Cell osteogenic bioactivity mediated precisely by varying scaled micro-pits on ordered micro/nano hierarchical structures of titanium

Abstract

Hierarchical surface structures with micro-nano scale play a crucial role in regulation of cell proliferation and osteogenic differentiation. It has been proven that cells are extremely sensitive to the nanoscaled structure and show multifarious phenotypes. Though a vital function of microstructure on osseointegration has been confirmed, the cell performances response to different microscaled structure is needed to be further dissected and in depth understood. In this work, the ordered micro-nano hierarchical structures with varying micro-scaled pits were precisely fabricated on titanium successfully by the combination of electrochemical, chemical etching and anodization as well. In vitro systematical assessments indicated that the micro-nano multilevel structures on titanium exhibited excellent cells adhesion and spreading ability, as well as steerable proliferation and osteogenic differentiation behaviors. It is shown that smaller micro-pits and lower roughness of the hierarchical structures enabled faster cell propagation. Despite cell growth was delayed on micro-nano titanium with relatively larger cell-match-size micro-pits and roughness, osteogenic-specific genes were significantly elevated. Furthermore, the alkaline phosphatase activity, collagen secretion and extracellular matrix mineralization of MC3T3-E1 on multi-scaled titanium were suppressed by a large margin after adding IWP-2 (an inhibitor of Wnt/β-catenin signal pathway), indicating this pathway played a crucial part in cell osteogenic differentiation modulated by micro-nano structures.

Keywords: Wnt/β-catenin; electrochemical self-organizing etching; osteogenic; varying micro-scaled pits.

Graphical abstract

Figure 1.

Schematic diagram of preparation process of ordered micro–submicro–nano hierarchical structures with varying scaled micro-pits on titanium.

Figure 2.

Morphology of varying micro-scale pits array on titanium upon different etching voltages.

Figure 3.

Schematic diagram of the formation process of micro-pits array on titanium.

Figure 4.

SEM and CLSM results of the prepared ordered micro–nano hierarchical titanium with varying micro-scaled pits.

Figure 5.

The results of static contact angles (a) and protein adsorption (b) on the as-prepared micro–nano hierarchical titanium. **P < 0.01.

Figure 6.

The results of cell fluorescence staining (a) and histogram of cell adhesion population (b) on hierarchical titanium with varying micro-scaled pits. *P < 0.05.

Figure 7.

Fluorescence staining results of cytoskeleton actin and nucleus of MC3T3-E1 incubated on hierarchically structured titanium with varying micro-scaled pits for 5 h and 24 h measured by CLSM (a), cell density (b) and cells area (c) measured by image J software.

Figure 8.

The MC3T3-E1 cell proliferation result on hierarchically structured titanium with gradient micro-scaled pits culturing for 1, 3 and 7 days. *P < 0.05 and **P < 0.01.

Figure 9.

Cell morphologies cultured on hierarchically structured titanium with varying micro-scaled pits for 3 days.

Figure 10.

Cell morphologies cultured on hierarchically structured titanium with varying micro-scaled pits for 7 days.

Figure 11.

ALP activity of cells incubated on different samples for 4 and 7 days. **P < 0.01.

Figure 12.

The mRNA relative expression for osteogenic genes of MC3T3-E1 cultured on the hierarchically structured titanium with varying micro-scaled pits for 5 and 10 days (a) ITG, (b) RUNX-2, (c) ALP, (d) OCN, (e) OPN, (f) Col-1. *P < 0.05 and **P < 0.01.

Figure 13.

The ALP staining result for MC3T3-E1 culturing with/without Wnt/β-catenin pathway inhibitor on hierarchically structured titanium with varying micro-scaled pits.

Figure 14.

Sirius scarlet staining result of MC3T3-E1 culturing for 14 days with/without Wnt/β-catenin pathway inhibitor on hierarchical titanium with varying scaled micro-pits.

Figure 15.

Alizarin red staining result of MC3T3-E1 culturing for 14 days with/without Wnt/β-catenin pathway inhibitor on the hierarchically structured titanium with varying scaled micro-pits.