Volume 39, Issue 5 , Pages 463-468, May 2010
The effect of injectable calcium phosphate cement on bone anchorage of titanium implants: an experimental feasibility study in dogs
Article Outline
- Abstract
- Materials and methods
- Results
- Discussion
- Funding
- Competing interests
- Ethical approval
- Acknowledgement
- References
- Copyright
Abstract
Calcium phosphate has high osteotransductive potential. The injectable form of calcium phosphate cement (ICAP) can be used as an adjunctive supportive agent for dental implants. The aim of this study was to assess the effect of an ICAP on the reverse torque resistance of titanium implants. Two implant beds (total 24) were prepared in each proximal tibia of 6 beagles. ICAP was injected into one of prepared implant beds (test) and the implant was inserted. The next implant was inserted without ICAP to serve as control. Three dogs were killed after 2 weeks and 3 after 12 weeks. Retrieved implants were subjected to reverse torque test. Results were analyzed with Student's t-test. Scanning electron microscope (SEM) was used for further evaluation. Mean torque values in 2-week healed implants were 52.48
Ncm and 50.57Ncm for test and control implants, respectively (p=0.4). 12-week healed implants showed 81.61Ncm and 76.71Ncm for test and control implants, respectively (p=0.14). Results indicated no statistical difference between test and control implants for either healing time. SEM images of tested samples revealed close contact between the bone–ICAP–titanium surface. ICAP must be tested on further developed experimental models.
Keywords: injectable calcium phosphate cement, bone anchorage, titanium implants, dogs.
Primary fixation is one of the perquisites in establishing adequate osseointegration between bone and fixture11. Lack of primary stability may lead to micromovement and implant loss, especially in orthopedic implants subjected to load bearing3. Polymethylmethacrylate (PMMA) is used as a gap filler between bone and implant to reinforce implant stability, but heat generation during polymerization (hardening), its inert nature (unable to resorb) and low biocompatibility compromize the success of implant treatment carried out with PMMA14. Calcium phosphates (CAPs) have been tried for orthopedic implant stabilization and as a bone graft17. Being a natural component of bone tissue, calcium phosphate offers positive potential in bone regeneration18. CAPs are reported to be suitable for peri-implant bone defects as well22.
Preparing CAP in a cement fashion; (liquid–powder process) it can set at room temperature allowing injection directly into the defect1. It can be shaped before the hardening phase. Injectable calcium phosphate cement (ICAP) forms a direct connection with living bone tissue allowing rapid osteogenesis10. Recently, ICAP has being used instead of PMMA for orthopedic implant fixation and the results are promising15.
CAPs were also used to coat the implant surface to enhance bone–implant contact and induce a positive osteogenic effect23. Comparative studies of CAP-coated implants with machined and blasted-acid etched surfaces revealed increased bone–implant contact percentage and higher torque resistance25. In review of these positive findings, it can be hypothesized that ICAP may be used in implant surgery as an adjunctive agent allowing increasing bone anchorage for dental implants. In order to verify the supportive effect and torque resistance, an animal model was designed to test ICAP applied to the osteotomy site before implant installation.
Materials and methods
6 beagles with a mean age of 36 months and of similar weight were used for this study. The study was approved by the Local Ethical Committee and the surgery was performed under the animal research guidelines. Prior to surgery, the animals were monitored for 2 weeks to ensure that they were healthy and stable.
Stepped cylinder design, titanium dental implants with sandblasted and acid etched surface (3.75mm Ø×13mm) were used (Frialit-II®, Dentsply, Friadent, Mannheim, Germany).
ICAP (Augmentech AT, Wetzlar, Germany) was present in a ready-to-mix tube with powder and liquid components. The powder consisted of tricalciumphosphate (TCP), magnesium phosphate, magnesium hydrogen phosphate and strontium carbonate. The liquid was a watery solution of diammonium hydrogen phosphate. The tube is placed in a mixing apparatus (Silamat, Vivadent, Schaan, Liechtenstein) and shaken for 15s (Fig. 1).
Fig. 1.
ICAP tube placed in an amalgamator for preparation. (a) Injection tip; (b) powder component; (c) liquid component.
Surgery and implant installation
The proximal tibia was chosen as the site of implantation because the risk of wound complication is less than in the oral environment and also because of its abundant trabecular bone volume so that test and control implants could be encouraged. This allowed the flow and entrapment of ICAP in the bone marrow around test implants. Before surgery, the areas in both left and right proximal tibias were shaved then washed and disinfected with betadine and draped for sterile surgery. As a premedication, Xylasin 1.5mg/kg (Rompun, Bayer, Germany) was injected intramuscularly (i.m.). Animals were anesthetized by ketamine 10mg/kg i.m. (Ketanest, Alfasan, The Netherlands). A full thickness flap was raised in the proximal region of the tibia. Implant bed preparation was performed according to the guidelines of the Frialit-II® dental implant system (Fig. 2). Two implant beds in each tibia were prepared in each dog at an approximate distance of 12mm from each other. The implant beds were irrigated with sterile saline to wash away the debris. Excessive hemorrhage was controlled with sterile gauze.
Fig. 2.
Preparation of the implant beds in proximal tibia.
ICAP was injected into one randomly selected implant beds in a retrograde manner beginning from the bottom of the cavity towards the top (Fig. 3). Immediately after the injection of ICAP, the implant was installed with the hand piece instrument and embedded in the bone. Cement that flooded out of the implant bed was cleaned from the area. In the other randomly assigned bed, the implant was inserted without ICAP to serve as a control (Fig. 4). 24 implants were placed with high primary stability, then cover screws were fastened. The flaps were sutured with layers using resorbable (4.0, Polyglactin 910, Ethicon, Johnson & Johnson, New York, USA) and non-resorbable silk (3.0, Dogsan, Istanbul, Turkey) sutures. Animals recovered without complications and received amoxicillin plus clavulanic acid 20mg/kg i.m. (Sysnulox, Pfizer, Belgium) for 7 days. The animals were fed with a standard diet during the healing period. They were killed following 2 and 12 weeks of healing with an intravenous (i.v.) injection of overdose sodium pentobarbital (65mg/kg, Dolethal, Laboratoire Vetoquinol, Lure, France).
Fig. 3.
ICAP was injected into one of prepared implant beds.
Fig. 4.
Implants were inserted into ICAP injected site (arrow) and empty bed (asterisk).
Removal torque test (RTT)
Tibias were dissected en bloc and implants were exposed and clinically examined. Tibia blocks inheriting implants were stabilized and cover screws were removed. The internal hexagon fitting apparatus of the Frialit-II implants system was connected to the implants and device testing arm. Standard torque testing was performed with 90° force application to the implant axis. Implants were subjected to increasing torque load using the Instron device (Instron, Canton, MA, USA) until the bone-to-implant interface ruptured. The torque force was used at a constant displacement speed of 0.5mm/min. At the point of implant failure, the test was stopped immediately to prevent complete damage of the interface. Measurements of peak torque to initiate reverse rotation were recorded by a computer.
Scanning electron microscope (SEM)
Following the torque test specimens were examined by SEM. Backscatter SEM (JEOL 6310, Jeol Ltd., Tokyo, Japan) images were obtained to evaluate the bone–cement–implant interface. All specimens were fixed and dehydrated by a graded series of ethanol and embedded in methylmethacrylate. After polymerization, samples were sectioned on the long axis in the center. All specimens were polished, carbon coated then investigated with the backscatter mode to examine the ICAP–bone–implant appearance and the localization of the rupture patterns in the interface.
Statistical analysis
Mean torque measurements were calculated for the implants in test and control groups. Differences in mean peak torque values for test and control implants were compared with paired Student's t-test. Values of p<0.05 were considered significant.
Results
Preparation and application of ICAP was convenient and easy. In the event of bleeding, ICAP was eroded by blood flow from the osteotomy sites; in previous studies this was described as a ‘wash-out effect’8, 9. Healing was uneventful in all dogs. Radiologic examination revealed no pathology around the implants. All implants were clinically stable and osseointegrated at both healing periods. Some implants were partially covered with bone and exposure of the implant shoulder was performed with a round bur after radiologic confirmation.
The mean removal torque values (RTV) at 2 weeks were 50.57 (±11.59)Ncm for the control implants and 52.48 (±11.94)Ncm for the implants inserted with ICAP. In the 12-week healing group, control implants showed 76.71 (±6.55)Ncm and test implants showed 81.61 (±7.9)Ncm torque resistance values. In neither healing period was statistical significance found (2 weeks: p=0.4; 12 weeks: p=0.14) between test and control groups (Table 1 and Fig. 5).
Table 1. Removal torque values in 2- and 12-week healing groups for test and control implants.
| 2 weeks (Ncm) | 12 weeks (Ncm) | ||||
|---|---|---|---|---|---|
| Control | Test | Control | Test | ||
| Dog 1 | Dog 4 | ||||
| (L) | 70 | 47.7 | (L) | 84.5 | 82.3 |
| (R) | 61.5 | 39.2 | (R) | 79.4 | 89.2 |
| Dog 2 | Dog 5 | ||||
| (L) | 49.4 | 70.4 | (L) | 78.3 | 67.4 |
| (R) | 50.1 | 57.9 | (R) | 65.4 | 79.8 |
| Dog 3 | Dog 6 | ||||
| (L) | 48.6 | 42.6 | (L) | 73.5 | 88.6 |
| (R) | 35.3 | 45.6 | (R) | 79.2 | 82.4 |
| Mean | 52.48 (±11.94) | 50.57 (±11.59) | 76.71 (±6.55) | 81.61 (±7.9) | |
| p | 0.4 | 0.14 | |||
Fig. 5.
Mean removal torque values (N
cm) after 2- and 12-week implantation periods.
New bone formation and good interfacial bone contact were observed in all samples. There were no signs of inflammatory response in any sample. New bone formation was evident in all samples with no visible gaps between ICAP, bone and implant. Fracture lines were visible in almost all samples.
In the 2-week healed implants, ICAP was visible in all test implants distributed into the surrounding trabeculae. ICAP was in direct contact with the native bone and implant surface. Trabeculae around the implant body were sparsely filled with ICAP. ICAP was not evenly spread on the implant interface in all implants. Rupture patterns were rare on the implant surface and mostly in the cement body as cracks and departed fragments showing the mechanical breakdown of the non-resorbed cement during reverse torque forcing.
The 2-week control implants showed direct bone contact. Detached implant body and bone was observed and a thin dark gap was visible at the bone implant interface (Fig. 6).
Fig. 6.
Backscatter SEM images of 2-week healed implants. (a) Implant inserted with ICAP. ICAP has penetrated into a large area through the trabecular network (light grey area) and in direct contact with the native bone (dark grey area). Crack lines are present in the ICAP core in contact with the implant surface (arrows). Resorption of the ICAP is not complete. (b) Implant inserted with ICAP. Non-resorbed ICAP can be observed in the apex of the implant left from the drill space. ICAP is in direct contact with the implant surface and native bone. Fracture lines within the ICAP body reveal the mechanical breakdown of the cement before implant surface rupture (arrows). (c) Control implant. Implant body has detached from native bone with a thin gap along the bone–implant interface.
In the 12-week healed implants, the thickness of the ICAP was visibly reduced and infiltrated by the new bone network compared with the 2-week images but resorption was not completed. The cement core was thinner and the border line with native bone was more indistinguishable compared with the 2-week group. Some slices showed very little remnants of ICAP. Bone-to-implant contact seemed denser in the 12-week group.
The 12-week control implants showed denser bone morphology and improved bone–implant contact. Implants were detached from the implant surface with a visible gap along the bone–implant interface (Fig. 7).
Fig. 7.
Backscatter SEM images of 12-week healed implants. (a) Implant inserted with ICAP. ICAP is still visible in the area but thinner compared with the level at 2 weeks. Crack lines are present in ICAP core and implant surface (arrows). (b) Implant inserted with ICAP. Cement is vastly resorbed with intact remnants still visible next to implant body (arrows). Implant body has detached from the bone surface area. (c) Control implant. Bone structure can be observed throughout the implant surface. Implant body has detached from the bone surface area.
Discussion
Oral implants installed in dog tibia were used to evaluate the behavior and supportive effect of an ICAP bone substitute. The histometric similarity of dog bone to human bone has been reported21 and CAP has been tested in dogs in similar studies1, 5. Bone grafts have been tested for their in situ behavior and suitability in dog tibia19. This study was performed on tibia to reduce the potential infection and wound-healing risks encountered in the oral environment. The cortical structure of the tibia was very dense which also provided high primary stability for all implants. The results of this study are limited by these bone characteristics.
RTTs were used to evaluate the strength of osseointegration due to the surface characteristics, thread geometry, modifications and additive/supportive agents applied at the bone–implant interface. Despite the fact that this method does not provide any information about the microstructural formation of the osseointegration, the force required to loosen the bond of bone-to-implant has been used as an objective criterion for comparison purposes4. In order to evaluate the microstructure of the bone–ICAP–implant, samples were investigated by SEM following the RTT.
SEM examination confirmed the bone biocompatibility and osteoconductive properties of the ICAP cement used. The material did not evoke any inflammatory response, but favored new bone formation at the bone–implant interface. These findings corroborate other studies of ICAP cement that reported excellent long-term behavior and stability5, 10, 18.
The use of ICAP to provide a rapid osteogenesis and fixation effect for dental implants inserted in dog tibia was assessed. Primary stability is one of the most important factors for a dental implant establishing osseointegration11. SEM evaluation revealed ICAP infiltrated into trabecular marrow around dental implants. This effect may also enhance the bone support around an implant, which is of great importance in primary implant stability and in predicting implant treatment outcome8.
The ICAP used in this study is provided in a ready-to-mix tube that facilitates application. This may be useful for clinicians to reduce the steps of a grafting procedure. Cement hardening takes about 10min, which seems long enough for grafting purposes.
The ICAP used in this study has a relatively low flexural strength of 2.3MPa compared with other ICAPs. This weakening could be related to the chemical formulation designed for faster biodegradation of the cement. It may also reduce the final strength of the material. A similar ICAP cement, with a compressive strength of 48.29MPa, was used with stainless steel screws and evaluated with RTT in a goat model. Statistically higher resistance was observed in the first week and after 6 months of healing with implants inserted with the ICAP cement rather than the screws alone16. Stainless steel implants have lower biocompatibility and are not suitable for comparison with the titanium implants used in this study. The use of less biocompatible stainless steel implants prevents comparisons being drawn with the present study.
The RTV of test and control implants in both groups were similar to those reported by other investigators. Gotfredsen et al.7 reported a mean 60Ncm RTV of TiO2 blasted implants placed in dog tibia which healed for 12 weeks. Another study used machined surface implants in rabbit tibia and reported 68Ncm, 77Ncm and 88Ncm RTV at the end of 3, 6 and 12 months of healing, respectively12. Klokkevold et al.13 placed 3.25mm Ø×4mm height titanium plasma sprayed implants in rabbit femur and found a mean 27Ncm reverse torque resistance after 2 weeks of healing. A human study using machined surface 3.75mm Ø×4mm implants inserted into temporal bone reported an average RTV of 42.7Ncm after 12–16 weeks healing24. RTV seems to be affected by many variants, such as implant diameter, length, surface healing period, bone type and animal model. The differences in the RTV required to loosen the fixture in the quoted studies must be considered in the light of these factors.
Growth factors and cytokines were also used to enhance the cellular response at the tissue–material interface. An example was bone morphogenetic proteins (BMP), a bone inductive stimulant that is a natural component of the fracture healing mechanism6. A study conducted by Bessho et al.2 applied lyophilized BMP in the cervical region of the prepared bone beds then inserted 3.75mm Ø×15mm machined implants, which were healed for 3 and 12 weeks. Implants were tested with RTT and RTV were significantly higher in implants applied with BMP in either healing periods. Another similar study achieved higher torque resistance by using specially formulated ICAP cement with Al2O3 blasted and titanium plasma spray coated press fit titanium implants. Implants placed with ICAP cement showed higher torque resistance (>212Nm) both in 2- and 10-week healing periods. As in the present study, crack lines were present in the cement body in contrast to the bone–implant interface in control implants20. Previous reports and the present results indicate that the final formulation of the cement has to be optimized and balanced to achieve high flexural strength and resorbability. Although the RTV of implants placed with ICAP was higher than that of regular implants in both healing periods, the differences were not statistically significant. Not only the ICAP but the use of a very dense recipient bone area might have influenced this outcome. The dense structure of the tibia may have hindered the possibility of observing any difference between test and control implants. The number of subjects in each healing period may not accurately represent the statistically comparable data.
SEM evaluation indicated that all implants showed interfacial bone contact proving biocompatibility and osteoconductivity of the ICAP. SEM evaluation also showed bone rupture patterns initiated from the non-resorbed cement body in control implants and from the bone–implant interface in test implants. This may also lead to the mechanical breakdown of the cement body resulting in the insignificance between test and control implants. Within the limits of this study it can be concluded that ICAP showed favorable handling properties, and did not evoke any adverse effects. The present torque data showed no significant difference between implants placed with ICAP and regular implants; most likely related to the chosen experimental model. Further studies performed on alveolar bone may better identify the anchorage effect of ICAP applied with dental implants.
Funding
This study was primarily funded by Turkish Society of Oral Implantology, Istanbul, Turkey. Implants were donated by Dentsply-Friadent Branch-Saglik Dis Deposu Istanbul, Turkey. Injectable calcium phosphate cement was provided by Augmentech AT, Wetzlar, Germany
Competing interests
Authors have no commercial or financial interest with the brands, materials and items mentioned in the study. All authors are independent academic staff.
Ethical approval
This study was approved by the Local Ethical Committee of Istanbul University (20/01/2002-001), Faculty of Dentistry and the surgery was performed under the animal research guidelines of Istanbul University, Faculty of Veterinary Medicine.
Acknowledgement
The authors would like to thank Professor Dr. John A. Jansen from University Nijmegen Medical Center, Nijmegen, The Netherlands for laboratory support in this study.
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PII: S0901-5027(10)00007-X
doi:10.1016/j.ijom.2010.01.004
© 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.
Volume 39, Issue 5 , Pages 463-468, May 2010
