Heat and radiofrequency plasma glow discharge pretreatment of a titanium alloy promote bone formation and osseointegration

Abstract

Orthopedic and dental implants manifest increased failure rates when inserted into low density bone. We determined whether chemical pretreatments of a titanium alloy implant material stimulated new bone formation to increase osseointegration in vivo in trabecular bone using a rat model. Titanium alloy rods were untreated or pretreated with heat (600°C) or radiofrequency plasma glow discharge (RFGD). The rods were then coated with the extracellular matrix protein fibronectin (1 nM) or left uncoated and surgically implanted into the rat femoral medullary cavity. Animals were euthanized 3 or 6 weeks later, and femurs were removed for analysis. The number of trabeculae in contact with the implant surface, surface contact between trabeculae and the implant, and the length and area of bone attached to the implant were measured by histomorphometry. Implant shear strength was measured by a pull-out test. Both pretreatments and fibronectin enhanced the number of trabeculae bonding with the implant and trabeculae-to-implant surface contact, with greater effects of fibronectin observed with pretreated compared to untreated implants. RFGD pretreatment modestly increased implant shear strength, which was highly correlated (r(2) = 0.87-0.99) with measures of trabecular bonding for untreated and RFGD-pretreated implants. In contrast, heat pretreatment increased shear strength 3-5-fold for both uncoated and fibronectin-coated implants at 3 and 6 weeks, suggesting a more rapid increase in implant-femur bonding compared to the other groups. In summary, our findings suggest that the heat and RFGD pretreatments can promote the osseointegration of a titanium alloy implant material.

Keywords: BONE MINERALIZATION; CELL DIFFERENTIATION; DENTAL IMPLANT; FIBRONECTIN; OSSEOINTEGRATION; OSTEOBLAST.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 1

Electron microscopic images of coronal sections 6 weeks following implantation. Implants were untreated (A,D) or pretreated with heat (B,E) or RFGD (C,F), left uncoated (A,B,C) or coated overnight with 1 nM fibronectin (D,E,F). Micrographs are shown for sections taken near the distal end of the femur. Red arrows show newly formed bone. Black arrows show areas in which newly formed bone has become anchored to trabeculae; green arrows show trabeculae that also appear to be joined to the outer band of cortical bone. Yellow arrows show a zone of demarcation between the bone surface around the implant’s circumference and the surface of the trabeculae with which it appears to be in contact. FN = fibronectin. HEAT = heat pretreatment; RFGD = radiofrequency plasma glow discharge pretreatment.

Figure 2

Effects of heat and RFGD pretreatment on the number of trabeculae joined to newly formed bone on the implant surface. (A) Number of trabeculae bonded to the implant per cross-section. (B) Number of trabeculae bonded to the implant / length (mm) of surface implant bone. ^{****, ***, **, *} Significantly greater (P<0.001, 0.005, 0.01, 0.05, respectively) than untreated and uncoated (No fibronectin) implants; ⁺ significantly greater (P<0.05) than untreated and fibronectin-coated (Fibronectin) implants; ^a significantly greater (P<0.05) than RFGD-treated and uncoated implants at the corresponding time points based on analysis of variance.

Figure 2

Effects of heat and RFGD pretreatment on the number of trabeculae joined to newly formed bone on the implant surface. (A) Number of trabeculae bonded to the implant per cross-section. (B) Number of trabeculae bonded to the implant / length (mm) of surface implant bone. ^{****, ***, **, *} Significantly greater (P<0.001, 0.005, 0.01, 0.05, respectively) than untreated and uncoated (No fibronectin) implants; ⁺ significantly greater (P<0.05) than untreated and fibronectin-coated (Fibronectin) implants; ^a significantly greater (P<0.05) than RFGD-treated and uncoated implants at the corresponding time points based on analysis of variance.

Figure 3

Effects of heat and RFGD pretreatment of Ti6Al4V on trabeculae – implant surface contact. (A) The length (in mm) of trabeculae-to-implant surface contact per cross-section. (B) The length (in mm) of trabeculae-to-implant surface contact / length (mm) of implant bone. ^{****, ***, **, *} Significantly greater (P<0.001, 0.005, 0.01, 0.05, respectively) than untreated and uncoated (No Fibronectin) implants; ^{++++, +++, +} significantly greater (P<0.001, 0.005, 0.05, respectively) than untreated and fibronectin-coated (Fibronectin) implants at the corresponding time points based on analysis of variance.

Figure 3

Effects of heat and RFGD pretreatment of Ti6Al4V on trabeculae – implant surface contact. (A) The length (in mm) of trabeculae-to-implant surface contact per cross-section. (B) The length (in mm) of trabeculae-to-implant surface contact / length (mm) of implant bone. ^{****, ***, **, *} Significantly greater (P<0.001, 0.005, 0.01, 0.05, respectively) than untreated and uncoated (No Fibronectin) implants; ^{++++, +++, +} significantly greater (P<0.001, 0.005, 0.05, respectively) than untreated and fibronectin-coated (Fibronectin) implants at the corresponding time points based on analysis of variance.

Figure 4

Effects of heat or RFGD pretreatment of Ti6Al4V implants on implant-femur shear strength for Ti6Al4V implants. ^**** Significantly greater (P<0.001) than untreated and uncoated (No Fibronectin) implants; ^{++++, +++} significantly greater (P<0.001 and 0.005, respectively) than untreated and fibronectin-coated (Fibronectin) implants; ^a,b significantly greater (P<0.001) than uncoated and coated RFGD-pretreated implants, respectively, at the corresponding time points based on analysis of variance.

Figure 5

Effects of heat and RFGD pretreatment of Ti6Al4V on the number of trabeculae bonded to the implant and trabeculae – implant surface contact normalized to the area (in mm²) of bone circumscribing the implant. (A) Number of trabeculae in contact with implant / area (mm²) of surface implant bone (B) The length of surface contact between trabeculae and implant / area (mm²) of implant bone. ^{***, *} Significantly greater (P<0.005, 0.05, respectively) than untreated and uncoated (No Fibronectin) implants; ^{++++, ++, +} significantly greater (P<0.001, 0.01, 0.05, respectively) than untreated and fibronectin-coated (Fibronectin) implants; ^a significantly greater (P<0.05) than RFGD-treated and uncoated implants at the corresponding time points based on analysis of variance. Inset for (A) and (B) : average shear strengths for the 6 experimental groups shown vs. number of trabeculae in contact with implant and trabecular surface contact with implant, respectively, each normalized to implant bone area (mm²). Correlation coefficients (r²) were calculated by linear regression.

Figure 5

Effects of heat and RFGD pretreatment of Ti6Al4V on the number of trabeculae bonded to the implant and trabeculae – implant surface contact normalized to the area (in mm²) of bone circumscribing the implant. (A) Number of trabeculae in contact with implant / area (mm²) of surface implant bone (B) The length of surface contact between trabeculae and implant / area (mm²) of implant bone. ^{***, *} Significantly greater (P<0.005, 0.05, respectively) than untreated and uncoated (No Fibronectin) implants; ^{++++, ++, +} significantly greater (P<0.001, 0.01, 0.05, respectively) than untreated and fibronectin-coated (Fibronectin) implants; ^a significantly greater (P<0.05) than RFGD-treated and uncoated implants at the corresponding time points based on analysis of variance. Inset for (A) and (B) : average shear strengths for the 6 experimental groups shown vs. number of trabeculae in contact with implant and trabecular surface contact with implant, respectively, each normalized to implant bone area (mm²). Correlation coefficients (r²) were calculated by linear regression.

Figure 6

Linear relationship between implant-femur shear strength and trabecular bonding to implant surface bone. Mean values at 6 weeks for implant-femur shear strength (obtained from Figure 4) for the untreated (UT), untreated + fibronectin (UT + FN), RFGD (RFGD) and RFGD + fibronectin (RFGD + FN) groups were plotted against the corresponding mean values at 6 weeks for the number of trabeculae bonding to the implant (A) and length (D) of trabecular surface contact with implant; and the number of bonded trabeculae and length of trabecular surface contacts normalized to the circumferential length (B and E, respectively) and area (C and F, respectively) of implant bone (obtained from Figures 2, 3 and 5). Correlation coefficients (r²) were calculated by linear regression.

Figure 7

FTIR spectral color maps of the acid phosphate levels and mineral:matrix ratio in bone adjacent to pretreated implants. (A.) Acid phosphate levels in bone that was in close proximity to the implant for sections from fibronectin-coated and uncoated heat and RFGD-pretreated implant specimens. (B.) Acid phosphate levels and mineral:matrix ratio in peri-implant bone for sections from fibronectin-coated and uncoated RFGD-pretreated implant specimens. White arrows denote focal areas of lower mineral:matrix ratio (blue areas) and higher acid phosphate content (red and yellow areas) compared to the surrounding trabecular or cortical bone. Mean values of acid phosphate and mineral:matrix ratio are shown below each spectral map color scale.

Figure 7

FTIR spectral color maps of the acid phosphate levels and mineral:matrix ratio in bone adjacent to pretreated implants. (A.) Acid phosphate levels in bone that was in close proximity to the implant for sections from fibronectin-coated and uncoated heat and RFGD-pretreated implant specimens. (B.) Acid phosphate levels and mineral:matrix ratio in peri-implant bone for sections from fibronectin-coated and uncoated RFGD-pretreated implant specimens. White arrows denote focal areas of lower mineral:matrix ratio (blue areas) and higher acid phosphate content (red and yellow areas) compared to the surrounding trabecular or cortical bone. Mean values of acid phosphate and mineral:matrix ratio are shown below each spectral map color scale.