Early Bone Formation at a Femur Defect Using CGF and PRF Grafts in Adult Dogs

A Comparative Study

Abstract

Purpose: 

The purpose of this study was to compare the predictability of new bone formation using an autologous concentrated growth factor (CGF) graft alone and platelet graft alone.

Materials and Methods: 

Four bony defects of 8 mm were formed, and 3.7- × 10-mm implants were placed in the right femur. The platelet-rich fibrin (PRF), CGF, and synthetic bone were grafted to the bone defect area. Enzyme linked immunosorbent assay quantitative analysis and microscopic analysis of the fibrinogen structure were performed.

Results: 

At 4 weeks, the comparisons of each experimental group showed a significant difference between the CGF group and the synthetic bone graft group. When comparing the CGF and allograft material groups, the allograft group showed significantly more new bone formation. In the case of vascular endothelial growth factor, CGF had 1.5 times more than PRF. CGF showed a fibrinogen structure with a constant diameter.

Conclusion: 

When applied to a clinical case, CGF is predicted to show better results than PRF.

As modern medicine develops, human life expectancy has been prolonged resulting in an increase of geriatric population and the requirement for prosthetic restoration for tooth loss. Because many of geriatric patients want fixed partial prosthetic, implant restoration has been widely used for restoration. In patients who lost their teeth because of periodontal disease, most of the cases are not suitable for implant placement because of alveolar bone destruction.

Especially if implants are placed immediately after tooth extraction, the difference between the diameters of the implant and the tooth can result in the creation of defects around the implants. In addition, bone defects are caused by periimplantitis near the installed implants. In most cases, such periimplant bone defects require guided bone regeneration. In cases in which the autogenous bone was used as donor material, clinical outcomes were shown to be superior.^1,2

To perform a bone graft, a donor area is required, and the ramus and chin are suitable. Nonetheless, for other donor areas, surgical treatment might be required, potentially causing discomfort and complications.^3,4 Therefore, recent efforts have been made to solve these problems by using concentrated growth factors (CGFs) obtained from autologous blood that do not cause infection and hypersensitive reactions, have a simple preparation, and are less invasive.⁵

Platelets contain transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and many other growth factors. These growth factors have been reported to accelerate the wound-healing process and tissue regeneration by accelerating cell division and promoting the migration of inflammatory and stem cells.⁶ Two representative growth factors that have been tested are TGF-β and VEGF, which help stimulate the proliferation of new blood vessels, critical factors in the regeneration of bone tissues and differentiation of osteoblasts.⁷

Recently, many techniques to concentrate platelets have been developed, and numerous materials that contain various concentrations of platelets, growth factors, leukocytes, and fibrin structure have been introduced.⁸ Initially, such materials were referred to as platelet-rich plasma (PRP).^9–12 Nevertheless, given that the duration of the release of these growth factors is short, the absence of a lasting effect on bone formation and soft tissue healing was reported as a shortcoming.^13–15 Subsequently, Choukroun¹⁶ developed and used a modified form of PPR, platelet-rich fibrin (PRF). The advantages of PRF are that the duration of the growth factor release from PRF is longer than that from PPR, it shows good results in the bone formation and soft tissue healing, and it is a very simple method. Since then, PRF has been used widely in the dental field. In 2006, Sacco¹⁷ introduced CGFs, which contain more growth factors and a harder fibrin structure; CGFs have recently been used in the dental field.

CGFs can be prepared by special centrifuges. Because platelets are concentrated by varying the centrifuge speed, CGFs are known to contain harder and more growth factors than those of previous preparations.¹⁷ Therefore, CGFs have been reported recently to be more effective in bone formation or soft tissue healing.¹⁸

This study evaluated whether PRF and CGF could replace bone graft materials by examining early bone formation capacities of PRF and CGF at periimplant defects. In addition, as representatives of the growth factors contained in PRF and CGF, TGF-β and VEGF were analyzed quantitatively by enzyme linked immunosorbent assay (ELISA), and the difference between CGF and PRF was evaluated by electron microscopic examination.

Materials and Methods

Surgical Procedures

Six adult male dogs, weighing approximately 15 kg each, were used. General anesthesia of the adult dogs was performed by injecting a mixture of 1.5 mL Rompun (Bayer Schering Pharma, Berlin, Germany) and 1.5 mL Zoletil (Virbac, Carros, France). Local anesthesia was performed by injecting lidocaine containing 1:100,000 epinephrine (Yuhan, Seoul, Korea). Using #15 surgical scalpels, the upper skin incision was performed, blunt dissection of the muscles was performed, and the femur was exposed. Four bone defect areas were created on the exposed right femur using a trephine bur 8 mm in diameter (Gebr.Brasseler, Lemgo, Germany), and periimplant bone defect areas were formed by placing implants 3.7 mm in diameter and 10 mm in length (Dentis, Deagu, Korea) (Fig. 1A). After the formation of the bone defect areas, autologous blood was collected from the carotid of the adult dogs, the PRF and CGF were prepared by the centrifugal process. PRF was prepared using the MF 300 (Hanil Science Medical, Daejeon, Korea). CGF was prepared using the Medifuse (Silfradent Srl, Sofia, Italy). After completing the centrifugation, except in one area of the control group, PRF, CGF, or synthetic bone (Osteon II; Dentium, Suwon, Korea) was grafted separately onto the periimplant defect areas (Fig. 1B). Layer-to-layer suture was performed completely. After 2 weeks, implants were placed in the left femur by the same methods. After another 2 weeks, the adult dogs were killed, tissue sections were prepared, and new bone formation (NBF) and the bone-to-implant contact rate were measured by a light microscope.

Fig. 1: Experimental design. A, Control group. B, Experimental group. The graft materials were grafted into the periimplant defect.

Histomorphometric Evaluation

The specimen preparation procedure followed the usual method. The specimen was dyed using Villanueva osteochrome staining. The prepared specimens were observed under light microscopy, and images were captured with the MagnaFire digital camera system (Optronics, Goleta, CA). The region of interest was measured and analyzed for the NBF rate, and bone-to-implant contact (BIC) were measured at the upper 4 threads of fixtures using the Visus Image Analysis System (Image & Microscope Technology, Daejeon, Korea). The total amount of NBF area was calculated according to the percentage of the entire region between the threads. The BIC was calculated by the percentage of the bone contact to the implant.

Detection of TGF-β and VEGF

The PRF and CGF obtained from the 10 mL of venous blood collected during the surgical procedure were stored in small bowls. After extraction of approximately 2 mL of liquid from the formed PRF and CGF, each extract was carried into the tube separately. The levels of TGF-β1 and VEGF were analyzed quantitatively using ELISA kits (Quantikine ELISA; R&D Systems, Inc., Minneapolis, MN). The ELISAs were performed according to the manufacturer's instructions, and the quantitative analysis was conducted using a wavelength spectrophotometer at 450 nm. This procedure was repeated 2 times.

Histomorphometric Evaluation With Scanning Electron Microscopy

The PRF and CGR obtained during the surgical procedure were fixed in a 2% glutaraldehyde solution. The samples were set by dehydrating with ethanol, and the fibrinogen structure was analyzed by a scanning electron microscope (SEM) (S-4800; Hitachi, Tokyo, Japan). The magnification was ×8,000.

Statistical Analysis

In the 2- and 4-week groups, 1-way ANOVA was conducted to assess the difference in the rate of NBF for each graft material and in the BIC rate. For the quantitative comparison of the growth factors contained in the CGF and PRF, an independent t test was conducted. The analysis was performed with the SPSS 17.0 program (SPSS Inc., Chicago, IL).

Results

New Bone-Forming Area

In the histological analysis of the 2-week group, the average NBF was 11.17% in the control group, 38.00% in the CGF group, 19.75% in the PRF group, and 49.75% in the synthetic bone graft group (Fig. 1). In the 4-week group, the NBF was 11.33% in the control group (Fig. 2), 52.33% in the CGF group (Fig. 3), 21.00% in the PRF group (Fig. 4), and 69.00% in the synthetic bone graft group (Fig. 5) (Table 1). In the 2-week group, the comparison of the control and experimental groups revealed no significant differences in the NBF area. In the 4-week group, significant differences were detected between the 4 groups. NBF of the CFG graft group and the synthetic bone graft group were higher than that of the control group and the PRF graft group.

Fig. 2: Histopathologic findings of the control group at 4 weeks show little BIC around the implant (asterisks) and NBF in the defect area (A). B, Higher magnification of the upper part of the left side of A. The arrows indicate the areas of NBF (Villanueva osteochrome stain. A: ×12.5, B: ×40).

Fig. 3: Histopathologic findings of the CGF graft at 4 weeks show good BIC around the implant (asterisks) and NBF in the defect area (A). B, Higher magnification of the upper part of the left side of A. The arrows indicate the area of NBF (Villanueva osteochrome stain. A: ×12.5, B: ×40).

Fig. 4: Histopathologic findings of the PRF graft at 4 weeks show relatively good bone-implant contact (BIC) around the implant (asterisks) and NBF in the defect area (A). B, Higher magnification of the upper part of the left side of A. The arrows indicate the area of NBF (Villanueva osteochrome stain. A: ×12.5, B: ×40).

Fig. 5: Histopathologic findings of the alloplastic bone graft at 4 weeks show better BIC around the implant (asterisks) and new bone formation in the defect area (A). B, Higher magnification of the upper part of the left side of A. The arrows indicate the area of NBF (Villanueva osteochrome stain. A: ×12.5, B: ×40).

Table 1: NBFA at 2 and 4 Weeks After Implant Placement (mean ± SD%)

Bone-to-Implant Contact

In the 2-week groups, the BIC was 11.83% in the control group, 32.50% in the CGF group, 22.75% in the PRF group, and 55.50% in the synthetic bone graft group. In the 4-week groups, the BIC was 12.50% in the control group, 53.33% in the CGF group, 30.60% in the PRF group, and 69.33% in the synthetic bone graft group (Table 2). The comparison of the 2-week control and experimental groups revealed no significant differences for the BIC. In the 4-week group, significant differences were detected between the 4 groups. When the 4-week groups were compared, BIC of the synthetic bone graft group was higher than that of other groups.

Table 2: BIC at 2 and 4 Weeks After Implant Placement (mean ± SD%)

ELISA Quantitative Analysis

In the ELISAs performed for the quantitative analysis of TGF-β and VEGF, TGF-β was produced minimally and could not be detected in either CGF or PRF. In the CGF, an average of 69.2 ± 47.0 pg/mL VEGF was detected. In the PRF, an average of 38.3 ± 22.7 pg/mL VEGF was detected. These differences were not significant (Table 3).

Table 3: Quantity of Released VEGF (mean ± SD)

Electron Microscopic Examination of Structures

The dehydrated CGF and PRF samples were examined by electron microscopy. The CGF contained more thick fibrinogen fibers per area unit and regular fibrinogen structures (Fig. 6).

Fig. 6: SEM analysis of PRF (A) and CGF (B). The CGF shows a thicker and more regular pattern of fibrin than PRF (magnification: ×8000).

Discussion

Implants have become a useful treatment method for the restoration of the edentulous jaw. The total treatment period was shortened by placing the implants as early as possible after tooth extraction. After tooth extraction, at the time of implant placement, bone defects can develop between the implants and extraction sockets because of differences in the shape or diameter of the tooth and implants. In addition, bone defect areas develop frequently because of the progression of periimplant inflammation. In such bone defect cases, guided bone regeneration has been shown to be a useful method for treating the bone defect areas.¹ However, in guided bone regeneration, although autogenous bones have been known to show good results, they have shortcomings that create other problems in the donor area. The shortcoming of allogenic bone grafts or xenogenic bone grafts is their high cost. To overcome such deficiencies, recently, treatment methods that show good results by grafting platelets obtained from the blood of the patient have been introduced. Among them, PRF showed good results and had thus been used widely in the dental field until recently.^18,19 The study for the advancement of application of PRF was developed. Song et al²⁰ reported that silk fibroin can be used as a scaffold of PRF for rabbit calvarial defect repair. However, after the method of concentrating platelets as CGF was introduced, it began to be used as a new material to replace bone grafts. CGF was formed using a special centrifugal machine, which can change speed from 2400 to 2700 rpm and in the case of PRF, it was formed from 12 minutes of the centrifuge at 3000 rpm. Although formed PRF and CGF have the same appearance, it had been reported to contain more growth factors than PRF and a harder fibrinogen structure Additionally, good results with respect to bone defects have been reported for CGF.²¹ Kim et al²² reported that maxillary sinus augmentation using fibrin-rich block with CGF alone without bone materials is successful and predictable.

Of the current autologous bone graft materials, PRF has been used most widely in the dental field. When applied to periimplant bone defect areas, it has been used as a material to replace bone graft materials, with good effects being reported.^21–25 However, studies reporting the clinical use of CGF are not abundant. In 2009, Sohn et al²¹ reported that in maxillary sinus augmentation and alveolar bone grafts, CGF was more effective in regeneration of blood vessels or NBF than PRF. Rutkowski et al²⁶ also reported that good results were obtained with CGF in the dental field. In our study, similar to the previous studies, CGF showed a better NBF rate.

TGF-β and VEGF are known to be representative growth factors contained in CGF that exert effects on the NBF or accelerate wound healing. By stimulating cell division, matrix remodeling, and new blood vessel formation, these growth factors accelerate wound healing.^27–29 Consequently, good clinical results applying the CGF or PRF, which contain such growth factors abundantly, have been reported. In our study, CGF and PRF exudates were harvested, and quantitative analysis of TGF-β and VEGF was performed by ELISA. The TGF-β levels were very low and thus could not be detected by spectrophotometry. This result is consistent with those of other studies, which also reported that the amount of TGF-β was very small.^30,31 Although not significant because the standard deviation was large and the sample size was small, CGF contained approximately 1.5 times more VEGF than PRF.

CGF has a complex 3-dimensional structure identical to PRF, composed of fibrinogen-containing platelets, leukocytes, and abundant growth factors.^32,33 CGF and PRF have been reported to show better outcomes than PRP, which was used in the early period because they contain a particular structure of fibrinogen and thus release growth factors for a longer period than does PRP. CGF and PRF release growth factors even approximately 7 days after grafting.²¹ Examining the structure in detail, red blood cells and platelets are embedded in the fibrinogen mesh, which has been shown to be a real skeleton to maintain growth factors.^34,35 In our study, SEM revealed similar dense and concentrated fibrinogen structures. CGF had a thicker and denser arrangement of fibrinogen fibers per unit area than PRF. When CGF was applied, more NBF was observed compared with PRF, and the bone formation was better than in cases without grafting. However, the amount of bone formation was smaller than in cases for which synthetic bones were grafted in the bone defect areas. Thus, CGF could not completely replace bone graft materials.

A limitation of our study was the small number of samples. In addition, in the detection of the growth factors contained in the CGF and PRF, the thickness or density of the fibrinogen was not analyzed by the application of appropriate programs when reverse transcription polymerase chain reaction (RT-PCR) (not ELISA) or other immunoblotting techniques were used or when the SEM analysis was conducted. Therefore, more accurate studies on the differences in the growth factors and fibrinogen structure of CGF and PRF would require more samples and the application of immunoblotting techniques.

Conclusion

CGF showed a better NBF rate in periimplant bone defects than PRF. In the SEM examination, similarly, fibrinogen structures that were thicker per unit area and had a more regular pattern were shown for the CGF compared with the PRF. Although more NBF was observed histologically with CGF, it is difficult to consider the difference between growth factor contents of each as the main reason. Thus, more research is needed. Because the volume of bone formation for CGF was smaller than that of the synthetic bone graft material, CGF could not completely replace the bone graft materials; however, when small-volume or early bone formation is required, CGF could be considered as a new graft material.

Disclosure

The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.

Supported by research fund from Chosun University, 2016.