The pathway to intelligent implants: osteoblast response to nano silicon-doped hydroxyapatite patterning

Abstract

Bioactive hydroxyapatite (HA) with addition of silicon (Si) in the crystal structure (silicon-doped hydroxyapatite (SiHA)) has become a highly attractive alternative to conventional HA in bone replacement owing to the significant improvement in the in vivo bioactivity and osteoconductivity. Nanometre-scaled SiHA (nanoSiHA), which closely resembles the size of bone mineral, has been synthesized in this study. Thus, the silicon addition provides an extra chemical cue to stimulate and enhance bone formation for new generation coatings, and the next stage in metallic implantation design is to further improve cellular adhesion and proliferation by control of cell alignment. Topography has been found to provide a powerful set of signals for cells and form contact guidance. Using the recently developed novel technique of template-assisted electrohydrodynamic atomization (TAEA), patterns of pillars and tracks of various dimensions of nanoSiHA were achieved. Modifying the parameters of TAEA, the resolution of pattern structures was controlled, enabling the topography of a substrate to be modified accordingly. Spray time, flow rate and distance between the needle and substrate were varied to improve the pattern formation of pillars and tracks. The 15 min deposition time provided the most consistent patterned topography with a distance of 50 mm and flow rate of 4 µl min(-1). A titanium substrate was patterned with pillars and tracks of varying widths, line lengths and distances under the optimized TAEA processing condition. A fast bone-like apatite formation rate was found on nanoSiHA after immersion in simulated body fluid, thus demonstrating its high in vitro bioactivity. Primary human osteoblast (HOB) cells responded to SiHA patterns by stretching of the filopodia between track and pillar, attaching to the apex of the pillar pattern and stretching between two. HOB cells responded to the track pattern by elongating along and between the track, and the length of HOB cells was proportional to the gaps between track patterns, but this relationship was not observed on the pillar patterns. The study has therefore provided an insight for future design of next generation implant surfaces to control and guide cellular responses, while TAEA patterning provides a controllable technique to provide topography to medical implants.

Figure 1.

Transmission electron microscopy micrographs of nanoSiHA particles synthesized for TAEA deposition.

Figure 2.

X-ray diffraction patterns of major HA on electrosprayed nanoSiHA on glass substrate.

Figure 3.

The relationship between the flow rate and applied voltage (AV) in maintaining the stable cone-jet mode (envelope) during TAEA spraying of 3 wt% nanoSiHA suspensions, the regions above and under the envelope are multi-jet and unstable cone-jet mode. For each flow rate, there is a minimum and maximum voltage value to maintain stable cone-jet mode. Typical jetting pictures for each region are shown. Diamonds with dotted lines, min AV; squares with dotted lines, max AV.

Figure 4.

Optical microscopy images of track patterns prepared by TAEA processing at the flow rate of (a) 4 µl min⁻¹; (b) 5 µl min⁻¹; (c) 6 µl min⁻¹; (d) relationship between template size and measured pattern size with the flow rates varied from 4 to 6 ml min⁻¹.

Figure 5.

The effect of the distance between nozzle and substrate, increased from 20 to 60 mm, on the track patterns prepared by TAEA.

Figure 6.

The morphology of pillar pattern with spraying time varied from (a) 5 min, (b) 10 min to (c) 15 min.

Figure 7.

Three-dimensional profile of track pattern created by TAEA, spraying for (a) 5 and (b) 15 min. (c) The effect of spraying time variation (from 5 to 15 min) on the height of various patterns (T1-short track, T2-long track, P1-pillar).

Figure 8.

SEM micrographs of pillar pattern (a) before heat treatment and (b) heat treated; (c) track pattern; (d) higher magnification of track pattern; (e) track produced under optimized parameters (insert shows high magnification of deposited SiHA).

Figure 9.

The changes of surface morphology of (a) nanoSiHA immersion in SBF. (b) EDX surface analysis shows the bone-like apatite formed after 3 days in SBF was rich in Ca and P.

Figure 10.

Confocal micrographs of HOB cells (nuclei, blue; actin, green) on (a) non-patterned and (b) pillar-patterned surface.

Figure 11.

Confocal micrographs of the actin cytoskeleton of HOB cells on (a) tracks (b) pillar patterns.

Figure 12.

(a) Optical microscopy image of HOB cells (live) on nanoSiHA pillar pattern; (b) and (c) SEM micrographs of HOB cells attachment on nanoSiHA pillar with small (b) and large (c) gap.