**2. Experimental study**

In order to optimize the biodegradation of the injectable CaP, three different formulations were deduced be tested experimentally to achieve an objective comparison. In this respect, a dog model was chosen due to the available bone volume. The tibia was the choice of defect creating since the oral cavity may pose significant infection and mastication forces risks. After the retrieval of an ethical approval, six beagle dogs were housed for the experiment, and the tibias were exposed after the general anesthesia. Three standard bone defects were created in the proximal tibia of animals, and the mentioned iCaP formulations were mixed and injected into the defects. One defect was left empty to serve as control. All animals were injected with fluorochrome labels in the first and last week of healing to discriminate the pattern of healing on the histology. The animals were sacrificed after 4 weeks, and bone biopsies were obtained from the defect sites. The samples were processed according to the non-decalcified histology protocol. The histologic slices were obtained and subjected to histomorphometric analysis for objective comparison.

#### **2.1. The injectable CaP cements**

The cements were founded as powder and liquid components in a capsule carrier and are readily available for mixing (**Figure 2**).

**Figure 2.** The injectable CaP was provided in a ready-to-mix tube. The powder and liquid form is separated by a plas‐ tic barrier. Prior to the placement to the shaker (i.e., amalgamator), the barrier plastic is removed thereby allowing the mixing of the components. The material is shaken for 30 s for proper viscosity.

After mixing, the iCaP cement can be injected to the defect and cured in a similar manner to others cement materials.

Tetracalcium phosphate (TTCP: Ca4(PO4)2O) and an hydrous di-calcium phosphate CaHPO4 are mixed 2:5 molar and obtained the Ca(H2PO4)2. H2O + Ca2NaK(PO4)2. The mixture is diluted with 0.5 ml distilled water. The powder was sterilized via 27 kGy gamma radiation. The hydrous part of cements was divided into two groups. In the group 1, the hemi-hydrate part excluded. In group 2, the hemi-hydrate part was included but the concentrations were 5.

The powder and liquid form was provided in an application tube ready to be inserted in to a mixer. The separator is removed, and the power and liquid forms are mixed for 20 s.

#### **2.2. Surgical procedures**

**Figure 1.** A dental implant placed into the alveolar crest should be surrounded by living bone tissue. Any lack of bone that is described as the dehiscence defects (intermittent lines) should be covered by a graft material with an osteogene‐

Many attempts have been undertaken to overcome the issues related to biodegradation of the iCaP. A series of powder and mixture settings was formulated and experimented on animal models to reveal the best configuration. The hydrous component allowed fine-tuning of the setting time and flowability characteristics, while the powder component was mainly related with the final hardness of the cement. Concomitantly, a porous character was obtained for gaining better flow and penetration by bodily cells and fluids. A delicate balance of the setting time, ease of injectability, and the final setting hardness was a constant challenge in the

In order to optimize the biodegradation of the injectable CaP, three different formulations were deduced be tested experimentally to achieve an objective comparison. In this respect, a dog model was chosen due to the available bone volume. The tibia was the choice of defect creating since the oral cavity may pose significant infection and mastication forces risks. After the retrieval of an ethical approval, six beagle dogs were housed for the experiment, and the tibias were exposed after the general anesthesia. Three standard bone defects were created in the proximal tibia of animals, and the mentioned iCaP formulations were mixed and injected into the defects. One defect was left empty to serve as control. All animals were injected with fluorochrome labels in the first and last week of healing to discriminate the pattern of healing

sis potential.

176 Dental Implantology and Biomaterial

**1.10. Biodegradation of injectable CaP**

**2. Experimental study**

development stage in many previous studies [17].

All surgical procedures were performed under general anesthesia. The left tibia of the dog was shaved and disinfected with povidone-iodine. During all surgical procedures, the animals were pre-anesthetized with Xylazine (Rompun/ Bayer, Germany) 1.5 mg/kg intramuscularly [i.m.] and anesthetized by Ketamine (Ketanest/ Alfasan, Holland) 10 mg/kg i.m. and main‐ tained by isoflurane 3.5% (volume/volume) (Forane, Abbott Laboratories, Rungis Cedex, France). They were administered through an endotracheal tube. Ten, the bone surface in the proximal tibia region was exposed by an incision followed by skin and periosteal elevation.

Three recipient sites were prepared, using a drill of 3.8 mm in diameter to a depth of 13 mm to obtain standard defect sites. A minimum of 1.5 mm was sustained between defects to provide adequate healing conditions for the defects.

**Figure 3.** Two standard bone defects (a diameter and depth of 3.8 and 13 mm respectively) were prepared for the injec‐ tion of groups 1 and 2 (middle and the right defect). One defect (on left side) was left empty to serve as control.

After achieving bleeding control, iCaP cement capsule placed in amalgamator for 20 s, after that iCaP cement injected in all defect except one defect for each animal left empty (**Figure 3**).

After the removal of the residual cement, 10 min time was exceeded for the setting of the material. Then, the periost and dermis sutured with vicryl 3–0 suture material (Ethicon, Polyglactin 910, Chicago, A.B.D). In order to monitor patterns of bone formation, hydrochloric tetracycline (at the 4th week (Tetra, Mustafa Nevzat İlaç Sanayı, Istanbul, Turkey) and Alizarin complexone (Sigma-Aldrich, Bonn, Germany); (at the 11th week) were injected intravenously (I.V.). The animals were fed with standard diet throughout the 12-week recovery period. At the end of the 12 weeks, the animals were sacrificed by an overdose of sodium pentobarbital (Abbot Lab. Chicago A.B.D).

#### **2.3. Histologic preparation and analysis**

The block biopsies taken from the proximal tibias, stored in phosphate-buffered solution and maintained at 4°C. The blocks were dehydrated with a series of alcohols and put into a transparent flask filled with methyl methacrylate. This was essential for the non-decalcified histologic sections. Following the polymerization, the blocks were removed from the flask and using the Donath technique, 395 μm non-decalcified sections were obtained. A total of three sections were taken from each defect. The first two sections stained with methylene blue and basic fuchsin, and the third section with the thickness of 390 μm was taken without staining for fluorescence analysis. For the analysis, the sections were magnified under ×100 light magnification of a stereo microscope. The new bone and residual area was calculated manually using dedicated software (Olympus Image Analyzer, Tanaka, Japan). The percentage of new bone and the residual graft area was calculated.

#### **2.4. Statistical analysis**

The normality of the results was controlled by the Shapiro–Wilk Normality test. Student t-test was used for the statistical analysis. All analyses were performed by the GraphPad Prism software (San Diego, California, USA).
