Additively manufactured Tantalum-titanium alloys with optimized osteogenic and immunomodulatory properties for load-bearing orthopedic implants

Abstract

The Tantalum-Titanium (TaTi) alloys demonstrate significant potential as an orthopedic implant material. This study presents the development and comprehensive evaluation of novel additive manufactured TaTi alloys for orthopedic implant applications. Through a combination of materials engineering and biological validation, we designed pre-alloyed TaTi spherical powders with varying compositions (Ta25, Ta55, and Ta75) and fabricated dense and porous structures via selective laser melting (SLM). The SLM Ta55 alloy (Ti-55 wt %Ta) exhibited optimal mechanical properties, including a tensile strength of 891 MPa and an elastic modulus of 74 GPa, closely matching cortical bone. Surface characterization revealed that oxide layer (comprising Ta₂O₅/TiO₂) of SLM Ta55 promoted osteoblast adhesion and focal adhesion signaling activation. In vitro studies demonstrated superior osteogenic differentiation of MC3T3-E1 cells on SLM Ta55, evidenced by upregulated alkaline phosphatase (ALP) activity, mineralization, and osteogenic gene expression (ALP, Col-1, OCN, OPN). Transcriptomic analysis linked these effects to enhanced extracellular matrix remodeling and integrin-mediated mechanotransduction. Immunomodulatory assessments showed SLM Ta55 facilitated M2 macrophage polarization by suppressing JAK-STAT1 and TNF/NF-κB pro-inflammatory pathways while activating JAK3/STAT6, creating an anti-inflammatory microenvironment conducive to bone regeneration. In vivo rabbit femoral defect models confirmed SLM Ta55's exceptional osseointegration, with 37 % new bone area at 12 weeks, outperforming pure Ti and other TaTi alloys. Histological and immunofluorescence analyses validated reduced inflammation and increased osteocalcin expression around SLM Ta55 implants. This work establishes SLM Ta55 as a promising next-generation orthopedic biomaterial, synergizing mechanical compatibility, osteogenesis, and immunomodulation to advance personalized bone repair strategies.

Keywords: Immunomodulation; Orthopedic implant material; Osteogenesis; Selective laser melting (SLM); Tantalum-titanium (TaTi) alloys.

Graphical abstract

Fig. 1

Characterization of spherical powders: Ti, Ta25, Ta55, Ta75, and Ta. (a) Representative SEM micrographs with corresponding EDS elemental mappings, the red dots represent Ti, and the green dots represent Ta. (b) EDS spectral profiles; (c) Particle size distribution curves; (d) Apparent density, (e) Tapped density, and (f) Hall flow rate measurements.

Fig. 2

Physicochemical characterization of SLM Ti, TaTi alloys, and pure Ta. (a) Representative SEM micrographs with corresponding EDS elemental mappings of SLM Ti, Ta25Ti75, Ta55Ti45, Ta75Ti25, and pure Ta. The red dots represent Ti, while the green dots indicate Ta. (b) Surface morphology of porous structures: SLM Ti, Ta25Ti75, Ta55Ti45, Ta75Ti25, and pure Ta. (c) EDS spectral profiles, (d) XRD diffraction patterns, and (e) measured densities for all SLM-fabricated samples.

Fig. 3

Surface characterization of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. (a–d) XPS spectra. (e) Images of water and DMSO droplets on the sample surfaces. (f) Water and DMSO contact angles and the calculated surface energy.

Fig. 4

Mechanical properties characterization. (a, b) Representative Vickers hardness images and corresponding quantitative values. (c) Tensile strength and elastic modulus of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. (d) Compressive yield strength and compression modulus of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta porous scaffolds.

Fig. 5

In-vitro biocompatibility and cell attachment assessment. Live/dead cell staining and CCK-8 assay of MC3T3-E1 cells (a, b) and Raw264.7 cells (c, d) cultured on the surface of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. (e, f) Morphologies and quantitative fluorescence intensity of MC3T3-E1 cells cultured on the surface of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, n = 3.

Fig. 6

Evaluation of osteogenic differentiation in MC3T3-E1 cells cultured on the surface of SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. (a) Representative images of osteogenic differentiation markers visualized by ALP fluorescence staining, ALP staining, and ARS staining. (b–d) Quantitative analysis of ALP activity and mineralization (ARS). (e–h) Relative mRNA expression levels of key osteogenesis-related genes. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, n = 3.

Fig. 7

The RNA sequencing analysis of MC3T3-E1 cells co-cultured with SLM Ti, SLM Ta25, SLM Ta55, SLM Ta75, and SLM Ta. (a) Pearson correlation between samples. The horizontal and vertical axes in the figure represent the squared correlation coefficients (R²) between respective samples. (b) Volcano plot showing the results of differential expression analysis of SLM Ta55 vs SLM Ta and SLM Ta75 vs SLM Ta. The red dots represent upregulated genes, while the green dots indicate downregulated genes (False discovery rate <0.05 and Fold change >2). (c) Results of GO enrichment analysis of SLM Ta55 vs SLM Ta and SLM Ta75 vs SLM Ta. The color and size of dots represent gene expression abundance and p-value, respectively. (d) Volcano plot showing the results of differential expression analysis of SLM Ta55 vs Ta25, SLM Ta55 vs SLM Ti, SLM Ta75 vs SLM Ta25 and SLM Ta75 vs SLM Ti. (e–f) Results of GO and KEGG enrichment analysis of SLM Ta55 vs SLM Ta25, SLM Ta55 vs SLM Ti, SLM Ta75 vs SLM Ta25 and SLM Ta75 vs SLM Ti.

Fig. 8

Focal adhesion gene expression and osteogenic mechanism of SLM Ta55 alloy. (a–e) Relative mRNA expression levels of key focal adhesion-related genes. (f) Schematic illustration showing the osteogenic mechanism of SLM Ta55 alloy.

Fig. 9

In vitro immune responses of macrophages on various groups. (a–c) RAW264.7 macrophages immunofluorescence images and quantitative fluorescence intensity of Arg-1 and CCR7 staining on different samples. (d) Attachment of RAW264.7 macrophages on different samples. (e) Polarization of macrophages cultured on SLM different samples evaluated using flow cytometry for CCR7 (M1) and Arg-1 (M2). (f) Relative mRNA expression levels of M1 macrophage-related genes (iNOS and IL-6) and M2 macrophage-related genes (Arg-1 and IL-10). ∗p < 0.05, ∗∗p < 0.01, n = 3.

Fig. 10

Immunomodulation mechanism of SLM TaTi alloys on the gene expression level. (a) Pearson correlation between samples. (b) Volcano plots of the upregulated and downregulated genes between SLM Ti, SLM Ta25, and SLM Ta55 group (False discovery rate <0.05 and Fold change >2). (c) GO biological processes analysis of the differentially expressed genes. (d) KEGG pathway analysis of the differentially expressed genes.

Fig. 11

Validation the role of JAK-STAT and TNF/NF-κB signaling pathways in the immunomodulation process of different samples on the macrophage polarization. (a, b)Western blot images and (c–j) semiquantitative analysis of the JAK-STAT and NF-κB signaling pathways. (k) Scheme illustration of the mechanism of SLM TaTi alloys promote M2 macrophage polarization. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, n = 3.

Fig. 12

In vivo osseointegration and bone regeneration in New Zealand white rabbit femur defects. (a–b) The VG staining and of semi-quantification of different groups in vivo bone regeneration at 4 weeks and 12 weeks postoperatively. (c–d) Representative immunohistochemistry OCN staining and quantification of OCN-positive cells per femur condyle area according to the immunohistochemistry results. (e) SEM images of osteocytes surrounding the implants following implantation periods of 4 and 12 weeks. ∗p < 0.05, ∗∗p < 0.01, n = 4.

Fig. 13

Inflammatory response in bone tissues surrounding implanted porous scaffolds across experimental groups. (a) Representative H&E staining images. (b) Immunohistochemical staining and (c, d) quantitative analysis of CD68 expression in peri-implant bone tissues at 4 and 12 weeks. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, n = 4.

Fig. 14

SLM TaTi alloys modulated immune microenvironment in bone defect repair. Immunofluorescence staining images (a) and quantification (b) of Arg-1, Immunofluorescence staining images (c) and quantification (d) of CCR-7. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, n = 4.