The role of titanium surface micromorphology in MG-63 cell motility during osteogenesis

Abstract

Different surface micromorphologies influence osteoblast movements and impact the osteogenesis around implants. In this study, a biomimetic chip that simulates the microenvironment of the implant and bone in vitro was developed (tissue-on-chip of group T and group C) to study the correlation of cell movement velocity (CMV), direction (CMD), acceleration (CMA), and cell attachment number (CA) with the surface micromorphology of the Titanium material. Computational fluid dynamics (CFD) was used for flow analysis. Changes in intraosseous pressure (IOP), local blood perfusion index (LBPI), new bone microstructure, microvessel density (MVD), and bone-implant contact (BIC) in beagle dogs were detected as implant surface alterations. Surface skewness (Ssk) and surface arithmetic mean height (Sa) were the most important negative factors for high CMV, accounting for 51% and 32%, respectively, of all the influencing factors. Higher Ssk (Ssk_T > 0, Ssk_C < 0) and Sa (Sa_T > Sa_C) resulted in lower CMV (CMV_T:CMV_C = 0.41:1), greater CA (CA_T:CA_C = 1.44:1), and higher BIC (BIC_T:BIC_C = 3.06:1) (P < 0.05). The surface micromorphology influenced the CMD of MG-63 cells within 20 μm from the material surface. However, it could not regulate the IOP, LBPI, MVD, new bone microstructure, or CMD (P > 0.05).

Figure 1

Variations in implant surface micromorphology impact bone-implant contact (BIC). (a) Four mandibular premolars of beagle dogs were extracted; SEM images of the surface micromorphology of the implants, etched by either formula T or formula C; the implantation sites. Ssk, Sa and hydrogen content of implant T and C. (b, c) Pathological features of new bone adjoined to the implants of groups T1 and C1. The BIC of group T1 was higher than group C1 at 4 w and 6 w after implantation; I: implant; B: bone tissue (P < 0.05). Ssk and Sa resulted in high BIC, accounting for 96% and 47%, respectively, of all factors (OR > 1, β > 0); the low hydrogen content led to high BIC (OR < 1, β < 0). (d) The quantity of the new bone formed surrounding the implants of group T1 was more than that of group C1 at 4 w and 6 w after implantation (P < 0.05). This figure was generated by Adobe photoshop CC 2015.

Figure 2

The variation of implant surface morphology had no impact on the intraosseous pressure (IOP), local blood perfusion index (LBPI), or microvessel density (MVD). (a) Histopathological images of the newly formed microvessel next to the titanium implants at a magnification of 200$\times$. The red arrows indicate the newly formed microvessel. (b) There was no significant difference in IOP and MVD between groups T and C (P > 0.05). (c) The major microstructure parameters of the new bone formed surrounding the implants of groups T and C showed no statistical differences. The statistical significance was set at P < 0.05. This figure was generated by Adobe photoshop CC 2015.

Figure 3

The variation of implant surface morphology affected the cell movement velocity (CMV). (a) Design of the tissue-on-chip (TOC). (b) The instrument connections of the microfluidic pressure and flow control system. (c) 3D reconstruction of the TOC; the velocity contour of the TOC-T and TOC-C, generated using Fluent 19.0. The red arrows indicate that CMV increases when the fluid passes through the smaller cross-sectional area of the bone cavity. (d) Live images and velocity vector diagrams of MG-63 cells traversing the TOC-T and TOC-C. (e) The CMV of group T was significantly lower than that of group C (P < 0.05); there was no significant difference in the CMA of groups T and C (P > 0.05). (f) The values of Sv, contact angle, and zeta potential of groups T and C; Factors contributing to high CMV: Sa and Ssk negatively influenced the high CMV of MG-63, accounting for − 32% and − 51%, respectively (P < 0.05). Sv, contact angle, and zeta potential did not affect CMV (P > 0.05); the hydrogen content was an indirectly positive factor for high CMV (P < 0.05). The statistical significance was set at P < 0.05. This figure was generated by Adobe photoshop CC 2015.

Figure 4

The cell movement velocity (CMV) down-regulated the cell attachment number (CA) of MG-63 cells. (a) Images and diagram of attached MG-63 cells at the bottom of TOC-T and TOC-C with a magnification of 20×. (b) Factors contributing to high CA: Sa and Ssk played positive roles in the high CA, accounting for 27% and 65%, respectively (P < 0.05). The CMV, contact angle, and hydrogen content were negative factors for high CA, accounting for − 42%, − 44%, and − 63%, respectively (P < 0.05). (c) The CA was a positive factor for high BIC, accounting for 64% (β = 0.64, P = 0.00); the CMV was a negative factor for the high BIC, accounting for − 50% (β =  − 0.50, P = 0.00). The statistical significance was set at P < 0.05. This figure was generated by Adobe photoshop CC 2015.

Figure 5

Schematic diagram of the processes of MG-63 cells attached to the surfaces of group T (a) and group C (b). The MG-63 cells were more likely to be adsorbed onto the material surface of group T than group C. v: cell velocity; P: air pressure. This figure was generated by Adobe photoshop CC 2015.