Dual-functional titanium implants via polydopamine-mediated lithium and copper co-incorporation: synergistic enhancement of osseointegration and antibacterial efficacy

Abstract

Introduction: Orthopedic implant failure due to inadequate osseointegration and infection remains a critical challenge. To address this, we engineered a polydopamine (PDA)-mediated dual-functional platform for lithium (Li⁺) and copper (Cu²⁺) co-incorporation on titanium alloy (Ti6Al4V) implants, aiming to synergize osteogenic and antibacterial properties through a scalable surface modification strategy.

Methods: PDA coatings were polymerized onto polished Ti64 substrates, followed by sequential immersion in LiCl (800 μM) and CuCl₂ (10 μM) solutions to construct Li⁺/Cu²⁺ co-doped surfaces (PDA@Li 800-Cu 10). In vitro assays assessed MC3T3-E1 pre-osteoblast proliferation (CCK-8), osteogenic differentiation (ALP activity, RT-PCR for ALP/Axin2), and antibacterial activity against S. aureus and E. coli (live/dead staining, CFU assays). In vivo efficacy was evaluated in a rat femoral defect model via micro-CT and histology.

Results and discussion: Li⁺-functionalized surfaces (PDA@Li 800) enhanced osteoblast proliferation and osteogenesis via Wnt/β-catenin activation. Cu²⁺-loaded coatings (PDA@Cu 10) eradicated >99% bacteria but moderately suppressed osteogenic markers. The dual-doped PDA@Li 800-Cu 10 surface resolved this bioactivity conflict, maintaining antibacterial efficacy comparable to PDA@Cu 10 while elevating the osteogenic capacity of Cu²⁺-only modified surfaces. In vivo, dual-modified implants eliminated bacterial colonization within 72 h and significantly increased peri-implant bone volume (BV/TV) in comparison to Ti64 controls, outperforming PDA-only counterparts. By harmonizing Li-driven osteoinduction and Cu-mediated bactericidal action through a scalable PDA platform, this work advances a transformative strategy for next-generation orthopedic and dental implants, simultaneously addressing infection risks and bone regeneration demands.

Keywords: Ti6Al4V; antibacterial; metal ions; osseointegration; surface modification.

FIGURE 1

Schematic and physicochemical characterization of titanium surface modification via a mussel-inspired ion immobilization strategy. (A) A scalable dip-coating technique to deposit a conformal PDA thin film on Ti-6Al-4V substrates, illustrating its integration into bone implant surface engineering. (B) SEM characterization of the nanoscale topography and uniformity of the PDA-coated surface following Li⁺/Cu²⁺ ion immobilization (scale bar: 2 μm). (C) EDX elemental mapping confirmed the successful incorporation and homogeneous distribution of Cu across the functionalized surface (scale bar: 25 μm). (D) XPS analysis validated the chemical composition of the modified surfaces. (E) Representative water contact angle images of different sufaces and measured results. (F) Cu²⁺ and Li⁺ release profiles of PDA@Li 800-Cu 10 over 4 days incubation in PBS.

FIGURE 2

Characterization of the effect of Li⁺-incorporated surfaces on MC3T3-E1 morphogenesis, proliferation, and osteogenic differentiation. (A) Representative fluorescence images of F-actin cytoskeletal organization (phalloidin, green) and nuclei (Hoechst 33258, blue) in MC3T3-E1 cells cultured on different surfaces at 1 and 7 days post-seeding. Scale bar: 1 mm. (B) ALP activity staining after 7 days of culture. Scale bar: 500 μm. (C) Quantitative cell viability was assessed via CCK-8 assay on days 1, 3 and 5. *p < 0.05, ****p < 0.0001 (n = 3). (D) Normalized ALP activity quantification at day 7. *p < 0.05¸**p < 0.01 (n = 3). (E) qRT-PCR analysis of ALP and Axin2 mRNA expression at days 4 and 7. *p < 0.05 (n = 3).

FIGURE 3

Characterization of cell morphology, proliferation and osteogenic differentiation of MC3T3-E1 on Cu²⁺-incorporated surfaces. (A) Fluorescence microscopy images of cytoskeletal architecture via phalloidin staining (F-actin, red) and nuclei (Hoechst 33258, blue) after 1 and 7 days of culture. Scale bars: 1 mm. (B) ALP staining at day 7. Scale bar: 500 μm. (C) Quantification of cell proliferation by CCK-8 assay on Cu-doped coatings over time. *p < 0.05, ****p < 0.0001 (n = 3). (D) Normalized ALP activity corroborates the staining results relative to unmodified groups at day 7. *p < 0.05 (n = 3).

FIGURE 4

Antibacterial efficacy of copper-incorporated functional surfaces. (A) Fluorescence micrographs of live/dead-stained Staphylococcus aureus and E. coli colonies cultured on modified surfaces for 24 h, with viable bacteria (green) and dead bacteria (red) visualized. Scale bars: 1 mm. (B) Representative photographic documentation of bacterial colonies harvested from surfaces. (C) Quantitative assessment of antibacterial efficacy, calculated via CFU reduction rates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 3).

FIGURE 5

Characterization of the effect of dual-functional Li⁺/Cu²⁺-incorporated coatings on MC3T3-E1 morphogenesis, proliferation, and osteogenic differentiation. (A) Representative fluorescence images of F-actin cytoskeletal organization (phalloidin, green) and nuclei (Hoechst 33258, blue) in MC3T3-E1 cells cultured on different surfaces at 1 and 7 days post-seeding. Scale bar: 1 mm. (B) ALP activity staining after 7 days of culture. Scale bar: 500 μm. (C) Quantitative cell viability was assessed via CCK-8 assay on days 1, 3 and 5. *p < 0.05, ****p < 0.0001 (n = 3). (D) Normalized ALP activity quantification (absorbance at 405 nm) at day 7. *p < 0.05¸**p < 0.01 (n = 3). (E) qRT-PCR analysis of ALP and Axin2 mRNA expression at days 4 and 7. *p < 0.05 (n = 3).

FIGURE 6

In vitro and in vivo antibacterial evaluation of dual-functional Li⁺/Cu²⁺-incorporated coatings. (A) Live/dead fluorescent staining of Staphylococcus aureus and E. coli cultured for 24 h on modified surfaces (green: live; red: dead). Scale bar: 1 mm. (B) Representative CFU harvested from surfaces after 24 h of culture. (C) In vitro quantitative analysis for dual-ion-modified surfaces. *p < 0.05, **p < 0.01, ****p < 0.0001 (n = 4). (D) Surface imprints and spread plate analyses of 3 days implanted samples. (E) Quantification of in vivo bacterial retention. *p < 0. 05 (n = 4).

FIGURE 7

Bone regeneration evaluation after 4 weeks implantation. (A) MicroCT reconstructed images of the bone formation around the implanted samples. (B) Quantitative results of microCT scanned data. *p < 0. 05 (n = 4). (C) HE staining to characterize the osteogenesis associated with various implants. Scale bar: 500 μm (upper), 100 μm (down).