The study consisted of two separate sub-studies, both designed as paired animal studies. A total of ten skeletally mature Labrador dogs were used. Mean body weight was 34 kg [range 25 kg: 39 kg]. Mean age was 18 months [range 14-23 months]. Each dog had two implants inserted medially in the proximal part of the tibia. Each dog had four implants, so a total of 40 implants were used. The implants were inserted press-fit in cortico-cancellous bone. In the upper tibia site, we investigated the effect of surface etching on grit blasted implants. At the more distal tibia site we investigated the effect of combining surface etching and plasma sterilization on grit blasted implants. Due to denser bone quality, the upper tibia surgical site offered a better biomechanical implant fixation compared to the lower site (p=0.0098). Hence, no cross comparisons between the two sub-studies was performed. The observation time was four weeks (Fig. 1).

Fig. (1). Schematic drawing of the placement of the implant pairs in the right and left tibia.

Under general anesthesia, using sterile technique, the proximal part of tibia was exposed through an anteromedial extraarticular approach. Two Kirschner (K) wires were inserted perpendicular to the surface with 15.0 mm in between. The most proximal K wire was inserted with the implant centre 15 mm from the tibia plateau. The K-wires guided the 5.5 mm cannulated drill creating 11.0-mm-deep holes. Drilling was performed at two rotations per second to prevent thermal trauma to the bone. All bone debris and soft tissue was removed from the drill hole before the implant was inserted (press fit). Finally, the soft tissue was closed in layers. Pre-and postoperatively, the dogs were given one dose of Cefuroxim, 1.5 g intravenously, as antibiotic prophylaxis. A fentanyl transdermal patch (75 μg/h) lasting three days was given as postoperative analgesic treatment. The dogs were allowed full weight bearing postoperatively. The dogs were bred for scientific purposes, and the study was approved and monitored by the Danish Animal Research Committee (Fig. 2).

Fig. (2). Peri-operative picture showing the two implants inserted medially in the proximal part of the right tibia.

The aluminum oxide (Al₂O₃) grit blasted implants (L = 10 mm, Ø = 6 mm) were made out of Ti-6Al-4V alloy (Depuy Inc., Warsaw, IN, USA). The implants had a central threaded hole which in combination with a threaded rod made it possible to handle and insert the implants without touching and thereby potentially polluting the implant surface. The implants were prepared as follows.

Acid etching: Acid etching was done at room temperature in an acidified NaF solution for six minutes. The implants were then rinsed with a 1% Alconox (Alconox, Inc., White Plains, NY, USA) and 2% Liquinox (Alconox, Inc., White Plains, NY, USA) detergent at 45ºC for 30 minutes. The implants were then rinsed three times in 15 minute cycles with RO-H₂O at 45ºC. Finally the implants were dried at 60ºC.

Plasma sterilization: The plasma sterilization with pure O₂ gas was performed in a 500 Watt plasma chamber (7200 RF Plasma Processing System; PVA TePla America, Inc.) with a gas flow rate of 250 standard cubic centimetres per minute (sccm) and a chamber pressure of 300 mTorr (40 x 10^-5 bar). The cycle time was 30 minutes.

Passivation/gamma sterilization: After standard cleaning in the lab passivation of the implants was carried out using the validated (ASTM A967-05) manufacturing passivation process. Parts were passivated using 35% Nitric Acid at 60°C. The cleaning process involved a series of temperature controlled water baths. The samples were dried and packaged by trained employees in the clean room. Parts were then Gamma Sterilized using the same processing plant used for standard DePuy (Depuy Inc., Warsaw, IN, USA) Implants. All of these processes are validated manufacturing processes used for actual DePuy (Depuy Inc., Warsaw, IN, USA) Orthopaedic Implants.

The implants were packaged individually in glass vials with screw lids. The glass vials were packaged in two layers of sterile packaging and one layer of unsterile packaging (a cardboard box).

The implants were attached to the lid with a threaded rod screwed into the hole in the implant. This way the implants did not touch the inside of the glass vial and could be handled and inserted using the rod, without risk of polluting it with endotoxins from sterile gloves or instruments (Fig. 3).

Fig. (3). SEM pictures of the implant surfaces: In picture d, crystals of aluminum oxide (Al2O3 particles) from the grit blasting are clearly visible. Pictures e and f show the pits created by the etching process and the pictures indicate that the majority of the particles has been removed by the etching process.

After four weeks of observation the animals were sacrificed with an overdose of hyper saturated barbiturate and the proximal part of tibia was excised and cleaned and thereafter stored at –20°C. The outermost 0.5 mm of the implant-bone specimen interphase was cut off and discarded. The rest of the implant with surrounding bone was divided into two sections perpendicular to the long axis of the implant with a water-cooled diamond band saw (Exact Apparatebau, Nordenstedt, Germany). The outermost section was cut to a thickness of 3.5 mm and stored at –20°C pending mechanical testing [19]. The innermost part 6.0 mm was prepared for histomorphometry. The specimens were dehydrated in graded ethanol (70-100%) containing basic fucsin, and embedded in methylmetacrylate (Technovit 7200 VCL; Exact Apparatbau, Nordenstedt, Germany). Four vertical, uniform, random sections were cut with a hard-tissue microtome (KDG-95; MeProTech, Heerhugowaard, The Netherlands) around the center part of each implant as described by Overgaard et al. [20]. Before making the sections, each cylindrical implant was rotated randomly around its longitudinal axis. The sections were cut parallel to this axis. The 40-μm-thick sections were counterstained with 2% light green (BDH Laboratory Supplies, Poole, UK) and then mounted on glass [21]. This preparation provides red staining of non-calcified tissue and green staining of calcified tissues such as woven and lamellar bone.

Blinded histomorphometric analysis was done using a stereological software program (C.A.S.T.-grid Olympus Denmark A/S, Ballerup, Denmark). Fields of vision from a light microscope were captured on a computer monitor and a user-specified grid was superimposed on the microscopic fields. Four vertical sections representative of each implant were analyzed and cumulated [20].

With the aid of the software, we defined two zones in the sections. Zone 1 stretches from the implant surface and 500 μm outwards into the surrounding tissue, and Zone 2 was defined starting 500 μm from the same implant outer surface line and extending another 500 μm outwards into the surrounding tissue.

In both zone 1 and 2 tissue volume fractions were quantified by point counting [22]. On the implant surface, line intercept-technique with sine-weighted lines was used to estimate the bone-implant contact [23].

Intra-observer variation was determined as coefficients of variation on double measurements on four randomly selected implants. All the coefficients were well within the expected range; however, defining small amounts of new bone on the implant surface and discriminating between new and lamellar bone close to the implant proved troublesome (new bone in zone 1 [24%], lamellar bone on the surface [52%]) (Fig. 4).

Fig. (4). (A) Picture showing a histological cross section used for Histomorphometry. The close-up picture (B) shows Lamellar Bone (L) and New Bone (N) in contact with the implant surface. Marrow is marked (M).

The thawed implants were tested for failure by axial push-out test on a MTS Bionics Test Machine (MTS, Eden Prairie, MN, USA). The specimens were placed on a metal support jig with a 7.4 mm diameter central opening. The implant was centralized over the opening, assuring a 0.7 mm distance between the implant and the support jig. The direction of loading was from the cortical surface inward. A preload of 2 N was applied to standardize contact conditions before initiating loading. We used a displacement rate of 5 mm/min with a 2,5 kN load cell. Load and deformation were registered by a personal computer. Each specimen length and diameter was measured with a micrometer screw gauge and used to normalize push-out parameters [24]. Ultimate shear strength (MPa) was determined from the maximal force applied until failure of the bone-implant interface. Apparent stiffness (MPa/mm) was obtained from the slope of the linear section of the curve. Energy absorption (kJ/m²) was calculated from the area beneath the curve until failure.

Histomorphometrical as well as mechanical data followed a normal distribution and a paired t-test was used to distinguish between groups. The data were presented as means with standard deviation (SD). Differences between means were considered statistically significant for p-values less than 0.05.

All dogs were fully weight bearing within three days of surgery. No postoperative complications were seen, and no dogs were excluded during the observation period. No signs of infection were seen during the harvesting of the specimens.

There were no statistically significant differences between Grit Blasted and Grit Blasted+Acid Etched (sub-study 1), or Grit Blasted and Grit Blasted+Acid Etched+Plasma Sterilized (sub-study 2) in regards to any of the tissues compared in any of the zones measured.

All implants were well osseointegrated, with almost 50% of the implant surface in direct bone contact in sub-study 1 (upper site) and almost 40% implant to bone contact in sub-study 2 (lower site). Furthermore, there was no fibrous tissue in the bone-implant interface in sub-study 1, and only 5% in sub-study 2.

By gross observation, all implants were well anchored in the bone. The mechanical testing confirmed this observation.

The implants in sub study 1, which were placed more proximally in the tibia were significantly better anchored in all three mechanical parameters (strength, energy and stiffness) than the implants in sub study 2 at the more distal tibial location. No statistically significant mechanical differences were found within the two individual sub studies.