Effect of micro-arc oxidation surface modification of 3D-printed porous titanium alloys on biological properties

Abstract

Background: Three-dimensional (3D) printing technology has been widely used in orthopedics; however, it is still limited to the change of macroscopic structures. In order to further improve the biological properties of 3D-printed porous titanium scaffolds, this study introduced micro-arc oxidation (MAO) technology to modify the surface of porous titanium scaffolds and construct bioactive coatings on the surface of porous titanium scaffolds to improve the biocompatibility and osseointegration ability of the material.

Methods: For in vitro experiments, human bone marrow stem cells (hBMSCs) were seeded onto untreated scaffolds (control group) and MAO-treated scaffolds (experimental group). After 24 h of co-culture, cytotoxicity was observed using live/dead staining, and cell/scaffold constructs were retrieved and processed for the assessment of cell morphology by using scanning electron microscopy (SEM). Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) assay after 3, 7, and 14 days of co-culture. The levels of alkaline phosphatase (ALP) in the cell supernatant were detected after 7 and 14 days of co-culture. For in vivo experiments, micro-computed tomography (micro-CT) and Masson Goldner's staining were used to evaluate bone ingrowth and osseointegration at 4 and 8 weeks postoperatively.

Results: In vitro experiment results confirmed that the two groups of scaffolds were non-cytotoxic and the cell adhesion status on the MAO-treated scaffolds was better. Over time, cell proliferation and ALP levels were higher in the MAO-treated group than in the untreated scaffolds. In the in vivo experiments, the MAO-treated scaffolds showed better bone ingrowth and osseointegration than the untreated group at different time points.

Conclusions: The MAO-treated porous titanium scaffold formed a uniform and dense bioactive coating on the surface, which was more conducive to cell adhesion, proliferation, and differentiation and showed better osseointegration and bone ingrowth in vivo.

Keywords: Three-dimensional printing (3D printing); biocompatibility; micro-arc oxidation (MAO); osseointegration.

Figure 1

Representative SEM images of the untreated and MAO-treated Ti₆Al₄V scaffolds. (A,B) Smooth surface of the untreated scaffold. (D,E) Microporous structure of the surface of the MAO-treated scaffold. (C,F) Thickness of the coating and pore sizes. SEM, scanning electron microscopy; MAO, micro-arc oxidation.

Figure 2

Representative EDS analysis of the MAO-treated Ti₆Al₄V scaffold. EDS, energy-dispersive X-ray spectroscopy; MAO, micro-arc oxidation.

Figure 3

XPS and XRD patterns for the MAO-treated scaffold. XPS, X-ray photoelectron spectroscopy; XRD, X-ray diffractometer; MAO, micro-arc oxidation.

Figure 4

Hydrophilic testing of untreated Ti₆Al₄V and MAO-treated Ti₆Al₄V. (A,B) Representative images of the contact angle test. (C) Statistical analysis of the contact angle of the two types of scaffolds. **, P<0.01. Sample number in each group is 4. MAO, micro-arc oxidation.

Figure 5

SEM observation after 24 h of cell culture. (A,C) Cells adhered to Ti₆Al₄V scaffold at different magnifications. (B,D) Cells adhered to MAO-treated Ti₆Al₄V scaffold at different magnifications. MAO, micro-arc oxidation; SEM, scanning electron microscopy.

Figure 6

Biocompatibility and osteogenic differentiation of hBMSCs on two groups of scaffolds. (A) Live/dead staining of the scaffolds after 24 h of cell culture. Calcein-AM solution stained live cells and PI solution stained dead cells. Staining plots of live cells on the untreated scaffold and MAO-treated scaffold, respectively (a,d). Staining plots of dead cells on the untreated scaffold and MAO-treated scaffold, respectively (b,e). Mixed plots of live and dead cells on the untreated scaffold and MAO-treated scaffold (c,f). There were no obvious dead cells (red staining) on either type of scaffolds. (B) Cell proliferation on the untreated and MAO-treated scaffolds. (C) ALP activity of the untreated and MAO-treated scaffolds after 7 and 14 days of cell culture. *, P<0.05; **, P<0.01. MAO, micro-arc oxidation; PI, propidium iodide; ALP, alkaline phosphatase; hBMSCs, human bone marrow stem cells.

Figure 7

Evaluation of bone ingrowth and osseointegration of scaffolds in in vitro experiment in two groups of scaffolds. (A,B) Quantitative results of bone fractions in the peri-scaffold region and intra-porous region of the scaffolds. *, P<0.05; **, P<0.01. Sample number =8 in each group. (C) Light optical micrographs of the histological sections stained with Masson Goldner’s trichrome of the untreated and MAO-treated scaffolds at 4 and 8 weeks after surgery. The red circles in (a,c,e,g) correspond to (b,d,f,h), respectively. The mineralized bone tissues are stained in green, the osteoid tissues are stained in red/orange, and the scaffolds appear in black. MAO, micro-arc oxidation.