**3. Results**

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

**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

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

provide adequate healing conditions for the defects.

178 Dental Implantology and Biomaterial

(Abbot Lab. Chicago A.B.D).

**2.3. Histologic preparation and analysis**

The injection and the manipulation of the CaP were easy, and the material was set in almost 15 min. However, this period of time can be considered long for the clinical work. Also, in the presence of bleeding, the setting of the material was compromised and particles washed away by the blood. After the self-hardening, the manipulation of the soft tissues for suturing was rather easy.

Throughout the healing period (up to12 weeks), no local or general problems or complications were observed in the animals. The surgical area healed quickly in all dogs without any signs of a developed pathology. Radiographic examination showed no pathological changes at the surgical zone.

#### **3.1. Light microscopic examination**

All the histological cross-section views of the CaP graft material have showed bone integration. A dense trabecular bone structure was also noticed around the grafted area. A thin cortical bone layer surrounded a trabecular woven bone. In this layer, the particles of the CaP cement were in the center of the defect and substituted by a thick layer of trabecular bone tissue. Almost in all the histological sections, the coronal aspect of the injectable CaP graft was surrounded by a layer of cortical bone. At high magnification, the Howship lacunae (osteoclast resorption lacunae) were seen at the zone adjacent to the graft material. A continuous layer of osteoid deposition followed biodegradation of the graft material. The outer part of the graft core was replaced with new bone, but the core part (inner part) of the cement was not biodegraded. In contrary to the outer part (the sections in contact with the native bone), the center section was still intact. The graft material was strictly placed into the defect which was in a complete contact with the surrounding bone tissue, and no fibrous tissue or inflammatory reaction was observed in any of the histological sections. At the early stage of healing active angiogenesis and osteogenesis have been shown, and no inflammatory symptoms were seen. (**Figures 4** and **5**)

**Figure 4.** Group 1. Four weeks healing. Basic fuchsin and toluidine staining. Original magnification ×10. The un-resor‐ bed bulk material can be seen in the middle of the defect. Osteoid deposition is evident in the borders of the material. No inflammatory evidence is visible.

**Figure 5.** Group 2. Four weeks healing. Basic fuchsin and toluidine staining. Original magnification ×10. The apical portion of the material has been replaced by living bone tissue characterized by large trabecular zones. The material is not biodegraded completely as marked by the dark area. However, the iCaP shows excellent integration with the host bone.

At 12th week, the process of new bone apposition and mineralization was still visible. Moreover, the graft found at the base of the defect and next to the cortical bone has been degraded by osteoclast-like cells. At higher magnification, both dense trabecular area and unlimited osteoblast like cells, primary, and secondary osteons were found in the space between the vessels at the regenerated bone. In this time frame, it was evident that the formation of osteoid tissue began growing from the defect walls and continues toward the center (**Figures 6** and **7**).

**Figure 4.** Group 1. Four weeks healing. Basic fuchsin and toluidine staining. Original magnification ×10. The un-resor‐ bed bulk material can be seen in the middle of the defect. Osteoid deposition is evident in the borders of the material.

**Figure 5.** Group 2. Four weeks healing. Basic fuchsin and toluidine staining. Original magnification ×10. The apical portion of the material has been replaced by living bone tissue characterized by large trabecular zones. The material is not biodegraded completely as marked by the dark area. However, the iCaP shows excellent integration with the host

No inflammatory evidence is visible.

180 Dental Implantology and Biomaterial

bone.

**Figure 6.** Group 1. Twelve weeks healing. Basic fuchsin and toluidine staining. Original magnification, ×100. The floor of the defect is focused. High magnification in the apical portion reveals direct contact the living bone tissue with vital cells and ongoing ossification. Osteoid deposition is characterized by osteocytes (small dots) lines at the iCaP border. The iCaP material is being penetrated by small indentations (Howship lacuna) derived from the host bone tissue.

**Figure 7.** Group 2. Twelve weeks healing. Basic fuchsin and toluidine staining. Original magnification, ×100. Coronal part of the defect is focused. In the coronal aspect, the iCaP material is covered with new bone tissue. Despite the frag‐ ments of small ossification points, the iCaP material has not biodegraded (white bulk in the lower aspect of the image).

In the control defect, the cervical part of the defect was filled with soft tissue. On the contrary, the apical part was filled with mature bone. Low-mineralized primary osteoid was also seen especially at the borders of the defect in both the control and test groups.

#### **3.2. Fluorescence microscopic examination**

The use of fluorochrome labels allowed observation of the bone growth and the position of the new bone that occupied the grafted space in relation with the time frame. The tetracycline HCL applied in the 4th week stains the mineralization by green color (Native bone in dark green and new bone in light green). The alizarin complexone applied in the 11th week stains the mineralized tissues in orange color (Native bone dark orange/red/brown and new bone in light orange). Accordingly, tetracycline HCL stain was seen at borders of the defect at the 4th week sections. This indicates that the formation of this bone started shortly after CaP place‐ ment. The histological cross-section of the CaP-grafted area at the 12th week showed a continuous formation of bone and remodeling. It was evident that a bone turnover was ongoing throughout both the intervals. The rate of new bone formation was within normal physiology limits. The formation of the bone initiated from the borders of the defect towards the center (**Figures 8** and **9**).

**Figure 8.** Fluorescence microscopy view of the 4 weeks healed defect in group 1. Light green staining reveals new bone formation. Original magnification, ×200. Coronal side of the defect is focused. Light green areas depicting ongoing os‐ teogenesis around the iCaP area (right bottom corner). It is obvious that the osteogenic activity started soon after the placement of the iCaP.

**Figure 9.** Fluorescence microscopy view of the 12 weeks healed defect in group 2. Light green and orange staining re‐ veals new bone formation. Original magnification, ×150. Apical side of the defect is focused. Orange circles and lines reveal bone active remodelation and formation in the 11th week. The iCaP is being replaced by trabeculas of new bone. The bone formation that is evident on the 4th week (light green staining) is being deposited by osteocyte lamellas (or‐ ange color) in the 11th week.

#### **3.3. Histomorphometric analysis**

In the control defect, the cervical part of the defect was filled with soft tissue. On the contrary, the apical part was filled with mature bone. Low-mineralized primary osteoid was also seen

The use of fluorochrome labels allowed observation of the bone growth and the position of the new bone that occupied the grafted space in relation with the time frame. The tetracycline HCL applied in the 4th week stains the mineralization by green color (Native bone in dark green and new bone in light green). The alizarin complexone applied in the 11th week stains the mineralized tissues in orange color (Native bone dark orange/red/brown and new bone in light orange). Accordingly, tetracycline HCL stain was seen at borders of the defect at the 4th week sections. This indicates that the formation of this bone started shortly after CaP place‐ ment. The histological cross-section of the CaP-grafted area at the 12th week showed a continuous formation of bone and remodeling. It was evident that a bone turnover was ongoing throughout both the intervals. The rate of new bone formation was within normal physiology limits. The formation of the bone initiated from the borders of the defect towards

**Figure 8.** Fluorescence microscopy view of the 4 weeks healed defect in group 1. Light green staining reveals new bone formation. Original magnification, ×200. Coronal side of the defect is focused. Light green areas depicting ongoing os‐ teogenesis around the iCaP area (right bottom corner). It is obvious that the osteogenic activity started soon after the

especially at the borders of the defect in both the control and test groups.

**3.2. Fluorescence microscopic examination**

182 Dental Implantology and Biomaterial

the center (**Figures 8** and **9**).

placement of the iCaP.

Mean new bone formation was 22.12 (SD, 15.68), 18.62 (SD, 13.11), and 9.56 (SD, 11.11)% in the groups 1, 2, and the control, respectively. Statistically, significant higher new bone formation was evident in the groups 1 and 2 as compared to the control group (p < 0.01). However, these differences were no more discernable after 12 weeks of healing (**Figure 8**).

The rate of residual iCaP was similar in both time intervals. In the 4 weeks healing group, almost half of the iCaP was still present in both groups and there were no statistically signif‐ icant differences between the groups 1 and 2. After 12 weeks, the biodegradation was evident. However, complete biodegradation of the iCaP was not evident in any groups 45.44 (SD, 22.16) and 41.20 (21.20)% for groups 1 and 2, respectively; (**Figure 9**).

Cellular evaluation of the histologic slices in the groups yielded no inflammatory response or foreign body reaction. Histomorphometry taken together with the fluorescence sections reveals that new bone formation initiated right after the surgery at the iCaP and host bone border. In both groups, the staining in the 4th and 11th weeks was similar. It is obvious that the cells involved in bone turnover infiltrate the iCaP body by small indentations and the biodegradation was sparsely conducted in the roots of these indentations. The bulk and nonporous nature of iCaP, unfortunately, inhibited the infiltration of cells responsible in the biodegradation. This was characterized by orange stains in the 11th week, and there were not visible differences between the groups (**Table 3**).


**Table 3.** Evaluation of biodegradation in groups 1 and 2 as compared to the control group.

### **4. Discussion**

The restoration of the lost bone volume would be one of the most concerned topics in the future of dentistry. Given the prevalence of edentulism and tooth loss, more people are likely to apply for a fixed prosthetic restoration approach. Dental implant is an excellent base for such purposes. Unfortunately, the bone volume rapidly decreases due to the atrophy in lack of the functional stimulus [16].

In the restoration of an alveolar bone defect, the autogenous bone transfers are regarded as the golden standard. However, they are difficult manage and prone to many complications in the short and long term. Hence, extensive surface resorption, especially in the iliac grafts, questions their effective use. Nevertheless, the contents of living bone cells are critical for rapid healing [3]. The iCaP lacks any living cells and does not incorporate relevant growth factors or boneinducing elements. Therefore, its application and efficacy fundamentally depend on the vascularization of the native bone [18]. Moreover, the open nature of the iCaP allows integra‐ tion of any desired elements, cells, or components that may be tailored according to the sitespecific needs. The results of the present investigation are promising, and the iCaP seems suitable for further development for efficient and inexpensive bone regeneration purposes (**Figures 10** and **11**).

**Figure 10.** New bone formation measured in the time intervals.

biodegradation was sparsely conducted in the roots of these indentations. The bulk and nonporous nature of iCaP, unfortunately, inhibited the infiltration of cells responsible in the biodegradation. This was characterized by orange stains in the 11th week, and there were not

**Group 1 Group 2 Control**

Osteoid deposition and new bone formation visible in all histologic

New bone formation and lamellar formation is evident in all sections. A thin layer of new bone is bridging the top of the defect. iCaP was not biodegraded and left intact, especially in the center of the defect

Osteoid deposition and new bone formation visible at bottom side of the defect. The coronal aspect of the defect is

lacking bridging

group 1 and 2

The bone defect is being filled with trabecular bone. The amount of new bone formation is less than the

sections. No significant biodegradation is visible

The restoration of the lost bone volume would be one of the most concerned topics in the future of dentistry. Given the prevalence of edentulism and tooth loss, more people are likely to apply for a fixed prosthetic restoration approach. Dental implant is an excellent base for such purposes. Unfortunately, the bone volume rapidly decreases due to the atrophy in lack of the

In the restoration of an alveolar bone defect, the autogenous bone transfers are regarded as the golden standard. However, they are difficult manage and prone to many complications in the short and long term. Hence, extensive surface resorption, especially in the iliac grafts, questions their effective use. Nevertheless, the contents of living bone cells are critical for rapid healing [3]. The iCaP lacks any living cells and does not incorporate relevant growth factors or boneinducing elements. Therefore, its application and efficacy fundamentally depend on the vascularization of the native bone [18]. Moreover, the open nature of the iCaP allows integra‐ tion of any desired elements, cells, or components that may be tailored according to the sitespecific needs. The results of the present investigation are promising, and the iCaP seems suitable for further development for efficient and inexpensive bone regeneration purposes

visible differences between the groups (**Table 3**).

Osteoid deposition and new bone formation visible in all histologic sections. Major mass of the iCaP is

all sections. A thin layer of new bone tissue is bridging over the iCaP. No sections reveal complete biodegradation of the iCaP

**Table 3.** Evaluation of biodegradation in groups 1 and 2 as compared to the control group.

4th week (tetracycline staining)

visible

184 Dental Implantology and Biomaterial

**4. Discussion**

functional stimulus [16].

(**Figures 10** and **11**).

11th week New bone formation is evident in

**Figure 11.** Residual graft area measured in the time intervals.

After 4 weeks of healing, a considerable amount of new bone was evident in both groups as compared to the control defect. Nevertheless, there were no statistically significant differences in between these two groups. This may render that the inclusion of hemi-hydrate groups did not yield any positive influence in terms of new bone formation.

After 12 weeks of healing, the level of new bone was above 50% in all the defects and there were no statistically significant differences in the groups or the control defect. This may point the long healing period or the insufficient dimensions of the graft. Even so, there is no consensus on the critical size defects of the tibia in terms of a dental implant osteotomy.

The residual graft was also investigated to corroborate the results of new bone formation. In the 4 weeks, more than 60% of the injectable CaP material was still present in the defects. After 12 weeks, the rate of residual CaP decreased below 40% in group 2. The difference was statistically significant in none of the time intervals. Histologic section yielded a core material was left non-resorbed right at the center of the defect. Ooms et al. [19] in a study on dog, placed an injectable porous form CaP graft material into the standardized defects in combination with an experimental titanium implant and found that this material was highly ossified in the whole area, especially in infection cases or in case of few walled bony defects, and also he found out that the material outcome might be high if vascularization is supported. It may be proposed that further porosity is required around the iCaP to allow vascularization and body-fluid penetration.

Many different attempts and experiments have been performed in a purpose to increase the biodegradation of CaP cement. It is thought that increasing the solubility of CaP inside the bone results in accelerating the period of biodegradation. Solubility increases in direct proportion to the surface area [20]. The body fluids and their contents such as phagocytic cells expedite the resorption period of the graft when the contact area between the fluids and the graft material increases. As a result, the hardened graft material porosity is expanded so to rise the infiltration of fluids and blood between the graft. Daculsi et al. [21] produced a porous CaP cement with interporous distance ranges between 100–500 micrometer to introduce a good environment for cell growth.

In the present experimental model, new bone regeneration was ascertained without any complications that might occur thereby of using a barrier membrane such as exposure of the soft tissue and infection. This may be attributed to site of the tibia as the area of the defect. Nonetheless, applying the graft into the oral bone might also have good prognosis with no complications as it was observed in the tibia. Some researchers reported that the use of membrane may be unnecessary as the material acts as a space maintainer. In this study, the four-walled bony defect was involved so that no barrier membrane was used. Hence, new bone formation was occurred in both groups.
