Effects of Autogenous Tooth Bone Graft and Platelet-Rich Fibrin in Peri-Implant Defects: An Experimental Study in an Animal Model

Implants are widely used in dentistry to restore missing teeth, but adequate thickness and height of bone are required for optimal implant therapy. However, alveolar bone thickness decreases after tooth extraction.^1,2  As much of the alveolar bone loss occurs in the first year after tooth extraction,²  the immediate implant technique has been adopted to prevent bone loss. Although immediate implant placement decreases treatment time, preserves alveolar bone, and improves esthetics,^3–5  buccal bone can resorb after immediate implant placement, and bony defects can form around the implants.^1,6–8 

In past decades, various grafting materials have been used to treat peri-implant defects. Autogenous bone graft has been accepted as the ideal material because of its potential osteoinductive, osteoconductive, and osteogenic properties. Additionally, it stimulates healing and is not rejected by the immune system. However, autogenous bone graft has disadvantages, including bone resorption problems, limited donor sites, and an additional wound site.^9,10  Therefore, allografts, xenografts, and synthetic grafts have been used as alternative graft materials.^11–13  As none of these graft materials has the desired osteoinductive properties, an alternative bone substitute has been sought.

Dentin, the main tooth structure, contains type I collagen, which promotes new bone regeneration.¹⁴  The chemical composition of bone is quite similar to that of dentin, consisting of approximately 70% hydroxyapatite, 20% collagen, and 10% body fluid.¹⁵  The dentin matrix also contains noncollagenous proteins, and these proteins include various growth factors that stimulate osteoinductive activity.^16,17  Furthermore, like dentin, cementum also contains growth factors.^18,19  Recently, tooth grafting materials that include dentin and cementum have been used to take advantage of these organic and inorganic components and growth factors.^20–22 

In dentistry, there has been increased use of biologic and synthetic molecules, various mediator cells, and platelet concentrates derived from blood to enhance periodontal regeneration and bone formation.^23,24  Platelet-rich fibrin (PRF), an autogenous platelet concentrate developed by Choukroun et al,²⁵  is a fibrin network involving platelets, growth factors, leukocytes, cytokines, and stem cells.^26,27  Additionally, PRF provides mechanic fixation by binding with the graft particles.²⁸  Autogenous PRF, alone or combined with different biomaterials, has been used to treat periodontal, bony, and peri-implant defects.^12,29,30  The purpose of this study was to test the hypothesis that autogenous tooth bone graft (ATBG) combined with PRF can improve implant-to-bone integration and bone formation in peri-implant osseous defects.

The protocol was approved by the Institutional Animal Care and Use Committee of University (approval protocol No: 2016/03). The authors applied the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines.

This study was designed for 18 adult male New Zealand White rabbits weighing between 3 and 3.5 kg. For sample-size calculation, we used new bone formation results from a reference study.³⁰  They found large effect size (f = 3.4) for new bone formation according to the independent group comparisons results. We used a lower effect-size level (f = 1) with power of 90% and a significance level of .05 for sample-size calculation, and we calculated that we needed at least 6 rats in each group. Therefore, we included 6 rats for each group (total of 18 rats) to study. In the present study, we found that the effect size for new bone formation results was f = 0.84 and this study reached 84.5% power with a 95% confidence level according to the effect size.

The animals were kept in a specially designed room and fed ad libitum on a standard diet and water. The rabbits were placed in appropriate cages in a room with an ideal daylight/darkness cycle and an ambient temperature. The animals were randomly assigned to 1 control and 2 experimental groups (n = 6 animals) and a statistical software program (SPSS Statistics v24.0; SPSS Inc, Chicago, Ill) was used for randomization. The osseous defects were created, and then implants were placed. The defects were treated with 3 methods: defects were empty around implants, defects were filled with only ATBG, and defects were filled with ATBG combined with PRF (1:1 ratio). All analyses were applied by 2 independent, calibrated examiners to determine the groups.

Before the surgery, 5 mL of blood was collected in a blood collection tube without anticoagulant from the central auricular artery of each sedated animal. The blood was centrifuged immediately (PRF: 2700 rpm for 12 minutes), and a fibrin clot formed and was extracted from the tube with forceps under sterile conditions. The PRF clot was cut into small pieces and combined with the ATBG.

A mandibular left first molar tooth (included as enamel, dentin, cementum, and pulp) was removed and placed in the sterile chamber of a newly designed Smart Dentin Grinder (KometaBio, Fort Lee, NJ) and was ground and prepared for 300-μm to 1200-μm grafting particles. The particles were collected and stored in basic alcohol (0.5 M NaOH and 20% alcohol) for 10 minutes to defat and dissolve all the organic debris and bacteria.²²  The solution was then drained, and the graft particles washed twice in sterile phosphate-buffered saline before being used as graft.²² 

All surgeries were performed under general anesthesia with 2% xylazine (Rompun 2%, Bayer, Istanbul, Turkey) and 1% ketamine (Ketalar, Eczacibaşi-Warner Lambert, Istanbul, Turkey). The site was shaved and cleaned with povidone-iodine. After an incision was made, the tibia was exposed by subperiosteal dissection. Bone defects (10-mm diameter, 4-mm depth) were created with a trephine drill under irrigation with pure saline solution. Implant cavities (3-mm diameter, 6-mm depth) were prepared in the center of each defect according to the recommendation of the implant system manufacturer. Then, the implant cavities were rinsed with pure saline solution, dental implants (NR Line, 3.0 × 10 mm, Dentium, Cypress, Calif) were placed (6-mm depth), and primary stabilization was controlled. The upper parts of the dental implants were isolated in the center of the defects. Cover screws were placed on the implants, and the peri-implant defect was grafted with ATBG and ATBG+PRF in the experimental groups (Figure 1). After the surgery, the tissues were tightly sutured in 2 layers with degradable sutures (Pegelak, poly [glycolide-co-lactide], Dogsan, Trabzon, Turkey). Postoperatively, all the animals received Ceftriaxone 50 mg/kg (Rocephine, Deva, İstanbul, Turkey) and Carprofen 4 mg/kg (Rimadyl, Pfizer, New York, NY) intramuscularly once daily for 3 days. The animals were euthanized 28 days after surgery. The bones with the implants were dissected, and any sign of unusual healing was documented.

The implants and surrounding bone tissue were removed en bloc and immediately immersed in formaldehyde for histologic evaluation. The specimens were dehydrated with ascending percentages of ethanol and embedded in a methylmethacrylate-based resin (Technovit 7200 VCL, Kulzer and Co, Wehrheim, Germany). All specimens were prepared with a sawing and grinding technique (Exakt Apparatebau, Norderstedt, Germany). The sections were stained with hematoxylin and eosin.

All the sections were evaluated with stereologic analyses in a workstation with stereology software (Stereo Investigator version 11.0, Microbrightfield, Colchester, Vt), a charge-coupled device digital camera (Optronics MicroFire, Goleta, Calif), a personal computer, a Mac 5000 motor stage control unit (Ludl Electronic Products, Ltd, Hawthorne, NY), and a light microscope (Leica DM-4000B; Leica Microsystems, Wetzlar, Germany). A pathologist who was blinded to the animal group information, evaluated the histologic sections.

The percentage of bone-to-implant contact (BIC) was calculated by measuring the linear distance around the entire implant in which bone was in direct contact with the implant and then dividing that linear measurement by the total linear distance all the way around the implant. Additionally, histomorphometric parameters for using the calculation of new bone formation were defined as follows:

1. Total augmented area was defined as areas including fibrovascular tissues, residual graft materials, and newly formed bone in bony defects around the implant.

2. New bone formation (%) was calculated using the following formula: New bone formation = New bone area/Total augmented area × 100.

Shapiro–Wilk test was used to assess the normality. After parametric test assumptions were satisfied, one-way analysis of variance (ANOVA) was used for comparisons among groups. The post hoc Tukey test was used when a significant difference was determined with ANOVA. Calculations were made using the statistical software (SPSS Statistics v24.0). A difference between the groups was considered significant at P < .05. Methodology, results, and conclusions were reviewed by an independent statistician.

Wound infection or dehiscence and formation of abscesses was not detected at any surgical area. None of the animals died during the experimental procedure. All implants were in situ when the animals were sacrificed.

Newly formed bone and fibrous tissue were observed in all groups (Figure 2). However, new bone formation was lower in the control group than the test groups (P < .05). New bone formation was significantly higher in the ATBG+PRF group than the ATBG group (P < .05) (Table).

Histomorphometric evaluations revealed that BIC differed significantly among the groups (Table). BIC was significantly lower in the control group than the test groups (P < .05). Additionally, BIC was significantly higher in the ATBG+PRF group than the ATBG groups (P < .05).

The effect of ATBG combined with PRF was evaluated on peri-implant defects created in rabbit tibia. Animals, especially rabbits and rats, have been widely used to examine new bone formation. These animals are easily obtained and less expensive than other species.³¹  Rabbits were used in the present study because PRF and ATBG cannot be prepared from rats because of inadequate blood supply and teeth. Peri-implant defects in rabbit tibia have been used in other studies.^30,32  Durmuslar et al reported that rabbit tibia has sufficient bone to create peri-implant defects.³²  In the present study, the defect model was prepared as described previously.³⁰  In this animal model, the healing of marginal defects around implants occurs after approximately 20 to 30 days.³³  Hence, a 28-day period was used to evaluate the healing of peri-implant bone defects as in a previous study.³⁰ 

Peri-implant defects should be grafted to increase BIC and new bone formation. The restoration of peri-implant defects with graft materials has been investigated. Schuler et al³⁴  reported that the use of autogenous bone grafts improved the BIC values in peri-implant defects. In another study, biphasic calcium phosphate bone substitute preserved the defect space and enhanced new bone formation.³⁵  However, these materials have clinical disadvantages.^9–11  Therefore, ground tooth structure has been used as a graft material in bone surgery.

The dentin matrix plays a critical role in new bone formation because of its osteoinductive properties,³⁶  and demineralized dentin has been widely used as a bone substitute. Dentin must be demineralized for growth factors to be used because dentin tubules are expanded after demineralization, and these proteins are released.³⁷  Reis-Filho et al¹⁷  reported that demineralized dentin matrix (DDM) stimulates new bone formation by elevating the growth factor level. Bakhshalian et al³⁸  indicated that DDM does not cause any reaction and that it accelerates the bony repair and enhances bone quality. However, the demineralization procedure is not practical for clinical application because of the length of time required for demineralization, thus preventing the graft being performed during surgery. Additionally, Ike et al³⁹  determined that demineralized dentin did not have osteoinductive activity, and the demineralization procedure decreases the release of growth factors, including bone morphogenetic protein-7.⁴⁰  For these reasons, autogenous mineralized tooth structure was used as the bone substitute instead of DDM. Binderman et al²²  reported that demineralized dentin forms inadequate bone for implant support and showed that mineralized dentin allows early loading in implant treatment because it adheres to the newly formed bone. Additionally, a mineralized tooth graft may have a beneficial effect on bone formation in implant treatment with a sinus lift operation. Autogenous tooth graft materials can be used as an alternative bone substitute.⁴¹  They do not transmit disease and can be combined with other graft materials and PRF membrane.⁴¹  In our study, according to the results of histomorphometric analysis, BIC and new bone formation was found to increase significantly in the ATBG group compared with the control group. These findings indicated that ATBG stimulates new bone formation around peri-implant defects, which is consistent with the results of previous studies.^22,41  The increase of bone formation may be related to the release of the growth factors from the ATBG. Therefore, future studies are needed to evaluate the growth-factor levels in ATBG.

PRF has frequently been used in combination with other bone grafting materials to treat peri-implant defects. Simsek et al³⁰  suggested that PRF combined with demineralized freeze-dried bone allograft significantly increased BIC and new bone formation in peri-implant defects. In another study, Melek and El Said⁴²  grafted defects with a combination of tooth graft and injectable PRF and concluded that a combination of these materials supported bone fill. In the present study, PRF was used to increase the efficacy of ATBG. BIC, and new bone formation were determined to be significantly higher with PRF combined with ATBG compared with the ATBG and control groups. These findings showed that PRF induces new bone formation when used with graft materials and are consistent with previous studies. ^12,30,31,42  Use of PRF may enhance bone healing by increasing the growth factor levels. Five milliliters of blood was received from all animals for standardization; however, the amount of PRF obtained from each animal and the growth factors in PRF may be different. This is a limitation of this study.

Grafting materials combined with growth factors can stimulate new bone formation but are very expensive. This study showed that using ATBG enhances new bone formation and BIC in peri-implant defects. However, teeth that have root canal fillings, extensive caries, or restorations are not suitable for obtaining the ATBG, and this is a limitation for clinical application. Additionally, there is no information about this graft resorption time and rate. Further studies are required to determine tooth graft resorption time and rate. Teeth include germ stem cells or pulpal stem cells, and these cells may be critically important for tissue engineering. Future studies are needed to evaluate the potential of this graft's stem cells. However, long preparation may be required for these procedures. Moreover, within the limits of this animal study, these findings suggest that ATBG is more useful in combination with a growth factor source such as PRF. Long-term studies, however, are needed to evaluate the effect of ATBG and PRF on bone formation in animals and humans.

Abbreviations

ANOVA:

one-way analysis of variance

ARRIVE:

Animal Research: Reporting of in vivo Experiments

ATBG:

autogenous tooth bone graft

BIC:

bone-to-implant contact

DDM:

demineralized dentin matrix

PRF:

platelet-rich fibrin

This study was funded by the Scientific and Technological Research Council Of Turkey (TUBITAK). The authors would like to thankfully acknowledge Hande Şenol for performing the statistical analyses, Department of Biostatistics, Faculty of Medicine, Pamukkale University, Denizli, Turkey.

The authors report no conflicts of interest related to this study.