2.1.

Preparation of Implants: Deposition Conditions

For deposition of hydroxylapatite films on titanium substrates a special stainless-steel chamber was used, and all films were formed under the same deposition conditions.⁷ Before deposition, the commercially available cylindrical dental implants, 12 mm long and 3.3 mm in diameter (Figure 1), made from Ti6Al4V alloy (with a sand-blasted surface) were cleaned in acetone, toluene and in ethanol in an ultrasonic bath. A KrF excimer laser (Lambda Physic LPX 200, Goettingen, Germany, wavelength λ=248 nm and repetition rate 20 Hz) was used. Both, a HA target and an implant were placed into the stainless-steel deposition chamber. The laser beam was focused onto the sintered hydroxylapatite target at an angle of 45° and the target was rotated to keep the same ablation conditions. In the beginning the chamber was evacuated up to 10⁻⁴ mbar by a turbomolecular pump, and then was filled with an Ar–water mixture (Ar flow of 12 sccm, water vapor flow of 10 sccm). The substrate was preheated to 490°C using a CO ₂ laser; the deposition time was 15 min. The thickness of the hydroxylapatite layers created varied from 1.00 to 1.20 μm.

Figure 1

Photo of the implant used (commercially available, used as a substrate under the coating).

2.2.

Biological Evaluation

The experimental set for osseointegration consisted of 20 specimens, cylindrical dental implants from Ti6Al4V alloy with hydroxylapatite coatings, which were formed by the pulsed laser deposition technique, and of four noncoated titanium alloy implants with the same shape as a control group.

In this investigation four minipigs, 2 years old, weighing on average 40 kg, were used, and 10 weeks before implantation their lower premolars were extracted.

After the healing process the implants were inserted using the following procedure: after inducing general anesthesia, Azaperon and Metronidat (Le´cˇiva, Praha, Czech Republic), a local anesthesia, Mesocain, dose 4 mL per half jaw (Le´cˇiva, Praha, Czech Republic) was applied. The minipigs were secured on the preparation table in a prone position with their mouths open. The soft tissues were incised on the crest of the premolar area of the lower jaw and the bone was denuded. The implant bed preparation was started using a round drill, followed by a pilot drill and finished with a full size drill (drilling machine Elcomed 100, W&H Dentalwerk, Burmoos, Austria). During preparation sterile saline solution was used to protect the bone from overheating. Similarly, the socket was rinsed with sterile saline solution to clean out bone detritus after which it was allowed to fill up with blood. The implant was plugged into the bone and the soft tissues were sutured in layers with plain catgut. The implants in each minipig were arranged in the following way: three coated implants were inserted into the left side of the lower jaw and two into the right side. The control implant was plugged as a middle sample on each right side to provide sufficient bone support, comparable mastication force distribution, and to be shielded by neighboring implants. Each experimental animal had his own control. The implants were allowed to heal without loading for 16 weeks.

After the healing period, the implants were uncovered using general anesthesia and metal-ceramic crowns were inserted. The crowns were made from a chromium–cobalt alloy (Wiron, Bego, Germany) and a ceramic material, Vita Omega (VITA, Germany), following the manufacturer’s directions. Ketac-cem Aplicap (ESPE, Germany) was used for cementing. At this stage the fluorescent solution Calcein DCAF was injected intramuscularly in a dose of 20 mg/kg to determine the state of the bone after the unloaded healing period.

After 6 months of loaded osseointegration the experimental animals were sacrificed and the blocks of bone with implants were soaked in Schaffer’s solution (36 formaldehyde neutralized over CaCO ₃ +80 ethanol in a ratio 1:2–3) for fixing. This type of fixing solution does not leach a fluorescent label from tissues.⁸ The vertical position of the implants was checked radiographically (Trophy, Paris, France). The specimens were embedded into methylmethacrylate resin (Merck, Darmstadt, Germany), transversal microscopic sections were cut using a diamond saw blade, and then ground (both Buehler, Lake Bluff, IL) with water as a coolant to a thickness of approximately 100 μm. Five to seven sections were prepared from each implant, and the sections were examined under polarized and fluorescent light (Nikon Eclipse 600, Tokyo, Japan) with a charge coupled device (CCD) camera (Mitsubishi, Tokyo, Japan).

Computer software system (Sigma Scan and Sigma Scan Pro, Jandel, Erkrath, Germany) was used to analyze the osseointegration. Each section was viewed and the percent of contact length was calculated for each section and for the whole implant. The data obtained were compared with those of the control implant from the same animal. The significance of differences between the experimental and control groups was calculated by Student’s t test at probability P=0.05 .

3.1.

Mechanical and Physical

For the whole set of experiments similar x-ray diffraction (XRD) spectra of samples were observed (Figure 2), obviously due to the reproducibility of the deposition conditions.⁷ The spectra had peaks of HA, tetracalcium phosphate (TeCP)–Ca ₄ O(PO ₄ ) ₂ , tricalcium phosphate (TCP)–Ca ₃ O(PO ₄ ) ₂ and peaks of CaO and TiO ₂ . The actual preference of hydroxylapatite film orientations was controlled by the deposition conditions. The surface of the layers was very smooth (Figure 3), however, sometimes a few spherical smooth droplets (average diameter 0.002 mm) were always present. The formation of films was regular and was not influenced by the implant shape.

Figure 2

XRD of an implant surface coat used in experiments.

Figure 3

SEM of implant surface used in experiments (magnification 400×, bar=0.025 mm).

3.2.

Histological Evaluation under Polarized and Fluorescent Light

Evaluations after 16 weeks of unloaded osseointegration and 6 months of loaded osseointegration in the lower jaw have confirmed the presence of newly formed bone around all the implants. Osteoclasts, macrophages or inflammatory reaction cells including phagocytes as well as regressive changes were not observed in any of the ground sections.

In the experimental group, with a laser-deposited coating, the layer of fibrous connective tissue occurred in about 22.5 of the implant body surface without making a continuous layer. Figure 4 shows visible firm contact between the bone and the implant surface with interposition of fibrous tissue (shown by the arrow). The same view under fluorescent light (Figure 5) shows a uniform distribution of the fluorescent label in the whole bone, probably as a result of a remodeling process of early formed bone. The released label can be distributed in the whole bone volume. These findings support the assumption that bone healing was already finished and at the time of sacrifice no new bone was formed. These results were supported by the observations made at higher magnifications (Figures 6 and 7); the yellow spots mark active bone cells.

Figure 4

Firm contact of bone to the HA coated implant surface with a small interposition (shown by the arrow) of fibrous tissue. B—bone, I—implant (polarized light, magnification 40×, bar=0.25 mm).

Figure 5

Equitable distribution of a fluorescent label in bone around the HA coated implant. B—bone, I—implant (fluorescent light, magnification 40×, bar=0.25 mm).

Figure 6

Similar to Fig. 4. B—bone, I—implant (polarized light, magnification 200×, bar=0.05 mm).

Figure 7

Yellow spots marking active bone cells. B—bone, I—implant (fluorescent light, magnification 200×, bar=0.05 mm).

In the control group (titanium implant without a cover), the fibrous connective tissue between the implant and the newly formed bone occupied 34.8 (Figure 8), i.e., more than in the experimental group which was 22.5, especially in the middle portion of the implant. However, these differences were not significant (Student’s t test with probability P=0.05 ). The fluorescent label (Figure 9) was localized on the margin of the bone socket facing the implant and the adjacent periosteum (shown by arrows). This may suggest that at the time of sacrifice the bone had been still active.

Figure 8

Fibrous connective tissue between the control implant and the bone is more extensive. B—bone, I—implant (polarized light, magnification 40×, bar=0.25 mm).

Figure 9

The yellow label is localized in the bone margin between the periosteum and the implant (shown by arrows). B—bone, I—implant (fluorescent light, magnification 40×, bar=0.25 mm).

3.3.

Quantitative Analysis of Percentage of Osseointegration

To simplify calculation of the percent of osseointegration, each section was divided into individual sectors. The length of osseointegrated and fibrointegrated surfaces was measured in each of these sectors, summarized for the whole implant and then calculated for the groups of experimental and control implants. Table 1 presents the contact circumferential lengths of osseointegrated and fibrointegrated surfaces of the samples and the control implants for each sector of the section.

Table 1

The calculated area of the bone/implant interface varied from 65.2 (SE 13,5) for titanium implants to 77.5 (SE 10,2) for hydroxylapatite films. There was no significant difference (Student’s t test with probability P=0.05 ) between the type of surface and therefore we believe that the osseointegration of all HA films and of the control titanium implants was similar.