Abstract
Objective
Alumina toughened zirconia (ATZ) is more fracture resistant than unmodified zirconia and has been shown to be a viable substrate for the growth of osteoblasts. In this study, we examined the histological and biomechanical behavior of moderately roughened ATZ implants in rat femoral bone.
Methods
Miniature implants made of ATZ with pore-building polymers sintered onto the surface and electrochemically anodized titanium (TiUnite ® ) were placed into the femurs of Sprague-Dawley rats. Implant surface topography was analyzed by 3D laserscan measurements and scanning electron microscopy (SEM). After a healing period of 14 and 28 days, respectively, histologic and biomechanical testing was performed.
Results
Under the SEM, the TiUnite ® surface could be clearly distinguished from the ATZ surface, but 3D laserscan measurements indicated a moderately rough surface topography for both, TiUnite ® ( S a = 1.31 μm) and ATZ ( S a = 1.51 μm). The mean mineralized bone-to-implant contact showed the highest values after 14 and 28 days for TiUnite ® (58%/75%) as compared to ATZ (24%/41%). The push-in values after a healing period of 14 and 28 days, respectively, increased from 20 N to 39 N for TiUnite ® and from 10 N to 25 N for ATZ.
Significance
Our findings suggest that the moderately roughened ATZ implant surface is well accepted by rat bone tissue. However, compared to titanium, the osseointegration-process of ATZ seems to proceed more slowly in that early phase of implant integration.
1
Introduction
Within the last decade, zirconia implants have become a focus of interest in dentistry and are strongly discussed as a metal-free and whitish alternative to titanium. It has been shown in laboratory studies that so far at least one piece zirconia implants exhibit the mechanical properties that are required to withstand masticatory forces in the oral cavity . Further, cell culture and animal studies have demonstrated that zirconia especially with a moderately rough surface is accepted by osteoblasts and integrates into bone tissue. Clinical data is also available but needs careful consideration since only a limited number amongst the publications meet sound scientific criteria .
Most commonly, yttria stabilized tetragonal zirconia polycrystal (Y-TZP) is used to manufacture oral zirconia implants. But Y-TZP has been reported to be prone to an aging process caused by a phenomenon called low temperature degradation (LTD) which may ultimately result in failures . The addition of alumina to the zirconia matrix leads to the composite material alumina toughened zirconia (ATZ) which is even more fracture-resistant than Y-TZP but also more resistant to LTD . Further, ATZ has been shown to be a viable substrate for the growth of osteoblasts in an in vitro study . However, the success of endosseous implants is directly related to the principle of osseointegration, a process of implant–bone interaction that finally leads to bone-to-implant anchorage and that is preferably evaluated in living bone. The degree of osseointegration is typically quantified by histomorphometry and/or biomechanical testing . A moderately rough surface topography is known to positively affect the interfacial tissue reaction . But surface modification of zirconia is challenging. Besides sintering particles onto the implant surface, nano-technology, sandblasting and acid etching, and laser technology have been used to produce a roughened zirconia surface . In a recent animal study, in vivo evidence was collected that ATZ is a suitable candidate as dental implant material and first clinical data is also promising .
The purpose of this study was to further evaluate ATZ implants vs. titanium implants both with a roughened surface in a rat femur model.
2
Materials and methods
2.1
Implant design and surface analysis
Miniature cylindrical implants with a length of 2 mm and a diameter of 1 mm were used in this study. The implants were either made of commercially pure titanium or alumina toughened zirconia (ATZ). The surface of the titanium implants was modified by electrochemical anodization (TiUnite ® , NobelBioCare, Gothenborg, Sweden), and the surface of the ATZ implants was treated by sintering pore-building polymers with a size of 50 μm onto the surface. These polymers burn out during the sintering process resulting in a porous surface topography (Zircapore ® , Metoxit, Thayngen, Switzerland).
The surface of the substrates was examined by scanning electron microscopy (SEM) (Zeiss Leo 32, Zeiss, Oberkochen, Germany) after being gold-palladium-sputtered, and by 3D laser scanning (3D Laser Microscope VK-9700K, Keyence Corp., Osaka, Japan). Average roughness ( S a ), average value in micrometers of the absolute departures of the five highest peaks and the five deepest valleys ( S z ), average mean spacing of profile peaks in the mean plane as expressed in the x direction ( S cx ), and the developed surface area ratio ( S dr ) were calculated.
2.2
Placements of miniature implants in the rat femur
The study protocol was approved by the University of Freiburg Animal Research Committee. All animals were handled according to the policies and principles established by the German Animal Protection Law (“Deutsches Tierschutzgesetz”). Twenty-eight 8-week-old male Sprague-Dawley rats were anesthetized with a 1–2% isoflurane inhalation. After the legs were shaved and disinfected using 0.2% chlorhexidine, an incision on the dorsal site of the femur was made and a full-thickness flap was reflected to expose the distal femur. One cylindrical implant per femur was placed approximately 7 mm from the distal edge of the femur. The implant site was prepared by sequential drilling using a 0.7 mm round bur under sterile saline irrigation. With an endodontic file, the osteotomy was enlarged to 0.9 mm. The implants were subsequently placed into the osteotomy and carefully pushed into place until the end of the implant was aligned with the femoral bone surface. After the correct implant position was achieved, the tissues were sutured in layers using resorbable sutures (Vicryl ® , Ethicon GmbH, Norderstedt, Germany). TiUnite ® and ATZ implants were placed into the left and right femurs, respectively, of each rat. Animals were randomly divided into 4 groups of 7 each, which were killed at weeks 2 (groups A and B), and 4 (groups C and D) of the healing period. In groups A and C histological evaluation, and in groups B and D biomechanical testing was performed.
2.3
Histological procedure and histomorphometric analysis
After rinsing the harvested implant-femur specimens of groups A and C with saline, they were immersed in 10% buffered formalin for 2 weeks at 4 °C. Afterwards, the specimens were dehydrated in an ascending series of alcohol (50–96%) and finally embedded in photocuring, one-component resin (Technovit 7200 VLC, Heraeus Kulzer, Wehrheim, Germany). After polymerization of the resin, the non-decalcified specimens were cut using a diamond saw and successively ground to a thickness of approximately 80–100 μm with a grinding system (Exakt Apparatebau, Norderstedt, Germany) . The specimens were then stained with basic fuchsine.
Histological observations and computer-assisted histomorphometric analysis were performed using a Zeiss Axioskop (Zeiss, Oberkochen, Germany) equipped with a video camera (ColorView III, Olympus, Münster, Germany) and the software program cell* (Olympus, Münster, Germany). The histomorphometric analysis comprised the evaluation of the fraction of the implant in contact to the cortical bone.
2.4
Implant push-in test
After harvesting, the implant-femur specimens of groups B and D were immediately embedded into an autopolymerizing resin (Technovit 4071, Heraeus Kulzer, Wehrheim, Germany) using a custom-made metal mold. The implants were then loaded axially in a universal testing machine by using a 10 kN load cell and a 0.8 mm diameter stainless steel pushing rod with a crosshead speed of 10 mm/min. The applied load and the displacement of the implant were monitored at a sampling rate of 4 Hz. The push-in value was determined by a sharp drop-down of the pressure load curve displayed on the computer monitor.
2.5
Statistical evaluation
Data of the histological evaluation and the push-in test were expressed as mean value (MV) ± standard deviation (SD). A paired-samples t test, at the 5% level, was applied to evaluate the differences of the bone–implant contact and the biomechanical strength between the TiUnite ® (left femur) and ATZ (right femur) implants after 14 days of healing (group A versus group B), and after 28 days of healing (group C versus group D). An independent-samples t test, at the 5% level, was applied to evaluate the differences of the bone–implant contact and the biomechanical strength between the TiUnite ® implants after 14 and the TiUnite ® implants after 28 days of healing (group A versus group C), and also between the ATZ implants after 14 and the ATZ implants after 28 days of healing (group B versus group D).
2
Materials and methods
2.1
Implant design and surface analysis
Miniature cylindrical implants with a length of 2 mm and a diameter of 1 mm were used in this study. The implants were either made of commercially pure titanium or alumina toughened zirconia (ATZ). The surface of the titanium implants was modified by electrochemical anodization (TiUnite ® , NobelBioCare, Gothenborg, Sweden), and the surface of the ATZ implants was treated by sintering pore-building polymers with a size of 50 μm onto the surface. These polymers burn out during the sintering process resulting in a porous surface topography (Zircapore ® , Metoxit, Thayngen, Switzerland).
The surface of the substrates was examined by scanning electron microscopy (SEM) (Zeiss Leo 32, Zeiss, Oberkochen, Germany) after being gold-palladium-sputtered, and by 3D laser scanning (3D Laser Microscope VK-9700K, Keyence Corp., Osaka, Japan). Average roughness ( S a ), average value in micrometers of the absolute departures of the five highest peaks and the five deepest valleys ( S z ), average mean spacing of profile peaks in the mean plane as expressed in the x direction ( S cx ), and the developed surface area ratio ( S dr ) were calculated.
2.2
Placements of miniature implants in the rat femur
The study protocol was approved by the University of Freiburg Animal Research Committee. All animals were handled according to the policies and principles established by the German Animal Protection Law (“Deutsches Tierschutzgesetz”). Twenty-eight 8-week-old male Sprague-Dawley rats were anesthetized with a 1–2% isoflurane inhalation. After the legs were shaved and disinfected using 0.2% chlorhexidine, an incision on the dorsal site of the femur was made and a full-thickness flap was reflected to expose the distal femur. One cylindrical implant per femur was placed approximately 7 mm from the distal edge of the femur. The implant site was prepared by sequential drilling using a 0.7 mm round bur under sterile saline irrigation. With an endodontic file, the osteotomy was enlarged to 0.9 mm. The implants were subsequently placed into the osteotomy and carefully pushed into place until the end of the implant was aligned with the femoral bone surface. After the correct implant position was achieved, the tissues were sutured in layers using resorbable sutures (Vicryl ® , Ethicon GmbH, Norderstedt, Germany). TiUnite ® and ATZ implants were placed into the left and right femurs, respectively, of each rat. Animals were randomly divided into 4 groups of 7 each, which were killed at weeks 2 (groups A and B), and 4 (groups C and D) of the healing period. In groups A and C histological evaluation, and in groups B and D biomechanical testing was performed.
2.3
Histological procedure and histomorphometric analysis
After rinsing the harvested implant-femur specimens of groups A and C with saline, they were immersed in 10% buffered formalin for 2 weeks at 4 °C. Afterwards, the specimens were dehydrated in an ascending series of alcohol (50–96%) and finally embedded in photocuring, one-component resin (Technovit 7200 VLC, Heraeus Kulzer, Wehrheim, Germany). After polymerization of the resin, the non-decalcified specimens were cut using a diamond saw and successively ground to a thickness of approximately 80–100 μm with a grinding system (Exakt Apparatebau, Norderstedt, Germany) . The specimens were then stained with basic fuchsine.
Histological observations and computer-assisted histomorphometric analysis were performed using a Zeiss Axioskop (Zeiss, Oberkochen, Germany) equipped with a video camera (ColorView III, Olympus, Münster, Germany) and the software program cell* (Olympus, Münster, Germany). The histomorphometric analysis comprised the evaluation of the fraction of the implant in contact to the cortical bone.
2.4
Implant push-in test
After harvesting, the implant-femur specimens of groups B and D were immediately embedded into an autopolymerizing resin (Technovit 4071, Heraeus Kulzer, Wehrheim, Germany) using a custom-made metal mold. The implants were then loaded axially in a universal testing machine by using a 10 kN load cell and a 0.8 mm diameter stainless steel pushing rod with a crosshead speed of 10 mm/min. The applied load and the displacement of the implant were monitored at a sampling rate of 4 Hz. The push-in value was determined by a sharp drop-down of the pressure load curve displayed on the computer monitor.
2.5
Statistical evaluation
Data of the histological evaluation and the push-in test were expressed as mean value (MV) ± standard deviation (SD). A paired-samples t test, at the 5% level, was applied to evaluate the differences of the bone–implant contact and the biomechanical strength between the TiUnite ® (left femur) and ATZ (right femur) implants after 14 days of healing (group A versus group B), and after 28 days of healing (group C versus group D). An independent-samples t test, at the 5% level, was applied to evaluate the differences of the bone–implant contact and the biomechanical strength between the TiUnite ® implants after 14 and the TiUnite ® implants after 28 days of healing (group A versus group C), and also between the ATZ implants after 14 and the ATZ implants after 28 days of healing (group B versus group D).
3
Results
3.1
Surfaces analyses of miniature implants
Under the SEM, the surface of the ATZ implants could be clearly distinguished from the TiUnite ® surface ( Fig. 1 A and C ). Whereas the TiUnite ® surface showed the known characteristic appearance with its pores with diameters of up to 10 μm ( Fig. 1 C), roughened ATZ produced an irregular network with elevations and undercuts ( Fig. 1 A). When evaluating the surfaces using 3D laserscans ( Fig. 1 B and D), the height variation ( S a , S z ) was quite similar but with respect to spatial distribution ( S cx ) and overall surface enlargement ( S dr ) both surfaces were also clearly different ( Table 1 ).
| S a (μm) (SD) | S z (μm) (SD) | S cx (μm) (SD) | S dr (%) (SD) | |
|---|---|---|---|---|
| ATZ | 1.51 (0.69) | 9.07 (2.57) | 0.10 (0.00) | 13.64 (1.84) |
| TiUnite ® | 1.31 (0.17) | 12.38 (0.7) | 1.13 (0.04) | 29.6 (0.23) |
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