding an appropriate amount of tantalum to form Ti-Ta alloys is a promising development direction for future biomedical metallic materials[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe studies on titanium-tantalum (Ti-Ta) alloys as biomedical metallic materials are currently limited[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the early stages, Ti-Ta alloys were typically obtained through casting. Variations in the tantalum (Ta) content significantly affect the phase composition and mechanical properties of the Ti-Ta alloys[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When the Ta content is approximately 30 wt%, Ti-Ta alloys exhibit high strength and low elastic modulus. The advent of additive manufacturing (AM) provides an important technological option for fabricating components with complex structures and has broad application prospects in the aerospace and medical fields. It is particularly suitable for the fabrication of personalized medical implants[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, AM has been used for novel alloy materials. Indranath Mitra utilized selective laser melting (SLM) with physically blended powders to fabricate Ti-10Ta and Ti-25Ta alloys and conducted systematic studies on their properties[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The as-printed Ti-Ta alloys demonstrated significantly higher strength than cast Ti-Ta alloys, while the Ti-25Ta alloy exhibited Young's modulus as low as 64\u0026thinsp;\u0026plusmn;\u0026thinsp;6 GPa. Coupled with the ability of 3D printing to control the porous structure and microstructure of the printed parts, 3D-printed Ti-Ta alloys are considered promising candidates for the next generation of biomedical metallic materials.\u003c/p\u003e \u003cp\u003e3D printing, with its unique advantages, has opened new pathways for the fabrication of titanium-tantalum (Ti-Ta) alloys. Currently, the 3D printing of Ti-Ta alloys is predominantly achieved through the physical powder-mixing method[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This approach offers flexibility and precision in material selection and design[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, it also faces challenges such as controlling powder uniformity, preventing phase separation during sintering, and ensuring consistency in mechanical properties. Additionally, the resulting alloys often exhibit defects such as unmelted particles and pores.\u003c/p\u003e \u003cp\u003eThis study aims to address this issue by developing high-performance Ti-25Ta spherical alloy powders using an innovative pre-alloying technique. Subsequently, porous Ti-Ta scaffolds with uniform physical and chemical properties are fabricated using SLM. The potential advantages and feasibility of porous Ti-25Ta alloys in bone repair and regeneration will be validated through a series of in vitro and in vivo experiments. This research not only aspires to provide safer and more effective bone implant solutions for clinical applications but also seeks to offer valuable insights and references for the development of other high-performance biomedical alloy materials, collectively advancing the progress and development of medical materials science.\u003c/p\u003e"},{"header":"2 Experimental method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Material Preparation\u003c/h2\u003e \u003cp\u003eSpherical powders of pure Ti, and Ti-25Ta alloy were prepared via inductively coupled plasma (ICP) spheroidization. The powders were used to fabricate solid disk samples (Φ10 mm\u0026times;2 mm) for material characterization via SLM using a 3D printer (Renishaw AM400, UK) under an inert argon atmosphere with an oxygen content below 0.1%. Additionally, porous trabecular-structured disk scaffolds (Φ10 mm\u0026times;2 mm, Φ30 mm\u0026times;2 mm) were fabricated for cytological evaluation, and porous cylindrical rod scaffolds (Φ3 mm\u0026times;10 mm) were produced for in vivo animal experiments. Following heat treatment, the samples were ultrasonically cleaned in anhydrous ethanol and deionized water for 30 minutes to remove residual surface-adhered powders. For ease of reference, the two sample groups\u0026mdash;pure Ti and Ti-25Ta alloy\u0026mdash;are designated as Ti and Ta25, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Material Characterization\u003c/h2\u003e \u003cp\u003eThe surface morphology of powders, solid disc samples, and porous cylindrical scaffolds was observed using a scanning electron microscope (SEM, Hitachi SU3500, Japan).\u003c/p\u003e \u003cp\u003eThe distribution of powder particle size was analyzed using a laser particle size analyzer (BT-9300SE, China).\u003c/p\u003e \u003cp\u003eThe elemental composition and distribution on the surfaces of the powders and porous cylindrical scaffolds were analyzed using an energy-dispersive X-ray spectrometer (EDS, Oxford X-Max, UK) attached to the SEM.\u003c/p\u003e \u003cp\u003eThe elemental composition of the solid samples was determined using an X-ray fluorescence spectrometer (XRF, Panalytical Zetium, Netherlands).\u003c/p\u003e \u003cp\u003eThe phase composition of the solid samples was analyzed using an X-ray diffractometer (XRD, Rigaku D/MAX 2500, Japan) with Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;).\u003c/p\u003e \u003cp\u003eThe elemental composition and chemical states on the surface of the solid samples were examined using an X-ray photoelectron spectrometer (XPS, Thermo Scientific ESCALAB 250Xi, USA) with Al Kα radiation.\u003c/p\u003e \u003cp\u003eThe density of polished solid disc samples (Φ10 mm\u0026times;2 mm) was measured using a density balance (Ohaus EX-125DZH, USA). Two samples were measured per group, and the mean value and standard deviation were calculated.\u003c/p\u003e \u003cp\u003eThe hardness (HV0.2) of the solid samples was tested using a Vickers hardness tester (Qness Q10A+, Austria) with a load of 0.2 kg and a dwell time of 10\u0026ndash;15 seconds. Three random measurements were performed for each group of samples, and the mean value and standard deviation were calculated.\u003c/p\u003e \u003cp\u003eThe contact angle and surface energy were measured using a contact angle goniometer (KR\u0026Uuml;SS DSA100, Germany). After polishing, the samples were sequentially cleaned with deionized water and anhydrous ethanol and then dried. A droplet (2 \u0026micro;L) of deionized water or dimethyl sulfoxide (DMSO) was dispensed onto the sample surface, and the contact angle was determined from images captured 5 seconds after droplet deposition. Each sample was tested five times with deionized water and DMSO, with the highest and lowest values removed before calculating the mean and standard deviation. Surface energy was calculated using the modified Owens\u0026ndash;Wendt method based on three sets of deionized water and DMSO test data input into the software accompanying the contact angle goniometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In Vitro Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Sample Preparation\u003c/h2\u003e \u003cp\u003eTwo groups of scaffolds were placed in a sterilization box and subjected to moist heat sterilization at 120\u0026deg;C for 20 minutes. After sterilization, the samples were dried using a forced-air drying oven. Once fully dried, they were cooled to room temperature for subsequent use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Cell Culture\u003c/h2\u003e \u003cp\u003eThe MC3T3-E1 cell line (mouse embryonic pre-osteoblasts) and RAW264.7 cell line (mouse mononuclear macrophage leukemia cells) were used for in vitro experiments. Both cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. MC3T3-E1 cells were cultured in DMEM/F12 complete medium (Hyclone, USA), while RAW264.7 cells were maintained in high-glucose DMEM (DMEM/High glucose, Hyclone, USA). Both culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin antibiotic solution (Hyclone, USA). The cells were incubated at 37\u0026deg;C in a 5% CO₂ humidified atmosphere and were used for in vitro experiments after being passaged twice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Live/Dead Cell Staining and Cell Proliferation\u003c/h2\u003e \u003cp\u003eScaffolds (Φ10 mm\u0026times;2 mm) from the two experimental groups were placed in a 48-well plate and co-cultured with MC3T3-E1 cells (2\u0026times;104 cells/mL) in 500 \u0026micro;L of complete culture medium. The plates were incubated at 37\u0026deg;C in a 5% CO2 atmosphere, with the culture medium being refreshed every two days. After 1, 4, and 7 days of culture, the cells were stained using a Live/Dead Cell Staining Kit (Dojindo, Japan) and observed under a fluorescence microscope (Olympus BX53, Japan).\u003c/p\u003e \u003cp\u003eCell proliferation on the scaffold surfaces was assessed using the Cell Counting Kit-8 (CCK-8, Japan). After 1, 4, and 7 days of co-culture in 48-well plates, the culture medium was aspirated, and 500 \u0026micro;L of fresh medium containing 10% CCK-8 reagent was added to each well. Three blank control wells without cells were also prepared. After incubation at 37\u0026deg;C for 2 hours, 100 \u0026micro;L of the supernatant from each well was transferred to a 96-well plate. The optical density (OD) at 450 nm was measured using a microplate reader (BioTek, USA). The mean OD value of the blank control group was calculated, and the final OD value for each sample was determined by subtracting the OD value of the blank control from that of the sample. The results were then subjected to statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Osteoblast Alkaline Phosphatase Immunofluorescence Staining and Extracellular Matrix Mineralization\u003c/h2\u003e \u003cp\u003eImmunofluorescence (IF) staining was performed to evaluate alkaline phosphatase (ALP) expression in MC3T3-E1 cells cultured on different scaffold surfaces. Two groups of scaffolds (Φ10 mm\u0026times;2 mm) were placed in 48-well plates and co-cultured with 500 \u0026micro;L of MC3T3-E1 cells (2\u0026times;104 cells/mL). On the following day, the medium was replaced with an osteogenic induction medium, which consisted of a complete DMEM/F12 medium supplemented with 10 nM dexamethasone, 50 mM ascorbic acid, and 10 mM β-glycerophosphate (Sigma, USA). The osteogenic induction medium was refreshed every two days. On days 7 and 14, the IF staining of ALP was conducted. The cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and blocked with 10% goat serum (Beyotime, China) for 20 minutes. Subsequently, the cells were incubated overnight at 4\u0026deg;C with rabbit anti-ALP primary antibody (1:500, Abcam, UK). After PBS washing, the samples were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500, Abcam, UK) at room temperature for 60 minutes. After PBS washing, nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 10 minutes, and images were captured using a fluorescence microscope. The fluorescent area was quantified using ImageJ software.\u003c/p\u003e \u003cp\u003eExtracellular matrix mineralization nodules were assessed using a tetracycline staining kit (Leagene, China). The scaffolds were co-cultured with MC3T3-E1 cells (1\u0026times;104 cells/mL) for one day, followed by the addition of tetracycline at a concentration of 3% in the culture medium. The culture medium was refreshed every two days, and incubation was continued for 14 days. On day 14, nuclei were stained with DAPI and fluorescence microscopy was used to examine osteoblast mineralization in vitro. The fluorescent area was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Macrophage Immunofluorescence Staining\u003c/h2\u003e \u003cp\u003eScaffolds (Φ10 mm\u0026times;2 mm) were placed in 48-well plates and co-cultured with 500 \u0026micro;L of RAW264.7 cells (2\u0026times;104 cells/mL) for three consecutive days, with high-glucose medium replaced every two days. On day 4, the IF staining was performed. The samples were fixed with 4% PFA for 15 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and blocked with 10% goat serum (Beyotime, China) for 20 minutes. The cells were then incubated overnight at 4\u0026deg;C with rabbit anti-Arginase-1 and rabbit anti-CCR7 polyclonal antibodies (1:500, Sangon, China). The samples were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies (Abcam, UK) at room temperature for 60 minutes. After PBS washing, nuclei were counterstained with DAPI for 10 minutes, and fluorescence microscopy was used to capture images. The fluorescent area was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6 Expression of Inflammation-Related Genes in Macrophages\u003c/h2\u003e \u003cp\u003ePorous scaffolds (Φ30 mm\u0026times;2 mm) were placed in 6-well plates and co-cultured with RAW264.7 cells (3\u0026times;104 cells/mL) to investigate the expression of inflammation-related genes. On day 3, cells were harvested from the co-culture system. Total RNA was extracted using an RNA extraction kit (Accurate Biology, China), and complementary DNA (cDNA) was synthesized using a reverse transcription kit (Takara, Japan). The expression of inflammation-related genes was then quantified by amplifying equal amounts of cDNA. The relative expression levels of IL-10 and ARG-1 genes were analyzed using 2⁻ΔΔCt.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 In Vivo Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Surgery\u003c/h2\u003e \u003cp\u003e This study was approved by the Animal Research Institute of Zhongshan Hospital, Dalian University (Approval No. 202404003). Eighteen male New Zealand White rabbits (6 months old, 2.5\u0026ndash;3 kg) were randomly divided into two groups. Anesthesia was induced with chlorpromazine hydrochloride at a dose of 0.1 mg/kg. The lateral condyle of the femur was located approximately 1 cm above the knee joint at the lateral femoral platform. A 2\u0026ndash;3 cm longitudinal incision was made along the femur, followed by stepwise dissection of the skin, subcutaneous tissue, and muscles until the bone surface was exposed. A bone defect (Φ4 mm\u0026times;10 mm) was drilled at the right femoral condyle, and a scaffold (Φ3 mm\u0026times;10 mm) was implanted. The surgical wound was then sutured. Postoperatively, gentamicin was administered for three consecutive days. Euthanasia was performed at 4, and 12 weeks post-surgery using CO2 asphyxiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Micro-CT\u003c/h2\u003e \u003cp\u003eMicro-computed tomography (micro-CT) was employed to scan and analyze the scaffolds in the two groups, using a voltage of 500 kV, a current of 80 mA, and a resolution of 10 \u0026micro;m. Three-dimensional reconstruction of the micro-CT images was performed. A region of interest (ROI) with dimensions Φ5 mm\u0026times;12 mm surrounding the scaffold was selected to assess the volume fraction of newly formed bone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Acid Etching\u003c/h2\u003e \u003cp\u003eBone specimens were fixed in 4% paraformaldehyde (PFA) for 7 days and dehydrated in a graded ethanol series. The samples were then embedded in polymethyl methacrylate (PMMA). Hard tissue sections were prepared using a microtome, cutting them into thin slices of 80\u0026ndash;100 \u0026micro;m. Further grinding and polishing were performed to achieve a final thickness of \u0026lt;\u0026thinsp;40 \u0026micro;m.\u003c/p\u003e \u003cp\u003eFor the acid etching process, the hard tissue sections were subjected to wet polishing using 400-, 600-, and 1200-grit sandpapers to refine one surface of each sample. Subsequently, the samples were polished using alumina suspensions with particle sizes of 1.0, 0.3, and 0.05 \u0026micro;m until a mirror-like surface was achieved. The polished samples were then etched with a 9% phosphoric acid solution for 20 seconds. After etching, the specimens were rinsed with deionized water and immersed in sodium hypochlorite solution for 5 minutes. Following bleaching, the samples were rapidly rinsed with deionized water and completely dried. Finally, gold sputter coating was applied before scanning electron microscopy (SEM) imaging to evaluate the tubule-lacunae network.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and statistically analyzed using GraphPad Prism 8 software. One-way analyses of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison tests were conducted. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterizations of the powders and scaffolds\u003c/h2\u003e \u003cp\u003eThe powders were characterized. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows the SEM images of the powders, where Ti and Ti-25Ta powders exhibit a spherical morphology with a uniform size distribution. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb displays the particle size distribution, revealing that all two powders follow a normal distribution with similar particle sizes ranging from approximately 10 \u0026micro;m to 100 \u0026micro;m. The average particle sizes of Ti and Ti-25Ta powders are 36.39 \u0026micro;m and 34.03 \u0026micro;m respectively. The EDS spectra of the powders are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, indicating that all two powders are composed of Ti and/or Ta, with no detectable impurities. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef illustrate the apparent density and tap density of the two powders, respectively. The above results confirm that all two powders meet the requirements for 3D printing.\u003c/p\u003e \u003cp\u003eThe surface morphology of the porous disk-shaped scaffolds was observed using SEM, and the elemental distribution on the scaffold surface was analyzed using EDS, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The printed porous scaffold exhibits a well-defined structure with uniform pore size and filament diameter, and the Ti and Ta elements are homogeneously distributed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb presents the XRD spectra of the two sample groups, showing that Ti exists as an α-phase pure titanium, while Ta25 is an α-phase titanium alloy. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows the XPS spectra of Ta25, revealing that the scaffold surface consists of Ta, Ti, and O. The spectra of Ta4f and Ti2p confirm the presence of Ta2O5 and TiO2 oxide layers. The O1s spectrum, attributed to Ta2O5 and TiO2, shows binding energies of 531.87 eV and 530.36 eV, respectively. The O1s peak area ratio of Ta2O5 to TiO2 is 1:4.57, corresponding to an atomic ratio of Ta to Ti of 1:5.71 in the oxide layer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe XRF analysis of Ta25 reveals that the Ta and Ti contents are 24.2 wt% and 75.8 wt%, respectively, corresponding to an atomic ratio of 1:11.84. A comparison of XPS and XRF results suggests that the Ta content in the oxide layer (14.90 at%) is significantly higher than in the alloy (7.79 at%), indicating that Ta is more prone to passivation than Ti.\u003c/p\u003e \u003cp\u003eThe densities, Vickers hardnesses, contact angles, and surface energies of Ti and Ta25 are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Compared to Ti, Ta25 exhibits an increased density; however, its density remains at only 39% of that of Ta. Ta25 shows significant improvements in hardness over both Ti and Ta. The contact angles of water and DMSO decrease with increasing Ta content, while the surface energy increases, suggesting that Ta exhibits better wettability and higher surface energy than Ti, laying a foundation for the excellent biocompatibility of Ta.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe densities, Vickers hardnesses, contact angles, and surface energies of the 3D-printed bulks of Ti and Ta25\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDensity (g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTa25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVickers hardness (HV\u003csub\u003e0.2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e171.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTa25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e295.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eContact angle of water (degree)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTa25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eContact angle of DMSO (degree)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTa25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSurface energy (mJ/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTa25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn summary, the Ta25 alloy demonstrates excellent printability and improvements in mechanical property and wettability over Ti without significantly increasing the density.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 In Vitro Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Cell Viability and Proliferation\u003c/h2\u003e \u003cp\u003eTo assess cell growth on the surfaces of two scaffolds, a live/dead cell staining assay was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The results demonstrate good biocompatibility across all groups, with minimal dead cells observed. After 7 days, the number of cells on the surface of the Ta25 group was higher than that of the Ti group. Cell viability and proliferation were further evaluated using the CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). At 1, 4, and 7 days, the absorbance of the Ta25 group was higher than that of the Ti group, but the difference between the two groups was not statistically significant at the first day. At 4 and 7 days, the difference between the two groups was statistically significant. These findings suggest that Ta25 has greater cell viability and proliferation than Ti, and incorporating Ta into Ti enhances cellular activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 ALP Activity and In Vitro Mineralization\u003c/h2\u003e \u003cp\u003eThe osteogenic differentiation of MC3T3-E1 cells in the two sample groups was systematically evaluated using IF staining of ALP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and an in vitro mineralization assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The results indicate that all groups exhibit a certain level of ALP expression, the ALP expression level of Ta25 group was significantly higher than that of Ti group, especially at 14 days, Ta25 group showed obvious advantages in promoting the differentiation of osteogenic precursor cells. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) further verified this conclusion, and the difference between the Ta25 group and the Ti group was statistically significant, indicating that the addition of Ta metal to Ti metal can effectively enhance the ALP activity of osteoblasts.\u003c/p\u003e \u003cp\u003eTetracycline staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) reveals that after 21 days of culture, MC3T3-E1 cells in both the Ta25 and Ti groups produced mineralized nodules, while the Ta25 group showed the strongest mineralization ability. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) further verifies this trend, showing statistically significant differences between all pairwise comparisons. These findings suggest that Ta25 alloy has a significant advantage in promoting the differentiation and strengthening of the bone precursor cells, and adding Ta can improve the salinity of osteoblasts in Ti.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Macrophage Immunofluorescence and qRT-PCR Analysis\u003c/h2\u003e \u003cp\u003eMacrophage polarization was assessed using IF staining for CCR7 and ARG-1 markers. The results showed that the fluorescence signal of the ccr7 immunofluorescence marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and Ti group was strong in the fluorescence signal of the inflammatory factors in the group of Ti group, which showed that the expression level of the inflammatory factors was higher, and the macrophages showed a typical activation state, and the cytoplastic volume increased and the pseudoponas increased. The fluorescence signals of the inflammatory factors in the Ta25 group of macrophages were weak, indicating that the level of the inflammatory factors was low, the cell morphology of the macrophages was normal, and the cytoplasm was smaller and the pseudoplanes were less. The quantitative analysis results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) further confirm this result. In the ARG-1 immunofluorescence marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), the fluorescence signal of the anti-inflammatory factors in the Ti group was weak, indicating that the expression level of anti-inflammatory factors was low. Macrophages may present a typical activation state. The fluorescence signal of anti-inflammatory factors in Ta25 macrophages was significantly enhanced, indicating that the expression level of the anti-inflammatory factor was higher, the formation of the cell morphology of the macrophages was more regular, and the state quantitative analysis of the more static rate was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, which further confirmed the conclusion. The anti-inflammatory factor of the Ta25 group was significantly stronger than the Ti group, indicating that the Ta25 alloy had a stronger anti-inflammatory effect. The Ta25 alloy may reduce the inflammatory response by regulating the polarization of macrophages and promoting its transformation of anti-inflammatory phenotype (M2).\u003c/p\u003e \u003cp\u003eTo evaluate gene expression differences between groups at the molecular level, qRT-PCR analysis was performed on macrophage-related genes IL-10 and ARG-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). GAPDH was used as the housekeeping gene for normalization, with gene expression levels in the control group set to 1. Ta25 group IL-10、ARG-1 gene expression was significantly higher than the Ti group. The differences in gene expression between the two groups were statistically significant. This result, further confirmed the anti-inflammatory effect of Ta25 metal from the gene expression level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 In Vivo Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Micro-CT Analysis\u003c/h2\u003e \u003cp\u003eMicro-CT reconstruction of the implants and surrounding bone tissue was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Compared with Ti scaffold, the repair effect of Ta25 scaffold on bone defects was significantly improved. There was no significant difference in bone defect repair at 4 weeks, and more newly formed bone tissue was observed on the Ta25 scaffold at 12 weeks. In addition, quantitative analysis of bone volume fraction (BV/TV) was performed on the 3D-reconstructed images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), which further confirmed the enhanced bone regeneration ability of the Ta25 scaffold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 SEM Observation of the Scaffold\u0026ndash;Bone Interface\u003c/h2\u003e \u003cp\u003eTo further evaluate the integration of the two scaffolds with bone tissue, acid etching, and SEM were used to observe cellular growth and interactions at the scaffold\u0026ndash;bone interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After 4 weeks of implantation in the lateral femoral condyle of rabbits, all groups exhibited similar material\u0026ndash;bone contact. After 12 weeks, a large number of osteocytes were observed around the Ta25 scaffolds, with their quantity significantly exceeding that around the Ti scaffold. The osteocytes formed tight connections with the material surface through canaliculi. Despite a\u0026thinsp;~\u0026thinsp;20 \u0026micro;m gap between the scaffold and the bone, numerous osteocytes are observed within the gap and interconnected via canaliculi that extend toward the material surface, facilitating direct bonding. These findings indicate that bone integration occurs through biological bonding, further confirming the excellent biocompatibility and osseointegration performance of porous Ta25.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThis study successfully fabricated porous Ti-25Ta alloy scaffolds using 3D printing. The Ti-25Ta alloy has excellent mechanical properties, good biocompatibility, and promising bone repair capabilities, indicating broad prospects in the orthopedic field.\u003c/p\u003e \u003cp\u003eCompared with inert metals such as titanium, tantalum exhibits superior stability, corrosion resistance, and biocompatibility[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Pure tantalum was first introduced into orthopedic implants in 1940. Porous tantalum prepared by chemical vapor deposition (CVD) was approved by the U.S. FDA for artificial acetabular prostheses in 1997[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After nearly 30 years of clinical application, porous tantalum has gained widespread recognition from physicians and patients due to its beneficial clinical performance. In recent years, 3D-printed tantalum implants have emerged as a novel alternative, overcoming the limitations of CVD-fabricated porous tantalum, such as forming difficulties, and significantly expanding the application of tantalum in orthopedic implants[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the high density of tantalum remains a major challenge restricting its widespread application. In this study, titanium was alloyed with tantalum to enhance the bioactivity of titanium alloys while mitigating the density issue[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirstly, compared to pure tantalum, the Ti-25Ta alloy significantly reduces the material density, achieving a lightweight structure. Additionally, the introduction of tantalum considerably enhances the mechanical properties of titanium alloys, with microhardness comparable to that of 3D-printed Ti6Al4V alloys, laying a solid foundation for orthopedic applications.\u003c/p\u003e \u003cp\u003eBiocompatibility is a critical criterion for evaluating the success of bone implant materials. In this study, the Ti-25Ta alloy exhibited excellent cytocompatibility. In vitro experiments demonstrate that osteoblast adhesion and proliferation on the alloy surface are superior to those on pure titanium, with enhanced mineralization capacity, indicating that the incorporation of tantalum improves cellular activity. Studies on macrophages revealed that tantalum possesses superior anti-inflammatory properties, and the incorporation of tantalum into the titanium alloy significantly enhances its anti-inflammatory capability. Improved anti-inflammatory effects help suppress immune rejection, thereby promoting osseointegration and bone repair. Therefore, tantalum metal exhibits superior \"bioaffinity,\" which has been confirmed by clinical studies on tantalum[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Particularly in cases involving inflammation or infection, the advantages of tantalum metal are fully demonstrated[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The improvement in cell activity of Ti-25Ta alloy is attributed to its surface oxide layer, which provides a suitable growth environment for cells. This study indicates that tantalum metal possesses higher surface energy and wettability compared to titanium metal, and the introduction of Ta element can enhance the surface energy and wettability of titanium metal. Furthermore, XPS analysis reveals that the content of Ta element in the surface oxide layer of the alloy is significantly higher than that in the alloy itself. This is due to the stronger passivation capability of tantalum metal compared to titanium metal, making it more prone to oxidation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, even though the atomic percentage of Ta in the alloy is only 7.79 at%, it can significantly improve the cell activity of titanium metal[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond the intrinsic bioactivity improvements of the Ti-25Ta alloy, the porous scaffold structure provides ample space for cellular infiltration, proliferation, and bone tissue regeneration[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The interconnected pores facilitate the migration of bone marrow and blood vessels to the implantation site, promoting new bone formation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This enhanced osseointegration underscores the significant potential of porous Ti-25Ta alloys in bone defect repair.\u003c/p\u003e \u003cp\u003eDespite the promising biological performance of the 3D-printed Ti-25Ta alloy scaffold, several challenges remain for its clinical translation. Firstly, more tests are required to confirm its mechanical properties and long-term effectiveness. Secondly, optimizing the 3D printing process is needed for better quality and wider application. Finally, the anti-inflammatory effect of the Ti-Ta alloy should be further investigated via animal models of inflammation or infection.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn this study, porous scaffolds of pure Ti and Ti-25Ta alloy were successfully fabricated using 3D printing. The spherical powders used in the 3D printing show excellent sphericity and purity. The physical properties, biocompatibility, macrophage polarization, and in vivo bone defect repair efficacy of the scaffolds were systematically evaluated. Compared to pure Ti scaffold, the Ti-25Ta scaffold exhibits superior mechanical properties, and enhanced cell adhesion, proliferation, and anti-inflammatory capability. This improvement is due to the incorporation of Ta, which is demonstrated by the highest cell proliferation, osteogenic differentiation, mineralized nodule formation, anti-inflammatory capability, and bone reconstruction of pure Ta scaffold. In summary, Ti-25Ta alloy remains a promising candidate for bone implant applications. Further optimization of its composition and microstructure is required to enhance its osteogenic properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present investigation was conducted in complete adherence to the guidelines outlined in the Guide for the Care of Animals. The research protocol through a rigorous evaluation process and received clearance from the Animal Research Institute of Affiliated Zhongshan hospital of Dalian University, with the assigned approval number 202404003.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares that there was no competing interests\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by the Liao Ning Revitalization Talents Program (project No. XLYC2203102), National Natural Science Foundation of China (project No. 82172398), Distinguished Youth Foundation of Dalian (project No. 2024RJ007).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eW.D. 、 L.Y.D. and Z.W.W. wrote the article and participated in all experimental projects. J.H.L.、G.B. and X.Q.W participated in the in vitro experiments, X.J.F. participated in the in vivo experiments, W.W.D., C.F. and Y.G.X. participated in the material characterization experiments, and LJL and ZDW provided the main experimental ideas, experimental plan funds and sites. All reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKaushik N, Nhat Nguyen L, Kim JH, Choi EH, Kumar Kaushik N. Strategies for Using Polydopamine to Induce Biomineralization of Hydroxyapatite on Implant Materials for Bone Tissue Engineering. Int J Mol Sci. 2020;\u003cem\u003e \u003c/em\u003e21. https://doi.org/10.3390/ijms21186544.\u003c/li\u003e\n\u003cli\u003eTur D, Giannis K, Unger E, Mittlb\u0026ouml;ck M, Rausch-Fan X, Strbac GD. 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Colloids Surf B Biointerfaces. 2021;\u003cem\u003e \u003c/em\u003e208:112055. https://doi.org/10.1016/j.colsurfb.2021.112055.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"3D printing, Ti-25Ta alloy, porous scaffold, biocompatibility","lastPublishedDoi":"10.21203/rs.3.rs-6134011/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6134011/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, a porous Ti-25Ta alloy scaffold was fabricated using 3D printing and compared with pure Ti to evaluate its biocompatibility and osteogenic potential. The scaffolds were fabricated using a selective laser melting (SLM) process, followed by investigations of physical properties, in vitro cytocompatibility, osteogenesis, macrophage polarization, and in vivo bone reconstruction. Pure Ti exhibits the highest wettability, cell proliferation, osteogenic differentiation, mineralized nodule formation, anti-inflammatory capability, and bone reconstruction. The adoption of Ta in the Ti-25Ta alloy significantly increases its wettability, osteogenesis, and anti-inflammatory without a great increase in density. In summary, the Ti-25Ta alloy has a favorable balance of physical properties and biological performance, making it a promising candidate as a bone implant material.\u003c/p\u003e","manuscriptTitle":"Fabrication and Biocompatibility of a 3D-printed Porous Ti-25Ta Alloy Scaffold","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 17:37:09","doi":"10.21203/rs.3.rs-6134011/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-18T11:53:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T18:05:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275800289499871895688872108369225554543","date":"2025-04-10T20:00:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188444692196836881520092337138992054692","date":"2025-04-10T09:24:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196467792541678654987952481042396688878","date":"2025-03-26T13:52:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-22T13:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109475992615543480827856653882634325869","date":"2025-03-21T13:18:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T12:52:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-19T09:41:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-18T06:00:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Applied Sciences","date":"2025-03-18T05:59:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Applied Sciences](https://link.springer.com/journal/42452)","snPcode":"42452","submissionUrl":"https://submission.springernature.com/new-submission/42452/3","title":"Discover Applied Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52151590-7682-4208-b406-044f9895f228","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-06-18T10:38:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 17:37:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6134011","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6134011","identity":"rs-6134011","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}