Biological and biomechanical evaluation of interface reaction at conical screw-type implants

The implants used in this study were newly developed parabolic screw-type implants (ILI) with a length of 10 mm and a diameter of 4.1 mm at the shoulder of the implant (Fig 1). The implants were made of pure titanium with a characteristic progressive thread design. The threads as well as the curvature of the implant provided a homogeneous strain distribution over the whole implant surface under vertical loading conditions (Fig 2), as revealed by finite element analysis [20]. The implants possess a microstructured texture of 20 – 30 μm deep grooves, where as the titanium surface it is smooth on a nanoscale level. The implant system consists of two parabolic burrs of different diameters and morphologies. The burrs are used subsequently to prepare the bony implantation bed. The diameter of the second burr is slightly smaller then the core diameter of the implant. Implants have a transversal core/thread relation of 1:1.2. Implant insertion is performed by manual tapping of the self cutting implants into the surgically created bony implantation bed.

Eight male Göttinger minipigs, 14 to 16 months of age and with an average body weight of 35 kg were used in this study. A total of 40 implants were placed into the cranial and caudal part of the tibia condoyle (Fig 3). This study was approved by the Animal Ethics Committee of the University of Münster under the reference number G 38/2003.

All surgery was performed under sterile conditions in a veterinary operating theatre. The animals were sedated with an intramuscular injection of ketamine (10 mg/kg), atropine (0.06 ml/kg) and stresnil (0.03 ml/kg). In the areas exposed to surgery 4 ml of local anaesthesia (2% lidocaine with 12.5 μg/ml epinephrine, Xylocain/ Adrenalin^®, Astra, Wedel, Germany) was injected. The tibias were exposed by skin incisions and via fascial-periosteal flaps. Thereafter, the implants were placed in the cranial and caudal part of the tibia. The implant sites were sequentially enlarged with both drills according to the standard protocol of the manufacturer. Implants with 10 mm in length and 4,1 mm in diameter were inserted by using continuous external sterile saline irrigation to minimize bone damage caused by overheating. At the surgical site, the skin and the fascia-periosteum were closed in separate layers with single resorbable sutures (Vicryl^®4-0, Ethicon, Norderstedt, Germany). Perioperatively, an antibiotic was administered subcutaneously (benzylpenicillin/dihydrostreptomycin, Tardomycel^®, BayerVital, Leverkusen, Germany), 2.5 ml every 48 h for 7days. After placement, the shoulder of each implant was 1 mm below the ridge crest to allow circumferential bone growth. Resonace frequency measurements (RFM) (Osstell, Integration Diagnostics, Gothenburg, Sweden) were made for each implant at the time of fixture placement and after euthanasia [24‐27]. The animals were inspected after the first few postoperative days for signs of wound dehiscence or infection and weekly thereafter to assess general health. Healing periods of 7 days and 28 days were allowed for half of the implants respectively. After 7 days and 28 days animals were sacrificed (4 minipigs each) with an overdose of T61 given intravenously. Following euthanasia, tibia block specimens containing the implants and surrounding tissues were dissected from all of the animals. The block samples were sectioned by a saw to remove unnecessary portions of bone and soft tissue and were prepared for the various investigations:

The removal torque test was performed by applying a counter-clockwise rotation to the implant, around its axis at a rate of 0,1°/s according to the experimental set up of Li et al. 2002 [28]. For each implant the torque rotation curve was recorded. The removal torque was defined as the maximum torque (Nmm) on the curve. The interfacial stiffness was defined as the slope (Nm/degree of the torque-rotation curve) calculated from a linear regression analysis of the data between 0,5° and 3°.

This method, as a non-destructive technique, evaluates the implant stability in term of interfacial stiffness. Resonance frequency measurements were made on each implant at the time of fixture placement and after the time of sacrifice (7 and 28 days) in all animals by attaching a 4-mm long standard transducer (Osstell, Integration Diagnostics, Gothenburg, Sweden) to the implant. The excitation sign was given over a range of frequencies (typically 5 kHz to 15 kHz with a peak amplitude of 1 V) and the first flexural resonance was measured [24‐27]. The frequency responses of the system were measured for each implant.

Block samples containing the implants were first divided into 2 halves, and then each sample was further dissected with a blade to obtain a sample containing the implant embedded in the alveolar bone and the corresponding bone sample detached from the implant (28 days after implant placement). Samples containing the implant were used for scanning electron microscopy (SEM). For SEM, glutaraldehyde-fixed specimens were critical point-dried. Samples were sputtercoated with gold for histological analysis. Specimens were examined under a fieldemission scanning electron microscopy (LEO 1530 VP, Oberkochen, Germany).

The implants were removed together with the surrounding bone and fixed in Schaffer's solution (ethanol (96%), formaldehyde (37%), ratio: 2:1). The specimens were dehydrated in a graded series of ethanol. Thereafter, samples were embedded in methylmetacrylate (Technovit^®7200, Heraeus Kulzer, Dormagen, Germany). Utilizing the 'sawing and grinding' technique, longitudinal sections were grounded to about 43–50μm for conventional microscopy (Exakt Apparatebau, Norderstedt, Germany). Five samples stained by Alizarin S 1% and Brilliant-Kresyl-blue 0,1% were prepared for each implant site. Histology was analysed by light microscopy (Zeiss, Axioplan 2, Göttingen, Germany).

Filters of wavelengths of 510–560 nm (green filter), 450–490 nm (blue filter), 355–425 nm (violet filter) and 340–380 nm (UV filter) (Zeiss, Göttingen, Germany) were utilized. The bone-to-implant contact ratio was defined as the length of bone surface border in direct contact with the implant (× 100 (%)). NIH-Imge software was used for image processing and analysis (National Institutes of Health, Bethesda, MD, USA)

Mean values and standard deviations (SD) were calculated for RFM, removal torque testing, interfacial stiffness and bone-to-implant contact ratio. Multiple comparisons between all groups were performed using two-way analysis of variance and the t-test. Difference was considered significant when p < 0,05. All calculations were performed through the use of SPSS for Windows (SPSS Inc., Chicago, IL, USA).

All implants were anchored monocortically. At placement and during healing, the implants remained clinically immobile. The animals recovered well after surgery and no signs of infection were noted at any time during the observation period.

Over the healing periods tested, the mean maximum torque of implants was 390.00 ± 148.32 Nmm after 7 days and 300.00 ± 69.22 Nmm after 28 days (Table 1). Statistically significant differences in the removal torque values were not observed between day 7 and day 28 (p = 0.351). The implant stiffness (Table 2), as assessed by the linear regression analysis, was higher after 7 days (0.3992 ± 0.063) than after 28 days (0.2648 ± 0.0257), but the difference was statistically not significant (p = 0.086).

The RFM demonstrated no significant change in the resonance frequency responses during the 28 days the experimental period. Implants had a high primary stability as revealed by RFM (6029 ± 458 Hz and 6057 ± 423) directly after insertion. The implant stability remained at this baseline level through the experimental course (6257 ± 229 Hz at day 7 and 5885 ± 367 Hz at day 28) (Table 3 and 4).

Dissection of the implant-containing bone by a blade confirmed the clinical finding that the implants were well osseointegrated after 28 days. There was intimate bone contact over the whole length of the implant (Fig 4). Typically, endosteal bone covered the implant surface. Collagen fibres and osteoblasts made up the bulk of the adjacent tissue layer. The collagen fibres appeared to be predominantly oriented perpendicular to the implant surface in the bulk bony tissue (Fig 5). Cells, extracellular matrix proteins, and mineralized bone tissue were in direct contact with the implant. In contrast to the collagen fibres in the original bone, which were oriented perpendicular to the implant, newly synthesized collagen in the vicinity of the surface appeared to form a felt-like matrix parallel to the surface. Intimate bone contact was presented at the neck of implants. Typically, cells (osteoblasts) were firmly attached to the implant surface. Probe processing by sample fracturing for the electron microscopic investigations suggested that the bond between the implant and the adjacent bone layer seemed to mimic the bond in the bone tissue itself. On the implant surface, cells and extracellular matrix remained attached following separation from the enveloping bone.

Direct bone-to-implant contact could be achieved during the healing period. There were no signs of inflammation. Histological analysis of the bone/implant interface revealed an intimate contact between the titanium surface and the bony implantation bed. At the bone-titanium interface, a thin tissue layer stained with Alizarin S and Brilliant-Kresyl-blue was seen in some areas coming into direct contact with the titanium. The bony apposition revealed a laminar structure containing individual osteocytes and Haversian canals at the neck of implants (Fig 6). The laminar bone demonstrated also an intimate contact between spongiosal trabecula and the implant surface at the body of implants (Fig 7 and 8). Quantitative histomorphometric analysis revealed an enhanced bone-to-implant contact for every healing period. After 7 days the bone-to-implant contact ratio was 35.8 ± 7.2% and after 28 days the bone-to-implant contact ratio was 46.3 ± 17.7%, but the difference in the bone to implant contact did not reach a level of significance (table 5). A direct bone – implant contact was documented especially at the cortical bone area (neck of implants) after 7 and 28 days (Fig 6).