Effects of titanium particle size on osteoblast functions in vitro and in vivo

Abstract

The formation of titanium (Ti)-wear particles during the lifetime of an implant is believed to be a major component of loosening due to debris-induced changes in bone cell function. Radiographic evidence indicates a loss of fixation at the implant-bone interface, and we believe that the accumulation of Ti particles may act on the bone-remodeling process and impact both long- and short-term implant-fixation strengths. To determine the effects of various sizes of the Ti particles on osteoblast function in vivo, we measured the loss of integration strength around Ti-pin implants inserted into a rat tibia in conjunction with Ti particles from one of four size-groups. Implant integration is mediated primarily by osteoblast adhesion/focal contact pattern, viability, proliferation and differentiation, and osteoclast recruitment at the implant site in vivo. This study demonstrates the significant attenuation of osteoblast function concurrent with increased expression of receptor activator of nuclear factor kappaB ligand (RANKL), a dominant signal for osteoclast recruitment, which is regulated differentially, depending on the size of the Ti particle. Zymography studies have also demonstrated increased activities of matrix metalloproteinases (MMP) 2 and 9 in cells exposed to larger Ti particles. In summary, all particles have adverse effects on osteoblast function, resulting in decreased bone formation and integration, but different mechanisms are elicited by particles of different sizes.

Fig. 1.

Particle size separation. (a) Alfa Aesar Ti particles: <1.5 μm (group I), ≥1.5 μm and <5.0 μm (group II), ≥5.0 μm and <10.0 μm (group III), and ≥10.0 μm and <15.0 μm, (group IV). (Scale bars, 20 μm.) (b) Left is an enlargement of a particle from aI, illustrating axial measurements (indicated by arrows) used in size determination of particles from group I. Right is an enlargement of a particle from aIV, illustrating axial measurements of a particle from group IV. The ratio of long to short axial measurement is 1 (Left) and 2 (Right).

Fig. 2.

Rat tibias with multiple insertion sites. The arrow indicates the selected position for pin insertions (7 mm proximal to the prominent point on the tibial crest). This location provided the optimal and most consistent integration when measured by extraction force. The arrowhead indicates the prominent portion of the tibial crest from which measurements were made. The holes, from left to right, are 12, 8.5, 4.8, and –1 mm relative to the point on the tibial crest (left leg).

Fig. 3.

Cross section of the proximal portion of a rat tibia. Distances proximal to the prominent point on the tibial crest and medial to the tibial crest are 8.1 ± 0.62 mm and 2.2 ± 0.27 mm, respectively (mean ± SD). Insertion depth and pull-out force for each particle-size-group are from control (no particles; 3.8 mm, and 9.44 N) (a), ≥1.5 μm and <5.0 μm (4 mm and 8.4 N) (b), and ≥5.0 μm and <10.0 μm (4 mm and 10.62 N) (c), respectively. Pin extraction was performed at 41 d after implantation.

Fig. 4.

Focal contact arrangement of normal osteoblast (Upper) in a well organized peripheral arrangement vs. Ti-treated osteoblast (Lower) in an unorganized random arrangement.

Fig. 5.

Effects of Ti-particle size on osteoblast proliferation and viability at 0.1 wt % for 72 h (n = 5). Four groups are particle size <1.5 μm (group I), ≥1.5 μm and <5.0 μm (group II), ≥5.0 μm and <10.0 μm (group III), and ≥10.0 μm and <15.0 μm (group IV), measured as percentage of proliferation and viable cells, normalized relative to control (without particle loading). Error bars, ±SD. *, Proliferation study done with P < 0.01; **, viability study done with P < 0.05.

Fig. 6.

Plot of real-time PCR data of RANKL levels after loading with 0.1 wt % Ti particles of three size-separated groups (relative to control) for 24 and 48 h. The number of assays measured for each data point ranges from n = 10 to n = 4. Error bars, ±SEM. *, P < 0.01 compared with control.

Fig. 7.

Results from zymography. The results present a clear increase in MMP-2 activity in groups III and IV at 48 and 72 h, but small particles (groups I and II) have no effect at 8 and 24 h. MMP-9 proand active-form activities are increased at 48- and 72-h incubation periods. (Particle concentration 0.01 wt %.)