Effect of micrometer-scale roughness of the surface of Ti6Al4V pedicle screws in vitro and in vivo

Abstract

Background: Titanium implants that have been grit-blasted and acid-etched to produce a rough microtopography support more bone integration than do smooth-surfaced implants. In vitro studies have suggested that this is due to a stimulatory effect on osteoblasts. It is not known if grit-blasted and acid-etched Ti6Al4V implants also stimulate osteoblasts and increase bone formation clinically. In this study, we examined the effects of micrometer-scale-structured Ti6Al4V surfaces on cell responses in vitro and on tissue responses in vivo.

Methods: Ti6Al4V disks were either machined to produce smooth surfaces with an average roughness (Ra) of 0.2 microm or grit-blasted, resulting in an Ra of 2.0, 3.0, or 3.3 microm. Human osteoblast-like cells were cultured on the disks and on tissue culture polystyrene. The cell number, markers of osteoblast differentiation, and levels of local factors in the conditioned media were determined at confluence. In addition, Ti6Al4V pedicle screws with smooth or rough surfaces were implanted into the L4 and L5 vertebrae of fifteen two-year-old sheep. Osteointegration was evaluated at twelve weeks with histomorphometry and on the basis of removal torque.

Results: The cell numbers on the Ti6Al4V surfaces were lower than those on the tissue culture polystyrene; the effect was greatest on the roughest surface. The alkaline-phosphatase-specific activity of cell lysates was decreased in a surface-dependent manner, whereas osteocalcin, prostaglandin E(2), transforming growth factor-beta1, and osteoprotegerin levels were higher on the rough surfaces. Bone-implant contact was greater around the rough-surfaced Ti6Al4V screws, and the torque needed to remove the rough screws from the bone was more than twice that required to remove the smooth screws.

Conclusions: Increased micrometer-scale surface roughness increases osteoblast differentiation and local factor production in vitro, which may contribute to increased bone formation and osteointegration in vivo. There was a correlation between in vitro and in vivo observations, indicating that the use of screws with rough surfaces will result in better bone-implant contact and implant stability.

Fig. 1

In vivo assessment of osteointegration. a: The macrostructure of a grit-blasted pedicle screw as demonstrated by scanning electron microscopy (×20). b: The positions of the pedicle screws were confirmed radiographically after implantation. The pedicle screws were inserted into the pedicular notch of L4 and L5 within the vertebral body. Fusion rods were connected vertically in order to achieve fixation and load-bearing.

Fig. 2

Experimental setup for assessing removal torque of pedicle screws in sheep spine. Left: A representative example. Right: The apparatus.

Fig. 3

Scanning electron micrographs showing the surface morphology of machined and grit-blasted Ti6Al4V substrates (×1200). The surfaces were machine-polished (a) or grit-blasted to an Ra of 2.0, 3.0, or 3.3 μm (b, c, and d, respectively).

Fig. 4

Scanning electron micrographs showing the surface morphology of machined pedicle screws (a [×240] and b [×1000]) and grit-blasted pedicle screws with an Ra of 3 μm (c [×240] and d [×1000]).

Fig. 5

Scanning electron micrographs showing the morphology of MG63 cells cultured for six days on a machined Ti6Al4V substrate (a) or grit-blasted Ti6Al4V substrates with Ra values of 2.0, 3.0, and 3.3 μm (b, c, and d, respectively) (×1000).

Fig. 6-AFig. 6-C Fig. 6-D

Figs. 6-A through 6-D Effects of surface structure on osteoblast cell number and differentiation. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 6-A The cells were harvested at six days, and the cell numbers were counted. Fig. 6-B Alkaline-phosphatase-specific activity was measured in isolated cell lysates. Fig. 6-C Osteocalcin levels were measured in the conditioned media of confluent cultures. Fig. 6-D Osteocalcin levels normalized by cell number were calculated.

Fig. 6-AFig. 6-C Fig. 6-D

Figs. 6-A through 6-D Effects of surface structure on osteoblast cell number and differentiation. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 6-A The cells were harvested at six days, and the cell numbers were counted. Fig. 6-B Alkaline-phosphatase-specific activity was measured in isolated cell lysates. Fig. 6-C Osteocalcin levels were measured in the conditioned media of confluent cultures. Fig. 6-D Osteocalcin levels normalized by cell number were calculated.

Fig. 6-AFig. 6-C Fig. 6-D

Figs. 6-A through 6-D Effects of surface structure on osteoblast cell number and differentiation. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 6-A The cells were harvested at six days, and the cell numbers were counted. Fig. 6-B Alkaline-phosphatase-specific activity was measured in isolated cell lysates. Fig. 6-C Osteocalcin levels were measured in the conditioned media of confluent cultures. Fig. 6-D Osteocalcin levels normalized by cell number were calculated.

Fig. 6-AFig. 6-C Fig. 6-D

Figs. 6-A through 6-D Effects of surface structure on osteoblast cell number and differentiation. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 6-A The cells were harvested at six days, and the cell numbers were counted. Fig. 6-B Alkaline-phosphatase-specific activity was measured in isolated cell lysates. Fig. 6-C Osteocalcin levels were measured in the conditioned media of confluent cultures. Fig. 6-D Osteocalcin levels normalized by cell number were calculated.

Fig. 7-AFig. 7-C Fig. 7-D

Figs. 7-A through 7-D Effects of surface structure on local factor production. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 7-A The total content of prostaglandin E₂ (PGE₂) in the conditioned media was determined with a radioimmunoassay kit. Fig. 7-B The amount of prostaglandin E₂ normalized by cell number was determined with a radioimmunoassay kit. Fig. 7-C Osteoprotegerin (OPG) in the conditioned media was measured with use of an ELISA kit. Fig. 7-D Transforming growth factor beta-1 (TGF-β1) in the conditioned media was measured with use of an ELISA kit.

Fig. 7-AFig. 7-C Fig. 7-D

Figs. 7-A through 7-D Effects of surface structure on local factor production. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 7-A The total content of prostaglandin E₂ (PGE₂) in the conditioned media was determined with a radioimmunoassay kit. Fig. 7-B The amount of prostaglandin E₂ normalized by cell number was determined with a radioimmunoassay kit. Fig. 7-C Osteoprotegerin (OPG) in the conditioned media was measured with use of an ELISA kit. Fig. 7-D Transforming growth factor beta-1 (TGF-β1) in the conditioned media was measured with use of an ELISA kit.

Fig. 7-AFig. 7-C Fig. 7-D

Figs. 7-A through 7-D Effects of surface structure on local factor production. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 7-A The total content of prostaglandin E₂ (PGE₂) in the conditioned media was determined with a radioimmunoassay kit. Fig. 7-B The amount of prostaglandin E₂ normalized by cell number was determined with a radioimmunoassay kit. Fig. 7-C Osteoprotegerin (OPG) in the conditioned media was measured with use of an ELISA kit. Fig. 7-D Transforming growth factor beta-1 (TGF-β1) in the conditioned media was measured with use of an ELISA kit.

Fig. 7-AFig. 7-C Fig. 7-D

Figs. 7-A through 7-D Effects of surface structure on local factor production. MG63 cells were cultured on tissue culture polystyrene (TCPS), machined Ti6Al4V substrates with an Ra of 0.2 μm, or grit-blasted Ti6Al4V substrates with an Ra of 2.0, 3.0, or 3.3 μm. Values are expressed as the mean and standard error of the mean of six independent cultures. Data were derived from one of two separate experiments, both of which had comparable results. Data were analyzed with analysis of variance, and significant differences between groups were determined with use of the Bonferroni modification of the Student t test. *p < 0.05 as compared with the tissue culture polystyrene, #p < 0.05 as compared with the 0.2-μm-Ra machined surface, and +p < 0.05 as compared with the 3.0-μm-Ra grit-blasted surface. Fig. 7-A The total content of prostaglandin E₂ (PGE₂) in the conditioned media was determined with a radioimmunoassay kit. Fig. 7-B The amount of prostaglandin E₂ normalized by cell number was determined with a radioimmunoassay kit. Fig. 7-C Osteoprotegerin (OPG) in the conditioned media was measured with use of an ELISA kit. Fig. 7-D Transforming growth factor beta-1 (TGF-β1) in the conditioned media was measured with use of an ELISA kit.

Fig. 8

Microcomputed tomography scans of machined (A, B, and C) and grit-blasted (D, E, and F) Ti6Al4V pedicle screws in the L4 vertebral bone of sheep.

Fig. 9

Undecalcified histological sections of peri-implant bone formation following osteointegration of smooth (a, b, and c) and rough (d, e, and f) Ti6Al4V pedicle screws in the L4 vertebra of sheep (hematoxylin and eosin; no magnification).