Material and methods

Three periodontally healthy, skeletally mature, male foxhound-mixed breed dogs, 1 to 2 years of age and weighing between 55 and 65 pounds, were used as the experimental model. The Institutional Animal Care and Use Committee at Texas A&M University Baylor College of Dentistry (Dallas, Tex) approved the care of the animals and the experimental protocols. Foxhounds are an established model for investigating peri-implant osseous dynamics; their large mandibles provide space between teeth for the placement of multiple MSIs.

The MSIs were Ancoragem Ortodontica screws (Neodent, Curitiba, Paraná, Brazil); they were self-drilling, made of titanium alloy, and 5 mm long, with a 1.6-mm outer diameter, a 1.1-mm internal diameter, and a 0.7-mm pitch. The head of the screw had a 1-mm-high collar and a 1.1-mm internal thread fabricated to accept the Osstell Mentor Smartpeg type A3 (Integration Diagnostics, Göteborg, Sweden), which was used to measure the implant stability quotient over time. The implant stability quotient/resonance frequency analysis method has proven to be a useful tool for determining the stability of MSIs and endosseous implants. Resonance frequency analysis uses a small transducer that is screwed onto the implant. The transducer has 2 piezoceramic elements attached to it; one vibrates the transducer with a sinusoidal signal, and the other measures the response. The data are represented as the implant stability quotient, which ranges from 1 to 100. Greater implant stability quotient values indicate increased stiffness or stability. Bone-to-implant contact was evaluated 3 dimensionally with microcomputed tomography (model 35; Scanco Medical, Bassersdorf, Switzerland).

The animals were quarantined for 10 days. On the day of MSI placement, they were sedated with a mixture of 2.2 mg per kilogram of body weight of ketamine (Bioniche Teoranta, Galway, Ireland) and 0.22 mg per kilogram of xylazine (IVX Animal Health, St Joseph, Mo). Prophylaxis was performed with ultrasonic scaling instrumentation. The surgical sites were irrigated with 0.2% chlorhexidine solution. The dogs were intubated and given 1% to 1.5% isoflurane (Butler Animal Health Supply, Dublin, Ohio) in oxygen at 1 L per minute. Atropine (0.05 mg/kg) (IVX Animal Health) was given subcutaneously to prevent bradycardia.

Using film size number 4, digital radiographs (Air Techniques, Melville, NY) were taken at various angles, and measurements were taken to determine MSI placement locations ( Fig 1 , A ). Gingival tissue around the surgical sites was irrigated and lavaged with 0.2% chlorhexidine before the administration of local anesthesia (2% lidocaine with 1:100,000 epinephrine; Patterson Dental, St Paul, Minn) with a 27-gauge needle. Radiographic measurements were transferred intraorally via a periodontal probe ( Fig 1 , B ), and implant locations were marked with a tissue marker (3M Unitek, Monrovia, Calif). Gingival tissue was removed using a 1.5-mm tissue punch (3M Unitek) ( Fig 1 , C ), and pilot holes were drilled through the cortical bone only at 16,000 rpm with a 1.1-mm surgical drill (Neodent) under copious irrigation with saline solution ( Fig 1 , D ).

A, Radiographic measurements of the MSI sites; B, transfer of radiographic measurements with a perioprobe; C, removal of gingival tissue with a 1.5-mm tissue punch; D, drilling of pilot holes with a 1.1-mm surgical drill under copious irrigation with saline solution at 16,000 rpm; E, injection of 50 μL of phosphate-buffered saline solution containing 16 μg of zoledronate (pH 7.0) or an equal volume of phosphate-buffered saline solution (pH 7.1) containing no zoledronate; F, postoperative radiograph used to ensure proper MSI placement.

Using random assignment, either 50 μL of phosphate-buffered saline solution (pH 7.1) containing 16 μg of zoledronate (Zometa; Novartis, Basel, Switzerland) or 50 μL of phosphate-buffered saline solution was injected by a blinded operator (C.C.) into bilateral pairs of pilot holes using a 3/10 mL syringe. To prevent contamination of adjacent tissues, the MSIs were spaced apart, the substances were carefully injected into the pilot holes, and gauze was used around every pilot hole to absorb any overflow ( Fig 1 , E ). Immediately after substance injection, the MSIs were hand-inserted until the head of the screw was within 0.5 mm of the cortical bone to standardize the depth of insertion and to prevent countersink friction. They were placed perpendicular to the cortical plate and parallel to the occlusal plane.

Twenty MSIs were placed in each dog. There were 30 experimental and 30 control MSIs. MSI placement was individually planned according to each animal’s anatomy. Ten of the mandibular MSIs were placed from the lingual aspect. All MSIs were placed in the interradicular and interdental areas around the second, third, and fourth premolars, and the first molars, in both jaws. Postoperative peri-implant sites were irrigated with 0.2% chlorhexidine. Periapical radiographs were taken to ensure proper MSI positioning and to verify that the roots had not been contacted ( Fig 1 , F ). Most of the buccal MSIs were placed in nonkeratinized gingiva because of the absence of attached gingiva.

Both analgesics (torbugesic, 0.2 mg/kg [2 mg/mL with 1 mL per animal]) and antibiotics (penicillin G, 60,000 units/kg) were administered postoperatively. Inspection of the MSIs, photographs, prophylaxis with a toothbrush and 2% chlorhexidine gluconate oral gel, and measurement of the implant stability quotient were performed weekly for 8 weeks. For the interim cleanings and measurements, the dogs were sedated with ketamine, 1.1 mL per kilogram, and xylazine, 0.1 mg per kilogram.

Before the study, the Osstell transducer was calibrated according to the manufacturer’s instructions. After MSI placement, the Smartpeg mount was magnetically connected to the Smartpeg type A3, screwed into the miniscrew’s head, and tightened with finger pressure according to the manufacturer’s instructions. To minimize possible MSI movement while screwing and unscrewing the Smartpeg, the MSI’s head was secured with a hemostat ( Fig 2 , A ). The Osstell’s transducer was oriented perpendicular to the long axis of the MSI ( Fig 2 , B ), and 4 blinded implant stability quotient measurements were made around the MSI, each 90° apart. The 4 measurements were averaged. Each MSI had 9 implant stability quotient values: the initial one, taken at placement, plus 8 weekly measurements. The reliability of the repeated measurements was high, with an intraclass correlation of 0.959.

A, Smartpeg mount and A3 Smartpeg screwed into the MSI’s head, which is being held by a hemostat to prevent movement of the screw; B, implant stability quotient measured perpendicular to the MSI from 4 perspectives (occlusal, distal, apical, and mesial) in a circular pattern.

Eight weeks after MSI placement, the animals were euthanized with 2 mL of Beuthanasia-D (Schering Corp, Kenilworth, NJ) given intracardially and perfused with 1 to 2 L of normal saline solution, followed by 1 L of 70% ethanol. The maxillae and mandibles were resected en bloc and stored in 70% ethanol until analysis.

All specimens underwent microcomputed tomography analysis. Each bone-implant specimen was retrieved with a 10-mm trephine bur (ACE Dental Implant System, Brockton, Mass) under copious irrigation. The specimens were trephined parallel to the long axis of the implant, ensuring that all 3 layers of the bone (cortical, medullary, cortical) remained intact and undamaged during the retrieval process, and stored in 70% ethanol. A maximum of 5 bone-implant specimens was placed (oriented parallel to the long axis of the holder) in each holder, along with 70% ethanol ( Fig 3 , A ). A 2-dimensional radiographic image of the specimens was used to determine the volume of interest to be scanned ( Fig 3 , B ). The specimens were then scanned using microcomputed tomography with a high isotropic resolution of 6 μm. X-ray energy levels were set to 70 kVp, current was set to 114 μA, and integration time was 800 ms. A 0.5-mm aluminum filter and a high-resolution setting of 1000 projections per 180° were used to ensure the highest-quality scans with minimal metal implant artifacts.

A, 12.3-mm cylindrical microcomputed tomography specimen carrier; B, original gray scale 2-dimensional image with the MSI in the center ( white ), surrounding bone ( gray ), and space ( black ); the volume of interest ( VOI ) was defined as a cylinder with a 430-pixel diameter with the MSI in the center; C, the coronal limit of the volume of interest visually defined as the first slice at which bone was observed to completely surround and contact the MSI. The apical limit was defined as 10 slices (0.06 mm) coronally from the MSI apex.

The volume of interest of each specimen was defined as a cylinder (430 pixel diameter), with the implant positioned in the center ( Fig 3 , C ). The apical limit of the volume of interest was set to 10 slices (0.06 mm) coronally from the MSI tip ( Fig 3 , D ). The average scanning time was 3.8 hours per specimen. After scanning, thresholds (1 gray scale or “brightness” number, above which all voxels were considered bone, and below which all voxels were considered nonbone) were determined using 10 randomly chosen specimens.

From the reconstructed 3-dimensional images, cortical and trabecular bone areas were delimited for each specimen; the mixed bone between was not evaluated ( Fig 4 , A ). Four Scanco Medical proprietary programming scripts with different parameters for bone-implant contacts were processed per sample. For each specimen, separate bone volume fractions were calculated for the cortical and trabecular layers. The coronal and apical limits of cortical bone within the volume of interest were visually defined as the first and last slices with cortical bone completely surrounding the MSI ( Fig 4 , A ). The same rules were used for the trabecular bone. The calculated areas of interest included 3 layers of bone, each 3 voxels (18 μm) thick, 6 to 24, 24 to 42, and 42 to 60 μm from the MSI surface ( Fig 4 , B ). The voxel of bone adjacent to the MSI (0-6 μm) was excluded because it was subject to metallic halation artifacts. The software made it possible to separate the MSI and the mineralized bone, allowing for better visualization of each layer ( Fig 4 , C ); a 3-dimensional image of bone-implant contact, with the MSI and supporting bone removed, was also obtained ( Fig 4 , D ).

A, Delimitation of the coronal and apical limits of the cortical and trabecular bone layers surrounding the MSIs; the zone between them that includes both cortical and trabecular bone was not evaluated; B, schematic depiction of the 3-dimensional bone layers evaluated, including 6-24 μm ( white ), 24-42 μm ( purple ), and 42-60 μm ( pink ) layers; C, titanium alloy MSI ( green ) and 6-24 μm layer of bone ( pink ); D, 3-dimensional image of a 6-24 μm layer of bone with the MSI removed.

Results

Of the 60 MSIs that were placed ( Fig 5 , A ), 2 failed (3.3%) during the study. Two (3.3%) more MSIs had to be removed a few days after placement because of root proximity, and prophylactic root canals had to be performed. Four (6.7%) MSIs were considered “mobile” or unstable, with implant stability quotient readings less than 25 and exhibiting clinical signs of mobility. Of the 58 MSIs that remained in place throughout the experiment, 52 (89.7%) showed implant stability quotient readings above 25 for all 8 weeks of the experiment.

A, Percentages of MSIs per group from the total placed (60); final stability was based on the 58 MSIs that remained in place (2 were removed) throughout the experiment; B, number of MSIs per dog (20 total) divided into categories and percentages per group.

Seventy percent of the MSIs placed in dog A (14 MSIs) showed implant stability quotient readings higher than 25 throughout the experiment ( Fig 5 , B ). Two MSIs (1 experimental, 1 control) in dog A failed. Both showed low implant stability quotient readings 4 weeks after placement and came loose during the fifth week while the Smartpeg was being unscrewed from the MSI head. Both had been placed in nonkeratinized tissue in the posterior region of the right mandible.

Four MSIs placed in dog A showed implant stability quotient readings below 25 and clinical signs of mobility. Two of those screws were placed in antimeric anterior regions of the maxilla between the lateral incisor and the canine (1 control, 1 experimental). Lower implant stability quotient readings were recorded as early as 1 week after placement. No implant stability quotient readings were obtained in the sixth and seventh weeks from 1 MSI because of mobility. The other 2 mobile MSIs were located in the posterior region of the mandible, in the furcation of the third molar (1 control, 1 experimental). Their implant stability quotient values dropped below 25 during the third and fourth weeks. Of the 14 MSIs that remained stable in dog A throughout the experiment, 2 that had been placed in nonkeratinized tissues in the maxillary first molar furcation area were buried because of excessive tissue overgrowth; both remained under the tissues for the rest of the experiment and showed excellent stability at the last week before the dog was euthanized.

There were no failures in dogs B and C. Two screws placed in dog B were removed because of root proximity. The remaining 18 MSIs (90%) in dog B had implant stability quotient readings above 25 throughout the experiment, as did the MSIs placed in dog C ( Fig 5 , B ).

The implant stability quotient values of the experimental and control MSIs were initially similar ( Table I ). Compared with their initial baseline values, the experimental MSIs showed a statistically significant ( P <0.05) loss of stability during the first week; after the first week, no implant stability quotient value differed significantly from baseline ( Fig 6 ). In contrast, all implant stability quotient values of the control MSIs were significantly less than the baseline values. The implant stability quotient values recorded at the end of the sixth week most closely approximated the baseline values. There were statistically significant differences in implant stability quotient values between the experimental and control MSIs after the fourth, fifth, sixth, and eighth weeks.

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