Effects of Injectable Platelet-Rich Fibrin on the Osseointegration of Dental Implants

Introduction

Tooth loss in the oral region is a common condition, resulting from oral diseases or maxillofacial trauma. It is estimated that more than 240 million people worldwide experience single or multiple tooth loss. The primary goal of dentistry is to restore the patient’s lost natural anatomy, aesthetics, function, and health to the highest possible level, regardless of the extent of material loss or injury. Throughout history, various methods have been used to replace missing teeth. Dental implants have been widely used in the treatment of tooth loss for many years and have continuously evolved [1]. Dental implants bond to the bone through osseointegration [2], which is defined as “a process in which clinically asymptomatic stable fixation is achieved and maintained with alloplastic materials during functional loading” [3].

The primary biological response to implant placement involves the formation of a hematoma, followed by mesenchymal tissue development, intramembranous woven bone formation, and lamellar bone formation [4]. The first biological component to interact with the endosseous implant is blood. Blood cells, including red blood cells, platelets, polymorphonuclear granulocytes, and monocytes, migrate from capillaries into the tissue surrounding the implant. These blood cells become activated at the implant interface and release various growth factors, such as transforming growth factor (TGF)-beta and platelet-derived growth factor (PDGF), as well as vasoactive factors, like serotonin and histamine. PDGF and TGF-beta have been shown to be not only mitogenic for fibroblasts but also chemotactic for neutrophils, smooth muscle cells, and osteogenic cells [3,4].

Implant stability plays a critical role in successful osseointegration, which is a prerequisite for functional implants. Continuous, objective, and qualitative monitoring of implant stability is essential. Implant stability is assessed in 2 different stages: primary stability and secondary stability. Primary stability arises from the mechanical connection between the implant and the cortical bone at the time of placement. Secondary stability develops through the regeneration and remodeling of bone and surrounding tissue after placement and is influenced by factors such as new bone formation and remodeling. The functional loading period depends on implant stability [5]. At the time of implantation, there is limited bone contact with the implant. Over time, newly formed bone fills the gaps in the interfacial region and integrates with the implant surface. Complete bone–implant contact rarely occurs, and clinically observed osseointegration corresponds to approximately 80% of bone–implant contact. However, more than 60% of contact is considered sufficient for implant stability [6].

Various methods are used to analyze implant stability. The resonance frequency analysis method evaluates the initial resonance frequency of a small transducer attached to an implant fixture or abutment [7]. In later models of resonance frequency analysis (Osstel; Osstell AB, Gothenburg, Sweden), a metal transducer (SmartPeg) is connected to the implant via a screw connection, and a wireless version has since been developed. The peg has a small magnet on top that is excited by magnetic frequencies from a handheld device. The peg vibrates in 2 directions: the first mode (highest resonance frequency) and second mode (lowest resonance frequency). As a result, 2 implant stability quotient (ISQ) values are obtained: one high, and one low [8]. The Hertz value obtained from Osstell measurements is converted into an ISQ value, a specific parameter representing implant stability. The ISQ scale ranges from 1 (minimum stability) to 100 (maximum stability), with each ISQ unit corresponding to a change of 50 Hz. Studies have classified implants with ISQ values between 60 and 80 as successful, while those with values below 50 are considered critical [9].

Recently, an injectable form of i-PRF was developed. To produce i-PRF, blood is collected in plastic tubes without any coating or anticoagulant and centrifuged at approximately 700 rpm for 3 min [10]. The plastic tubes have hydrophobic surface properties and do not effectively activate the coagulation process. The separated plasma and platelets form a light yellow layer at the top of the tube, which is then aspirated and used in an active injectable form. Due to the low centrifugation speed and duration, i-PRF contains higher percentages of leukocytes and blood plasma proteins [11].

Based on this information, we hypothesized that the application of i-PRF to the implant socket will result in greater osseointegration than in untreated implant sockets. This study was designed to evaluate the effects of i-PRF application on early-stage implant osseointegration. Additionally, we aimed to evaluate the effect of i-PRF on the osseointegration of dental implants in 20 healthy individuals measured by resonance frequency analysis.

Material and Methods

PARTICIPANTS:

The study included 20 patients (8 men and 12 women) aged between 25 and 69 years who presented for treatment at the Department of Periodontology at Izmir Katip Celebi University Faculty of Dentistry. After being informed about the nature, duration, and potential risks of the study, the participants signed an informed consent form. Ethical approval for this study was granted by the Local Ethics Committee of Clinical Research at Izmir Katip Celebi University Faculty of Medicine (10.11.2019-02).

The inclusion criterion was patients with at least 2 missing teeth that did not require advanced surgery for implantation. The exclusion criteria were as follows: any systemic disease, regular systemic medication, smoking, and pregnancy.

Of the 40 total implants, 20 made up the test group (i-PRF+) and 20 made up the control group (i-PRF−); 16 implants were placed in female patients and 24 in male patients, with 2 implants placed in each patient. Each patient required rehabilitation with dental implants in both the right and left half-jaws of either the maxilla or mandible. Four of the implants were placed in the anterior region and 16 in the posterior region. In the study, 2 of the implanted bones were D1 bone, 16 were D2 bone, and 2 were D3 bone. A detailed intraoral and extraoral examination was conducted for all patients. At least 6 months had passed since the extraction of teeth in the implant sites. Panoramic and periapical radiographs were taken to evaluate the implant sites. Anatomical structures, such as the mandibular nerve and maxillary sinus, which could restrict implant placement, were assessed, and patients requiring advanced surgical procedures were excluded from the study. Patients received oral hygiene training, including brushing and interdental cleaning instructions. In the following session, polishing was performed to remove surface discoloration, and oral hygiene was checked.

PREPARATION OF I-PRF:

For blood collection, 10-mL white vacuum tubes, needles, and holders were used. To prevent platelet activation and red blood cell hemolysis, blood was drawn quickly and placed into a specially designed centrifuge (EBA 20 S, Hettich, Zentrifugen, Tuttlingen, Germany). The collected blood samples were centrifuged at 700 rpm for 3 min. After centrifugation, the upper yellow liquid layer, containing PRF, was aspirated into a sterile 10-mL syringe. After the implant cavity was opened, 10 mL of i-PRF was placed into the cavity.

IMPLANT PLACEMENT:

A total of 40 SLA surface BLT (Straumann AG, Switzerland) dental implants were used. The preparation of i-PRF followed the same procedure mentioned above. This study was conducted as a randomized, split-mouth design. With patients under local anesthesia, a surgical flap was raised, and implant sites were prepared. The implant sites were randomly assigned to either the test group (i-PRF+) or the control group (i-PRF−). In the test group impants, i-PRF was applied to the implant site before implant placement. The control group implants were placed via the conventional method, without any biomaterial application. Three months after implantation, the ISQ values of the implants were measured by the primary and assisting researchers, to evaluate implant stability, and statistical relationships were assessed. Resonance frequency analysis using the Osstell device was used to determine ISQ values.

The Hertz value obtained from the Osstell device is converted into a specific parameter, the ISQ, which is based on the underlying resonance frequency and ranges from 1 (minimum stability) to 100 (maximum stability). Each unit change in the ISQ value corresponds to a change of 50 Hz. Implants with an ISQ value between 60 and 80 are considered successful, while implants with values below 50 are considered critical [9].

Implant placement followed the standard protocol of the implant system. The implant sites were prepared with the same surgical protocol. Bone resistance to drilling was classified according to Lekholm and Zarb’s classification [12], rated from 1 to 4, where “1” represents the hardest and most resistant bone and “4” represents the softest, least resistant bone. To prevent overheating, continuous sterile saline irrigation was used during the procedure. Before implant placement, both prepared sockets of each patient were irrigated with sterile saline to remove blood clots, bone particles, and tissue debris.

The implant sites of the test group implants were rinsed with i-PRF, under gentle pressure. Then, the appropriately sized implant was placed using a sterile transport mechanism. In the control group implants, no additional treatment was applied to the implant sites, and the implants were placed conventionally. After implant placement, the transport component was removed, and a transducer was attached for resonance frequency measurements. Once the necessary measurements were taken, the transducer was removed, and the implant cover screws were placed.

RESONANCE FREQUENCY ANALYSIS MEASUREMENT:

Measurements were performed for the test and control groups. The numerical values obtained were recorded as ISQ units. To avoid bias, care was taken to ensure that the doctor conducting the study was different from the doctor measuring ISQ. The first measurement was taken at the time of implant placement, and the second measurement was taken 3 months after the surgery. All resonance frequency analysis evaluations were performed by the same investigator. The transducer was placed into the implant site and screwed in until the first point of resistance was met using a plastic placement key. For each implant, resonance frequency values were measured at 4 different angles: buccal, lingual, mesial, and distal. The arithmetic mean of these 4 values was calculated to determine the ISQ value for each implant.

STATISTICAL ANALYSIS:

The normality of the measured data was evaluated using the Shapiro-Wilk test, and variance homogeneity was assessed using the Levene test. Descriptive statistics are presented as mean and standard deviation. Since the data showed normal distribution and homogeneity, implant stability and differences between test and control groups were analyzed using the ANOVA test. Periodic differences between groups with and without i-PRF application were analyzed using the paired t test, which was preferred in the study because the test and control groups were in the same individual, and 2 different times were considered. A P value of <0.05 was considered statistically significant. Data analysis was conducted using SPSS (version 22.0, IBM Corp, Armonk, NY, USA). Power analysis indicated that with an effect size of 0.89 and a significance level of α=0.05, a sample size of 40 implants from 20 patients provided an 85% study power. Therefore, including 20 patients with 2 different groups was deemed appropriate.

Results

ISQ VALUE IN I-PRF+ AND I-PRF− GROUPS:

The study included 20 patients (8 women and 12 men), with an age range of 25 to 69 years and a mean age of 47.2±13.6 years. The mean age of female patients was 51.1±11.9 years, while the mean age of male patients was 44.6±14.5 years. A total of 40 implants were placed in 20 patients (2 implants in each patient: 1 with i-PRF and 1 without i-PRF), with 20 implants (50%) assigned to the test group and 20 implants (50%) to the control group. Of the implants, 16 (40%) were placed in female patients and 24 (60%) in male patients.

The initial mean ISQ value for implants in the i-PRF+ group was 68.875±9.571, increasing to 74.237±8.283 at the 3-month evaluation. In contrast, the initial mean ISQ value for implants in the i-PRF− group was 73.275±7.699, with a 3-month ISQ value of 73.662±8.089. A statistically significant difference was found between implants in the i-PRF+ and i-PRF− groups in both the initial and 3-month ISQ measurements (P=0.004). Figure 1 shows the observation of changes in the test group (i-PRF+) and control group (i-PRF−) at different times.

ISQ VALUES IN FEMALE AND MALE PATIENTS:

For implants in male patients in the i-PRF+ group, the initial mean ISQ value was 70.854±2.741, increasing to 73.042±2.416 at the 3-month measurement. In the i-PRF− group, the initial mean ISQ value for implants in male patients was 73.771±2.276, with a 3-month value of 73.688±2.688. For implants in female patients in the i-PRF+ group, the initial ISQ value was 65.906±3.357, increasing to 76.031±2.959 at the 3-month measurement. In the i-PRF− group, the initial ISQ value for implants in female patients was 72.531±2.788, with a 3-month value of 73.625±2.938. Implants in female patients in the i-PRF+ group exhibited significantly higher ISQ values than did those of male patients (P=0.004), whereas no significant difference was found between implants in men and women in the i-PRF− group (P=0.399). Figure 2 shows the observation of changes in the test group (i-PRF+) and control group (i-PRF−) by patient sex.

ISQ VALUES IN DIFFERENT BONE STRUCTURES:

In the i-PRF− group, the initial mean ISQ value of implants placed in type 1 bone was 71.750±5.261, while the mean ISQ value at the 3-month evaluation was 72.375±5.816. In the i-PRF− group, the initial mean ISQ value of implants placed in type 2 bone was 74.563±1.860, whereas the mean ISQ value at the 3-month evaluation was 74.594±2.056. In the i-PRF− group, the initial mean ISQ value of implants placed in type 3 bone was 64.500±5.261, while the mean ISQ value at the 3-month evaluation was 67.500±5.816. Regarding bone density, no implants were placed in type 4 bone in either group. No statistically significant differences in ISQ values were observed among different bone densities in the test (P=0.109) and control (P=0.430) groups (Figure 3).

Discussion

Various invasive and non-invasive methods are used to evaluate the level of osseointegration of dental implants, during placement and in later stages. Histological and histomorphometric methods as well as invasive techniques, such as removal torque testing, and non-invasive approaches, like radiographic evaluation, Periotest, Implatest, and percussion tests, are used. Since the resonance frequency analysis method can be used to assess implants at different time intervals, it has become an essential and widely adopted tool [13]. This study evaluated the effects of i-PRF on implant osseointegration during the early healing period and found that i-PRF positively influenced early osseointegration.

Resonance frequency analysis was selected to assess implant stability, due to its ease of use, reproducibility, non-invasive nature concerning bone-implant contact, and ability to provide numerical values. The Osstell device was used for implant evaluation. Studies using resonance frequency analysis have shown significant increases in resonance frequency over time. Meredith et al placed implants in self-polymerizing polymethyl methacrylate in vitro to simulate bone around the implant during healing and observed significant increases in resonance frequency at intermediate stages [7]. These in vitro findings were later confirmed through in vivo measurements in which Meredith et al concluded that resonance frequency analysis, being non-invasive, is clinically convenient and provides quantitative data on implant stability and stiffness [8].

Conversely, Bischof et al found that the resonance frequency analysis method does not provide information about the bone–implant interface, unlike the torque test. In their study of 106 SLA implants in 36 patients, they observed a negative correlation between resonance frequency analysis and reverse torque. They did not find any effects of implant position, length, or diameter on primary stability [14]. However, Horwitz et al identified a positive correlation between implant ISQ and implant diameter, and a positive correlation between ISQ and insertion torque [15].

A simulation and histomorphometric animal study by Ito et al demonstrated the superiority of resonance frequency analysis in monitoring the implant treatment process. In vivo measurements under controlled boundary conditions showed significant repeatability of the Osstell device [16]. Literature reviews have identified numerous studies examining the factors influencing the widespread use of resonance frequency analysis in implant stability assessments [17–19].

Different researchers have reported ISQ values ranging between 52 and 90 [20–22]. In the present study, ISQ values ranged from 52 to 86. Implants with ISQ values above 50 are considered to have successful stability [23,24]. In the present study, the mean ISQ value for implants with i-PRF was 69 during surgery and 74 in the postoperative assessment. For implants without i-PRF, the mean ISQ value remained at 73 during both surgery and the postoperative evaluation. These findings are consistent with those in the literature [21,23].

When the test and control groups were evaluated, ISQ values in both groups were consistent with bone types, with type 1 bone exhibiting higher mean ISQ values than type 2 bone, although this difference was not statistically significant. A significant increase was observed in type 3 bone implants from baseline to final values, but due to the limited number of type 3 implants, a definitive evaluation could not be made. Studies investigating the relationship between bone quality and implant stability typically analyze a larger number of implants. The lack of a significant difference in this study may be attributed to the small sample size and uneven distribution of implants across different bone types.

For weekly resonance frequency analysis of the implants, healing abutments had to be removed and the appropriate transducer of the resonance frequency analysis device screwed in during each evaluation session. This process introduced micromovements in the implants, particularly during the early healing period, potentially affecting osseointegration negatively in implants with weak primary stability. Shobara et al demonstrated this effect in their study on mouse tibias [24].

Osseointegration of dental implants is critical for long-term success and stability. Various strategies have been proposed to enhance and accelerate osseointegration by increasing bone–implant contact [25,26]. Surface modifications can improve osseointegration, while another approach involves modulating the healing response after implantation [26]. This modulation has been achieved using biologically active molecules that induce osteoconductivity, enhance osteoblastic differentiation, and promote peri-implant bone healing [27,28].

One such study evaluated the effect of platelet-rich plasma (PRP), which contains growth factors, on implant osseointegration. Casati et al used 10 adult male hybrid dogs for their research. Three months after tooth extraction, osteotomies were performed on both sides of the jaws, with PRP applied to one socket and none to the other. Bone density and bone–implant contact assessments showed no significant effect of PRP on osseointegration [29].

Since i-PRF is a relatively new material, a limited number of studies have been conducted on it so far. Wang et al compared the effects of this new i-PRF on osteoblast behavior with those of conventional PRP. Human primary osteoblasts were cultured with i-PRF or PRP and compared with control cultures. Cell viability, migration, adhesion, and proliferation capacities were examined. Additionally, osteoblast differentiation was assessed using alkaline phosphatase (ALP), alizarin red, and osteocalcin staining, along with real-time polymerase chain reaction for genes encoding Runx2, ALP, collagen 1, and osteocalcin. The results showed high survival rates for all cells throughout the study. Compared with PRP, i-PRF resulted in a 3-fold increase in osteoblast migration and significantly higher proliferation rates on days 3 and 5. Moreover, i-PRF induced significantly higher ALP staining on day 7 and alizarin red staining on day 14. Additionally, i-PRF led to significant increases in ALP, Runx2, and osteocalcin mRNA levels and osteocalcin immunofluorescence staining, compared with PRP. These findings support the use of naturally formulated i-PRF over the use of anticoagulated PRP and recommend further studies on i-PRF [30]. The main cell responsible for the biological activity of PRF is the platelet. Platelets contain granules, including alpha granules, dense granules, and glycogen granules. Alpha granules are the main granules that contribute to wound healing, due to the various growth factors they contain, such as PDGF, TGF-beta, vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and epidermal growth factor (EGF). They then reach the target cells, bind to transmembrane receptors, and activate various intracytoplasmic proteins for movement-dependent gene expression, such as cell mitosis or collagen production. All these growth factors are responsible for the accelerated healing events mediated by PRF [31].

When used in conjunction with implant placement, i-PRF promotes the release of growth factors, such as PDGF-AA, PDGF-AB, EGF, and IGF-1, which remain significantly elevated even after 10 days. Additionally, i-PRF significantly induces TGF-β and collagen-1 mRNA expression by day 7. The positive effects of i-PRF on healing are thought to be due to its high platelet, leukocyte, monocyte, and granulocyte content. Furthermore, the resistance of i-PRF to infection, due to its cellular composition, can positively affect osteogenesis at the bone–implant interface, accelerating osseointegration.

Our review of the literature showed that numerous studies have tested various methods to enhance implant osseointegration. Among these, autologous blood products have shown the most promising results. In our study, we evaluated the liquid form of one of the newest autologous blood concentrates, i-PRF, and observed positive effects on implant osseointegration. Based on these findings, we believe that i-PRF, due to its fully autologous nature, ease and speed of preparation, and absence of adverse effects, could be beneficial in cases in which bone–implant contact could be low.

The limitations of the present study are the small number of patients, lack of sufficient standardization of implant areas, and differences in bone structure.

Conclusions

To gain a more comprehensive understanding of the effects of i-PRF on implant surface variations, implant morphology, and patient-related factors affecting stability, further clinical studies with larger sample sizes, longer follow-up periods, and a variety of implant brands are needed. Additionally, histological section analyses in animal studies would provide more detailed insights into changes at the bone–implant interface. Implants treated with i-PRF showed significantly higher stability in resonance frequency analysis than did untreated implants. Moreover, the stability increase was significantly greater in female patients than in male patients, while bone density had no effect on implant stability.

Availability of Supporting Data

The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request.