Evaluation of Regenerative Potential of Autologous Platelet Rich Fibrin in Horizontal Bone Defects in Chronic Periodontitis Patients

Introduction

Periodontitis is a complex disease which is associated with inflammation of the gingiva that results in periodontal pocket formation, with loss of supporting periodontal ligament and alveolar bone around teeth. In addition to the reduction of height, morphologic features of bone are also affected. The rate of alveolar bone loss can be slow and continuous or episodic. Clinicians often face the challenge of regenerating horizontal bone loss, which is regarded as the most prevalent bone destruction pattern in periodontitis. This condition leads to a decrease in bone height parallel to the tooth’s cementoenamel junction, while the bone margins remain at a right angle to the tooth’s surface.

The ultimate goal of periodontal therapy is to stop the progression of periodontal disease and regenerate structures lost due to pre-existing disease processes.¹ The important wound-healing events associated with periodontal development are recapitulated during periodontal regeneration to restore lost tissues to their original function and architecture.² For periodontal regeneration to occur, a number of biologic events including cell migration, adherence, multiplication, and differentiation, need to occur in a well-orchestrated sequence. In conventional open flap debridement, periodontal tissues destroyed by disease are not completely regenerated and current regenerative procedures have limited potential for restoring these tissues.³

Vertical bone loss, which occurs in 7.8% of cases, has a treatment rate of 96.8%, while horizontal bone loss, more prevalent at 92.2%, is only treated in 3.2% of cases.⁴ Open flap debridement has traditionally been employed as one of the standard approaches for addressing horizontal bone defects. Over the years, several treatment options have been investigated such as grafts, Guided Tissue Regeneration (GTR) membranes, Enamel Matrix Protein (EMP), recombinant human Bone Morphogenic Protein (rhBMP), etc. But the results of these studies were not promising; thus more studies were advocated to include newer treatment modalities or regenerative materials for regeneration in horizontal bone defects.⁵

Recent innovation in the field of dentistry is the development of autologous Platelet Rich Fibrin (PRF). Platelet Rich Fibrin (PRF) is a second-generation platelet concentrate widely used to accelerate soft and hard tissue healing and is a strictly autologous fibrin matrix containing a large quantity of platelet and leukocyte cytokines.⁶ It was first developed by Joseph Choukroun in 2001 in France. The combined properties of fibrin, platelets, leukocytes, growth factors and cytokines make platelet rich fibrin a healing biomaterial with tremendous potential for bone and soft tissue regeneration.⁷

Several studies have evaluated the effectiveness of PRF in intrabony defects, furcation defects, gingival recession, sinus augmentation, etc. and have found a positive outcome. However, regarding regeneration in horizontal defects, the data available is scarce. Therefore, the main objective of the present study was to assess the potential of autologous PRF to bring about periodontal regeneration in horizontal defects.

Materials and Methods

A prospective, interventional, single-arm study was conducted in the Department of Periodontics and Implantology, Vydehi Institute of Dental Sciences and Research Centre, Bangalore. A total of 20 sites with horizontal bone defects in five patients were taken for the study according to the sample size calculation which showed a power of 80%. Ethical clearance was obtained from the Institutional Ethical Committee.

This study included participants in the age group of 20-60 years, systemically healthy patients with chronic periodontitis clinically diagnosed based on American Academy of Periodontology (1999) classification having clinical attachment loss ≥ 3 mm in 30% of the sites and probing pocket depth ≥5 mm with at least six teeth, presence of horizontal bone loss in the radiograph. Medically compromised patients, patients who underwent periodontal surgery in the last one year, teeth with grade III mobility, smokers, pregnant and lactating women were all excluded from the study.

After enrolling participants who met the inclusion and exclusion criteria, a comprehensive explanation of the surgical procedure and its advantages was provided to them. Each patient then provided a signed informed consent form.

Clinical Parameters

All the parameters were recorded at baseline and at nine months by a single examiner. This included Gingival Index (GI),⁸ Plaque Index (PI),⁹ Probing Pocket Depth (PPD) and Clinical Attachment Level (CAL). For easy reproducibility, all the measurements were made using a surgical stent.

Radiographic Assessment Radiographic parameters were assessed using radiovisiography (RVG) at baseline and at nine months. RVG used in this study was SOPIX2 ACE and CARESTREAM CS2100 X-ray unit functioned at 60 kvp and 7 mA having a radiation exposure of 0.250 milliseconds. Charged coupled device (CCD) sensor of the RVG was covered with a plastic sleeve for each patient to ensure best possible hygiene. The dimension of the defect was assessed from cementoenamel junction (CEJ) to the crest of alveolar bone (CAB). From the CEJ of one tooth to the adjacent tooth, a line was drawn. Similarly another line was drawn from CAB joining adjacent two teeth. The distance from the midpoint of these two lines was calculated. RVG taken at baseline and at nine months were transferred to Digimizer image analysis software. Images were then resized to 600×400 pixels. The measurements which were attained were transferred to the master chart.

Initial Therapy

To ensure proper plaque control, thorough scaling and root planing was performed. Instructions were given regarding maintenance of oral hygiene. Reevaluation was done at four weeks after the completion of phase-I therapy (SRP) and baseline parameters were recorded. Sites in which persisting probing depth of more than 5 mm were included for surgical treatment using PRF. Cast was prepared and occlusal stent with grooves was made using acrylic resin and all the clinical parameters were recorded.

Preparation of Platelet Rich Fibrin

Collection of blood and preparation of PRF was delayed until the bone defects were adequately exposed so that the prepared PRF could be used immediately without requiring storage. Through venipuncture of the right antecubital vein, 10 mL blood was drawn from each patient in sterile glass test tubes without addition of any anticoagulants. It was centrifuged immediately on a tabletop centrifuge at 3000 rpm for 10 min. Due to differential densities, three basic fractions were separated: red blood cells (RBCs) at the bottom, top layer of platelet poor plasma (PPP), and middle PRF. PRF gel was separated from the underlying RBC layer using a sterile stainless steel scissors after aspirating the PPP and discarding it.

Surgical Technique

On the selected surgical teeth, occlusal stent was placed and a UNC 15 probe was used through the groove on the stent to measure and record probing depth, and clinical attachment level (Figure 1). Probing depth was measured from the gingival margin to the base of the pocket; clinical attachment level was measured from the cement enamel junction (CEJ) to the base of the pocket.

The surgical procedure was performed under local anesthesia using 2% Lignocaine Hydrochloride containing adrenaline at a concentration of 1:2,00,000. Full thickness Kirkland flap was raised (Figure 2 and 3), followed by meticulous debridement and root planing. In order to prevent the displacement of the graft material (PRF gel), pre-suturing was performed. A contra-angled micromotor handpiece with a round carbide bur (1 mm diameter) was used to penetrate the marrow space: multiple perforations were made not closer than 1 mm from each other and deep enough to induce bleeding. The graft material was filled close to 2 mm below the CEJ (Figure 4). Internal vertical mattress suture was placed using 3-0 silk suture (Figure 5). Sutures were then covered by periodontal dressing (Coe-Pak) on the operated area

Patients were prescribed antibiotics - Amoxicillin, 500 mg thrice daily for five days or Erythromycin, 500 mg twice daily for five days, (in case of penicillin allergy) and analgesics, Diclofenac sodium and Paracetamol combination after the surgery for three days. 0.2% Chlorhexidine mouthwash was prescribed to be used twice daily, from the day after the surgery, for a period of 14 days. The periodontal dressing and sutures were removed one week post-operatively. Patients were followed-up for nine months at every three months interval. Deplaquing was done if needed, and instructions regarding oral hygiene maintenance were re-instated at the recall visits and through telephonic conversation. Clinical and radiographic evaluations were repeated at the nine month recall visit (Figures 6-8). The flowchart describing the study design is given in Figure 9.

Statistical Analysis

IBM SPSS statistics software 23.0 Version was used to analyse the collected data. To describe about the data, descriptive statistics frequency analysis was used for categorical variables. Mean and standard deviation were used for continuous variables. Significant difference between the bivariate samples in paired groups was calculated using the Paired sample t-test. Probability value less than 0.05 was considered as significant.

Results

In total, 20 sites with horizontal bone loss were treated, and all patients were consistently followed up until nine months after treatment. There were no postoperative complications and healing was satisfactory.

Plaque index

There was a statistically significant reduction in mean plaque index from 2.41±0.44 at baseline to 0.92± 0.24 at nine months (Table 1).

Gingival index

Mean gingival index reduced from 2.26±0.35 at baseline to 0.66±0.21 at nine months. This was found to be statistically significant at p <0.05 (Table 1).

Probing pocket depth

At baseline, mean probing depth was 6.10±0.57 mm which reduced to 2.80±0.79 mm at nine months followup period. This was found to be statistically significant at p <0.05 (Table 1).

Clinical attachment level

Clinical attachment level showed a statistically significant improvement from baseline to nine months (Table 1).

Radiographic assessment

At baseline, mean radiographic defect depth was 6.95±0.74 mm which reduced to 4.81±0.75 mm at nine months follow-up period. There was a gain of 2.14 mm which was statistically significant.

Discussion

Periodontitis is a disease associated with complex etiology which alters the normal architecture of bone leading to different patterns of bone loss. Periodontal therapy aims at restoring the normal form and function of periodontium. Clinicians find it often difficult to regenerate horizontal pattern of bone defects. Therefore in this study, we aimed to evaluate the regenerative capacity of autologous PRF to bring about periodontal regeneration in horizontal defects.

According to Lindhe et al.,¹¹ a probing depth of > 5 mm after SRP requires surgical intervention. In this study, sites with persisting probing depth of more than 5 mm 4-week post-SRP were included for surgical treatment using PRF. It was performed at 20 sites with horizontal bone defects. All the periodontal parameters were assessed before and after regenerative periodontal therapy using PRF.

In this research, the noted enhancements in PI and GI were consistent with the findings of earlier classical studies conducted by Lindhe and colleagues in 1982 and 1984. These improvements were primarily attributed to the encouragement and reinforcement of oral hygiene practices.^10,11 Additionally, there were improvements observed in PPD and CAL, which align with previous research findings.¹²

Radiographic assessment showed a significant amount of defect depth reduction from baseline (6.95±0.74) compared to nine months (4.81±0.75) which was statistically significant.

The significant defect depth reduction from baseline could be attributed to the regenerative capability of PRF gel due to the optimal release of growth factors. This is also in accordance with the previous studies.

In comparison to normal human blood clots, the autologous biomaterial platelet-rich fibrin matrix (PRFM) contains dense concentrations of platelets, which may explain the significant improvement observed. The alpha granules within PRF house growth factors that influence every cell and contribute to the formation of all tissues engaged in the wound healing process. PRF’s substantial release of these growth factors, essential elements in the wound healing process through signaling transduction mechanisms, demonstrates its significant potential in the regeneration of both soft tissue and bone, making it a valuable candidate for regenerative therapy.¹³

The current study was a single-arm trial, where parameters were measured against their baseline values. Further studies are recommended to evaluate the potential of PRF in horizontal defects using relevant controls.

Conclusion

Despite the fact that horizontal defects are the most common alveolar bone defects, they have received least attention towards treatment and regeneration. Here we have tried a treatment modality for horizontal defect which has shown a significant improvement in clinical as well as radiographic parameters within the limitations of this study. Further research is required with larger sample size and control group to analyze the regenerative capacity of this graft material.

Conflicts of Interest

Nil