Copper‑Doped Microporous Titanium Coatings Enhance Osteoblast Activity and Bone Integration

Introduction

Titanium and its alloys remain the gold standard for load‑bearing implants because of their excellent mechanical performance and inherent biocompatibility. Yet, their chemically inert surfaces lack osteoinductive cues, relying solely on mechanical interlocking for stability, which can fail under physiological loading. Surface engineering—particularly micro‑arc oxidation—has emerged as a versatile technique to introduce porosity, roughness, and bioactive ions, thereby enhancing protein adsorption, cell adhesion, and ultimately osseointegration.

While numerous coating strategies exist (plasma spraying, electrophoretic deposition, sol–gel, etc.), they often suffer from low crystallinity, weak adhesion, or complex processing. MAO, in contrast, offers rapid, scalable deposition of robust, highly porous oxide layers with tunable chemistry.

Copper, a trace element essential for bone metabolism, stimulates osteoblast proliferation, upregulates VEGF, and confers antibacterial activity at physiologic concentrations. Incorporating Cu into TiO₂ matrices has shown synergistic effects on osteogenesis and microbial inhibition.

In this work, we present a Cu–TiO₂ microporous coating fabricated by MAO, evaluate its physicochemical properties, and assess its influence on osteoblast behavior in vitro and bone integration in a rabbit model, establishing a foundation for clinical translation.

Materials and Methods

Sample Preparation and Characterization

Commercial Ti alloy rods (12 mm × 1 mm for surface analysis; 3 mm × 8 mm for implantation) were ground to 600 grit, ultrasonically cleaned, and subjected to MAO. A low‑power MAO system operated at 450 V (constant‑current mode, 5 min, 1 kHz). The electrolyte comprised 1 wt% Ca(CH₃COO)₂ and 0.5 wt% Ca₃(PO₄)₂, with or without CuO additions (0.1 wt% for TCP Cu I and 0.3 wt% for TCP Cu II). Samples were labeled Ti, TCP, TCP Cu I, and TCP Cu II.

Surface morphology was examined by FE‑SEM; elemental mapping via EDS; roughness via a profilometer (Ra values). Phase identification used XRD (Cu Kα, 2θ = 10–80°); chemical states were probed by XPS (binding energies 520–1100 eV).

Cell Culture

MC3T3‑E1 murine pre‑osteoblasts were cultured in MEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO₂. Cells at passage 3 were used for all assays.

Live/Dead Staining

Cells (1.5 × 10⁴ cells cm⁻²) were seeded onto each substrate, incubated 3 days, and stained with calcein AM/ethidium homodimer. Live/dead ratios were quantified via fluorescence microscopy.

Cell Adhesion and Proliferation

Adhesion was assessed at 1, 2, and 6 h using DAPI nuclear staining and confocal imaging. Proliferation was quantified by CCK‑8 assay on days 1, 3, and 10. Data were expressed as absorbance at 450 nm.

Osteogenic Gene Expression

At days 1, 3, and 10, total RNA was extracted, reverse‑transcribed, and qRT‑PCR performed for BMP‑2, COL‑I, ALP, and OCN, with GAPDH as the internal control. Primer sequences are listed in Table 2.

Animals and Surgery

Sixteen adult New Zealand white rabbits (3.6 kg) were randomized into experimental (Cu‑coated) and control (TCP) groups. Under sodium pentobarbital anesthesia, a 2.7 mm × 6 mm defect was created in the lateral femoral condyle, and corresponding rods were implanted. Post‑operative care included penicillin for 3 days.

Micro‑CT Analysis

At 4 and 8 weeks, eight rabbits per group were euthanized. Excised femoral condyles were fixed, scanned at 9 µm voxel size, and BV/TV (%), trabecular thickness, and connectivity were quantified using commercial software.

Histology

Specimens were dehydrated, resin‑embedded, sectioned (20–30 µm), and stained with toluidine blue and fuchsin‑methylene blue. Bone‑implant interfaces were examined under light microscopy for new bone formation and fibrous gaps.

Statistical Analysis

Data were expressed as mean ± SD. One‑way ANOVA followed by the SNK test assessed group differences (p < 0.05 considered significant). SPSS 16.0 was used for analysis.

Results

Surface Morphology and Composition

FE‑SEM images (Figure 1) revealed a uniform, interconnected microporous network on TCP, TCP Cu I, and TCP Cu II surfaces, absent in bare Ti. EDS mapping (Figure 2) confirmed homogeneous distribution of Cu, Ca, P, Ti, and O, with no toxic contaminants. XRD patterns (Figure 3) showed TiO₂ (rutile/anatase) peaks and CuO in Cu‑doped samples. XPS spectra (Figure 4) corroborated the presence of TiO₂ and CuO (Cu 2p ≈ 932.7 eV).

Surface Roughness

Profilometry (Figure 5) indicated Ra values of 1.2 µm for TCP, 1.3 µm for TCP Cu I, and 1.4 µm for TCP Cu II, significantly higher than Ti (0.4 µm). Copper addition did not alter roughness appreciably.

Cell Adhesion and Proliferation

DAPI staining (Figure 6a) showed increasing cell numbers over time. Quantification (Figure 6b) demonstrated the order: TCP Cu II > TCP Cu I > TCP > Ti, with significant (p < 0.01) improvement on Cu‑doped surfaces. Proliferation assays (Figure 6c) mirrored adhesion trends, confirming the promotive effect of Cu‑doped porosity.

EdU Incorporation

EdU staining (Figure 7) revealed a higher percentage of proliferating nuclei on TCP Cu II, followed by TCP Cu I, TCP, and Ti, reinforcing the proliferative advantage of copper incorporation.

Cytotoxicity

Live/Dead assay (Figure 8) displayed minimal red (dead) cells across all groups, indicating no significant cytotoxicity from Cu doping.

Osteogenic Gene Expression

qRT‑PCR (Figure 9) showed progressive up‑regulation of BMP‑2, COL‑I, ALP, and OCN over time, with the highest expression on TCP Cu II, followed by TCP Cu I, TCP, and Ti. These differences were statistically significant (p < 0.05).

Micro‑CT and Histology

Micro‑CT reconstructions (Figure 10) revealed greater BV/TV in the Cu‑coated group at both 4 and 8 weeks (p < 0.01). Histological sections (Figure 11) showed a narrower fibrous gap and more mature bone around Cu‑coated implants, confirming enhanced osseointegration.

Discussion

The present study demonstrates that MAO‑fabricated Cu–TiO₂ microporous coatings provide a robust, bioactive surface that promotes osteoblast adhesion, proliferation, and differentiation, leading to superior bone integration in vivo. The strong bonding between the ceramic coating and Ti substrate—rooted in the high‑temperature plasma‑sintered TiO₂ matrix—ensures long‑term stability, addressing a common limitation of conventional coatings.

Cu’s dual role—stimulating osteogenesis and providing antibacterial activity—adds therapeutic value. The uniform distribution of CuO within the porous TiO₂ network allows sustained ion release at physiological concentrations, aligning with previous findings that low‑level Cu (≤ 0.1 wt%) enhances osteoblast function without cytotoxicity.

The interconnected pore architecture mimics native bone microstructure, increasing surface area for protein adsorption and cell ingrowth, while the elevated roughness further encourages focal adhesion formation. Together, these physicochemical cues synergize to accelerate bone apposition, as evidenced by the increased BV/TV and direct bone‑implant contact observed in the rabbit model.

Limitations include the short implantation period and the use of a single animal species. Future studies should evaluate long‑term stability, mechanical loading, and translation to larger animal models.

Conclusion

Micro‑arc oxidation of Ti alloys to produce a Cu‑doped TiO₂ microporous coating yields a robust, bioactive surface that enhances osteoblast activity and bone integration without cytotoxicity. These findings support the clinical potential of Cu–TiO₂ MAO coatings for next‑generation orthopedic implants.

Availability of data and materials

Not applicable.

Abbreviations

MAO

micro‑arc oxidation

Cu

copper

Cu–TiO₂ coating

copper–titanium dioxide coating

BV/TV

bone volume/total volume

ALP

alkaline phosphatase