Bioactive–hybrid–zirconia implant surface for enhancing osseointegration: an in vivo study

Preparation of zirconia implants

CAD/CAM zirconia milling blocks (NobelBiocare, Göteborg, Sweden) were used for preparation of zirconia implants (cylinders 3.7 mm × 8 mm). The milled implants were sintered according to manufacturer recommendations (1350 °C for 6 h) [24]. To produce a nano-porous surface, all specimens were subjected to selective infiltration etching (SIE) technique to modify their surface topography through the creation of a nano-porous zirconia surface that extends few microns deep below the surface. Further details are mentioned elsewhere [31]. The prepared implants were divided into two groups (n = 12), according to the coating used to fill surface porosities, while uncoated surface served as a control.

Characterization of zirconia surface

Several surface characterization techniques were implemented to study the proposed hypothesis. Mercury porosimetry was performed for testing the surface nano-porosity and its relevant parameters including the total porosity percent and the average pore diameter in nanometers. Poresizer (Porosimeter, Micromeritics 9320, USA) was used for testing the nano-porosity created on the surface covering pore diameter range from approximately 0.006 to 360 μm. Atomic force microscopy (AFM) was used to confirm surface topography on the nano-scale. High-resolution X-ray diffraction analysis (XRD) for thin coats (PANalytical, X Pert PRO) with Cu target (λ = 1.54°A), 45 kV, 40 mA, and 2Ɵ (10°–80°) was performed to investigate the crystallographic structure of the implants. Zirconia implants were gold sputter-coated (fine coat, JEOL JFC-1100E, Japan) before scanning electron microscopic examination (JEOL, JSM, 5410, Japan) at an accelerating voltage of 25 kV. Energy dispersive X-ray analysis (EDX) was used (INCA Penta FETX3, OXFORD Instruments, Model 6583, England) to study the elemental composition of the specimens.

Preparation of HA coating material

Natural hydroxyapatite was extracted from femoral bones of line V Spain white rabbits (the animal models of the experimental study) through two stages: the first stage was the deproteinization process that was conducted to eliminate all organic and protein components of bone, followed by a heat treatment (calcination) process of the inorganic bone salts for elimination of all phases other than HA [32]. EDX and XRD analysis were accomplished to characterize the extracted natural HA for its chemical and crystal phase purity. The prepared powder was firstly ground to reduce its particle size to the sub-micron scale using ultrasonic vibration (ESPE.CAPMIX 410630, W-Germany) with a ceramic ball. Afterwards, high-energy ball milling (8000 M Mixer/Mill, SPEX Sample Prep, USA) was used for grinding of the produced micro-particles to reduce their size to the nano-scale (100 nm). The produced nano-particles were collected using high-speed centrifugal unit.

A hydroxyapatite suspension was prepared by weighing 0.2 g of the milled nano-particles using a calibrated digital balance (OHAUS, CT 1200-S, USA) and adding them to 20 ml absolute ethyl alcohol, followed by shaking the suspension in ultrasonic shaker for 5 min to achieve even distribution at room temperature. Immersion coating was performed by completely immersing the prepared implants in the suspension for 3 min, followed by heat drying at 150 °C for 10 min (Nuova II, Sybron/Thermolyne, Spain). This immersion cycle was repeated three times. Finally, all specimens were heated at 850 °C for 3 h to fuse the particles with the surface of zirconia.

Preparation of platelet-rich plasma coating material

This study was approved by the ethics committee of Science and Technology Development Fund (STDF-389, the academy of scientific research) regarding using animals in research studies. Autologous platelet-rich plasma (PRP) was freshly prepared from each animal independently just before insertion of zirconia implants into the rabbit femurs. Protocol of blood collection from rabbits was approved by the ethics committee of Alexandria University for studies involving animal models [33]. The collected blood was centrifuged at 4000 rpm for 8 min at room temperature. The centrifuged blood was separated into platelet-poor plasma (PPP), PRP (buffy coat), and the more dense red blood cells. The PRP-coating solution was prepared by collecting the buffy coat in another sterile graduated tube, and 0.5 ml of 10% w/v calcium chloride was added to each 0.1 ml of separated PRP [34]. Each zirconia implant was completely immersed into the prepared PRP solution for 10 min at room temperature immediately before its insertion into its surgically prepared socket that was completely filled with the prepared PRP solution.

Surgical phase

Twenty-four male line V Spain white rabbits were obtained from the Poultry Research Center, Faculty of Agriculture, Alexandria University (6 months old and 3 kg), in good health. The rabbits were kept at the animal house and provided soft diet enriched with vitamins (General Pharma Group, Egypt) and selenium (Mchandes Pharma Veterinary, Egypt) which were added to the drinking water with dose of 1 ml/l for about 2 weeks before surgery. All surgical procedures were performed under general anesthesia and aseptic conditions; the same surgeon completed all surgical procedures. Animals were randomly divided into two groups (n = 12), in each group; each animal received one hybrid–zirconia implant surface coated with either HA or PRP in the right femur head while the left side received uncoated zirconia implant used as a control.

Before surgery, surgical sites were shaved to expose the skin that was coated with antiseptic iodine-based solution. Rabbits were anesthetized with intramuscular injection of ketamine in combination with xylazine at a dose of 35 and 5 mg/kg of body weight respectively. Surgical flap was made and reflected to expose the distal head of the femur; then, sequential drilling of implant socket was carried out under sufficient cooling at room temperature and with an absolute minimum amount of trauma; and afterwards, the implant was inserted into its created socket. Finally, the surgical flap was repositioned and sutured. Postoperative intramuscular injection of broad-spectrum antibiotic (Cefotax 250 mg; Egyptian INT; Pharmaceutical Industrial Co., Egypt) and analgesic (Voltaren 75 mg/3 ml; Novartis Pharma S.A.E. Egypt) were administrated daily for 10 days to avoid any infection and to relief pain. Rabbits were monitored daily for weight gain and cage behavior. The wounds were allowed to heal for 6 weeks before sacrifice by injection of an over dose of intravenous anesthetic agent [35, 36].

Histomorphometric analysis

Six weeks after insertion of the implants, bone blocks were collected, subjected to fixation and dehydration, imbedded in transparent methyl methacrylate monomer, and finally sectioned in a precision cutting machine using a diamond-coated disc producing 150-μm-thick sections. Sections were polished using silicon carbide and stained using Stevenel’s Blue and van Gieson picro-fuchsin. Histomorphometric analysis and determination of bone-to-implant contact percentage (BIC%) was performed on the mid section of each implant using digital images obtained from a stereo stereomicroscope (Olympus imaging digital camera, model E.330 DC 7.4 V, Japan). The images were then analyzed using computer software program (Olypus. Cell ˆA). Mature bone stained red in contact with implant diameter was measure as a percentage of the entire implant diameter to calculate bone implant contact percent of each test group. Data were fed to a statistical software (SPSS 14.0, SPSS Inc). One-way analysis of variance (ANOVA) and Bonferroni post hoc tests were used to analyze the data based on power analysis test [37, 38].