A comparative evaluation of platelet-rich plasma in combination with demineralized freeze-dried bone allograft and DFDBA alone in the treatment of periodontal intrabony defects: A clinicoradiographic study.

BACKGROUND: The aim of the present clinical trial was to compare PRP combined with a DFDBA to DFDBA mixed with a normal saline solution in the treatment of human intrabony defects.
MATERIALS AND METHODS: Twenty interproximal intrabony osseous defects in twenty non-smoking, healthy subjects diagnosed with chronic periodontitis were treated in this study. Ten subjects each were randomly assigned to the test group (PRP + DFDBA) or the control group (DFDBA + saline). Clinical and radiographic measurements were made at baseline, three month and at six-month evaluation.
RESULTS: The results at three and six months, when compared to the baseline, indicated that both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level and gingival recession; P < 0.01) and radiographic parameters (hard-tissue fill and bone-depth reduction; P < 0.01). However, the test group exhibited statistically significantly greater changes compared to the control group in plaque index at three months (P = 0.00), probing depth reduction at 6 months (P = 0.02) and the radiographic defect fill at 6 months (P = 0.01).
CONCLUSIONS: Treatment with a combination of PRP and DFDBA led to a statistically significantly greater improvement in plaque index at 3 months, probing depth at 6 months and radiographic defect fill at 6 months in intrabony periodontal defects as compared to DFDBA with normal saline.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Periodontal therapy aims to arrest disease progression and regeneration of structures lost to disease.[1] Periodontal regeneration requires a sequence of cell migration, adherence, growth and differentiation, which have potential to increase regeneration.[2] To be considered a regenerative modality, a material or technique must demonstrate that bone, cementum and a functional periodontal ligament can be formed on a diseased root surface. Only autogenous bone grafts provide viable osteogenic cells.[3] Regenerative treatment with bone grafting leads to some degree of regenerated bone, cementum and periodontal ligament.[4]

Platelet-rich plasma (PRP) is an autologous source of PDGF and TGF-β that is obtained by isolating and concentrating human platelets to a high percentage by gradient density centrifugation. PRP has been used successfully to enhance clinical outcome obtained with guided tissue regeneration (GTR) and bone grafts in treatment of intrabony defects.[56] Platelet gel has been used successfully in combination with DFDBA for treatment of periodontal osseous defects.[7] It eliminates concerns about immunogenic reactions and disease transmission.[8]

Although complete periodontal regeneration is unpredictable with any regenerative therapy currently used, periodontal bone grafts show strong potential. A large body of clinical evidence clearly indicates that grafts consistently lead to better bone fill than non-grafted controls.[4] DFDBAs have repeatedly demonstrated significant improvements in soft and hard clinical tissue parameters for the treatment of intraosseous periodontal defects. Substances present in the demineralized bone graft material, i.e., bone morphogenic proteins, stimulate local cell cycles to produce new bone. The freeze drying process destroys cells while maintaining cellular morphology and chemical integrity. These two factors, in conjunction with other materials, enhance periodontal regeneration and/or bone fill.[1]

Therefore, the purpose of this study was to compare clinical and radiographic outcomes obtained with combination of PRP and DFDBA to those obtained with DFDBA mixed with saline solution in treatment of periodontal intrabony defects 6 months after flap surgery. Membrane was not used in this study because it was desired to study the effect of PRP on the bony regeneration by DFDBA.

MATERIALS AND METHODS

Study population

Twenty patients diagnosed with chronic periodontitis (11 males and 9 females), aged between 20 and 50 years, who reported to the Department of periodontology, I.T.S Centre for Dental Studies and Research, Muradnagar, Ghaziabad, Uttar Pradesh, India, having a total of 20 intrabony defects sites were selected for this comparative clinico-radiographic study. The study subjects signed an informed consent form approved by the ethical committee of I.T.S Centre for dental studies and research.

Study design

The sample size of 20 patients was determined by a statistical software. Study was designed as a randomized clinical trial comparing periodontal clinical outcomes using DFDBA in combination with autologous PRP preparation (test) or a saline solution (control) in treatment of intrabony defects. Criteria for inclusion were individuals who were systemically healthy, no contraindications to surgery, no abnormal platelet counts, not received antibiotic therapy six months prior to treatment, not received any periodontal therapy for two years, non-smokers, had at least one vertical osseous defects with residual probing pocket depth of >5 mm with radiographic evidence of vertical/angular bone loss in the affected sites (At atleast one site in the quadrant) and had at least 2 mm of keratinized gingiva on facial aspect of selected tooth. Criteria for exclusion were patients allergic or sensitive to any medication and/or local anesthesia. Patients showing unacceptable oral hygiene compliance, pregnant and lactating mothers and teeth with severe attrition and excessive mobility.

Initial therapy (pre-surgical therapy)

Initial therapy consisted of oral hygiene instructions, scaling and root planing. Patients who met all criteria for entry into surgical phase of therapy were then randomized by a coin toss to test (PRP + DFDBA) or control (DFDBA) study groups.

Measurements of clinical parameters

At baseline, 3 and 6 months after surgical procedure, plaque index, gingival index, probing pocket depth, clinical attachment level and gingival marginal position were recorded by a single blinded observer using a University of North Carolina 15 (UNC 15) probe and a customized acrylic stent with a guiding groove.[9] All customized acrylic stents were stored on prepared study casts throughout study period to minimize distortion.[9] Probing pocket depth was recorded by noting the difference between measurements from fixed reference point to gingival margin and to the base of the pocket. Changes in clinical attachment level were recorded by noting the difference between measurements from the distance from fixed reference point to the base of the pocket and from fixed reference point to the cementoenamel junction. Gingival margin position was recorded by noting the difference between measurements from the distance from fixed reference point to the gingival margin and to the cementoenamel junction.

Radiographic assessment

Intraoral periapical radiographs were taken by long cone/extension cone paralleling technique using a positioning devices and an IOPA X-ray film in an X-ray unit (70 kVp, 4-8 mA, 0.630 mAs). A single grid was used along with all IOPA radiographs for measuring radiographic defect depth. Processing and viewing of the X-ray films were standardized [Figures 1 and 2].

Figure 1

Test Group (PRP+DFDBA) - Preoperative and 6 months postoperative radiograph

Figure 2

Control Group (DFDBA + Saline) - Preoperative and 6 months postoperative radiograph

Bone defect depth was measured as the distance from the alveolar crest to the base of the bone defect. The base of defect (BOD) was defined as the most coronal point where the periodontal ligament space showed continuous width. The alveolar crest (AC) level was taken as the crossing of the alveolar crest with the root surface.[10] The amount of defect fill was the difference between the initial and final defect depths at recalled time interval. Percentage (%) of defect fill was the amount of defect fill/baseline defect depth × 100.[11]

Preparation of PRP

PRP was prepared as described by Marx et al.[12] Sixteen milliliter of blood was drawn from anticubital region of patients’ forearm in vaccutainer containing heparin sodium as an anti-coagulant. Whole blood was then centrifuged at 1000 rpm for 10 minutes. The supernatant formed was PRP. PRP, buffy coat and upper 1-2 mm of R.B.C layer was collected in a fresh vaccutainer and again centrifuged at 1000 rpm for 10 minutes. Upper half of supernatant was discarded and lower half was mixed thoroughly to yield PRP. 2.5 ml of PRP was thoroughly mixed with 0.08 ml of CaCl2. This resulted in a clot formation and thrombin formation. The clot was discarded and thrombin was used for preparation of PRP. PRP + thrombin at 37°C for 2-3 min led to formation of PRP gel. Human DFDBA with a particle size 500-1000 μm was used as bone graft for all patients.

Treatment procedure

A standardized surgical procedure using a conventional periodontal flap surgery was performed by a single operator. Site was anesthetized using 2% lignocaine hydrochloride with adrenaline 1:200,000. Intracrevicular incisions were made, mucoperiosteal flaps were elevated and intrabony defects fully exposed. Meticulous defect debridement and root planing were carried out. Care was taken to keep the area free of saliva. Immediately before application PRP was activated by autologous thrombin with 1 ml of 10% CaCl2. Within a few seconds, the PRP preparation assumed a sticky gel consistency. Depending on the extent of intrabony osseous defect, coagulated PRP + DFDBA was placed up to vertical height of corresponding adjacent bone level. Surgical flaps were repositioned to presurgical level and sutured with 4-0 silk suture. A periodontal dressing was placed on surgical area. Postoperative medications included a single standard regimen of oral administration of Amoxycillin 500 mg thrice daily for 5 days, Metronidazole 400 mg thrice daily for 5 days and Ibuprofen 400 mg + Paracetamol 325 mg thrice daily for 5 days, along with 0.2% chlorhexidine gluconate rinse twice daily for a period of 2 weeks.

Post-surgical care and post-treatment assessments

Ten days following surgery, dressings and sutures were removed and patients were asked regarding discomfort, pain and sensitivity. All patients were recalled at one-month interval for check-up. After 3 months and 6 months postoperatively, parameters were recorded again.

Statistical Analysis

All the clinical parameters and radiographic measurements were subjected to statistical analysis using the statistical package SPSS v. 19. Clinical and radiographic parameters were subjected to Student's ‘t’ test and the ‘P’ values were obtained with appropriate levels of significance i.e., 5%. (statistically significant for P ≤ 0.05).

RESULTS

In the present study, 20 patients of Indian origin (11 males and 9 females, Graph 1) with ages ranging from 20 to 47 years, fulfilling the inclusion and exclusion criteria, contributing to a total of 20 intrabony defects were recruited. These 20 periodontal intrabony defects were then randomly assigned into experimental and control sites. All the preoperative variables were not statistically significant on comparison between the test and the control group, therefore assuring homogenicity between the two groups.

Graph 1

Age and sex of the study sample patients

The Silness and Loe plaque index values reduction achieved in the test group at 3rd and 6th months post-surgery was 0.753 ± 0.1265 (40.39%), and 1.158 ± 0.1671 (62.12%), respectively. The reduction achieved in the control group at 3rd and 6th months post-surgery was 0.633 ± 0.0769 (34.87%) and 1.066 ± 0.1414 (58.72%), respectively. The difference between the experimental and control groups with respect to these measurements was statistically highly significant at 3 months (P = 0.00) but not significant at 6 months (P = 0.45) [Table 1].

Table 1

Parameters recorded

The Loe and Silness gingival index values reduction achieved in the test group at 3rd and 6th months post-surgery was 0.712 ± 0.1476 (35.02%) and 1.269 ± 0.1513 (62.41%), respectively. The reduction achieved in the control group at 3rd and 6th months post-surgery was 0.548 ± 0.1199 (30.02%) and 1.033 ± 0.1008 (56.59%), respectively. The difference between the experimental and control groups with respect to these measurements was statistically not significant at 3 months (P = 0.59) and at 6 months (P = 0.35) [Table 1].

The mean probing depths reduction achieved in the test group at 3rd and 6th months post-surgery was 2.90 ± 0.9944 (41.43%) and 4.1 ± 0.9944 (58.57%), respectively. The reduction achieved in the control group at 3rd and 6th months post-surgery was 2.2 ± 1.0328 (22.84%) and 2.6 ± 1.1738 (38.81%), respectively. The difference between the experimental and control groups with respect to these measurements were not statistically significant at 3 months (P = 0.47) but highly significant at 6 months (P = 0.02) [Table 1].

The mean clinical attachment level reduction achieved in test group at 3rd and 6th months post-surgery was 1.9 ± 0.5676 mm (27.94%) and 3 ± 1.0541 mm (44.12%), respectively. Reduction achieved in control group at 3rd and 6th months post-surgery was 1.5 ± 0.7071 mm (22.39%) and 2 ± 0.6667 mm (29.85%), respectively. Difference between experimental and control groups with respect to these measurements were not statistically significant at 3 months (P = 0.6008) and at 6 months (P = 0. 134) [Table 1].

Preoperative mean gingival margin position did not have any significant differences (P = 0.7263). The reduction achieved in test as well as control groups at 3rd and 6th months post-surgery was 0.6 ± 0.9661 mm (600%). The difference between experimental and control groups with respect to these measurements were not statistically significant at 3 and 6 months (P = 0. 24) [Table 1].

There was no statistical significant difference in radiographic bone level between the groups when the baseline (P = 0.62) and 3 months (P = 0.45) measurements were compared. However, there was a statistically significant difference in the value at 6 months (P = 0.01). Mean percentage of bone fill in control group obtained was 24.63%, whereas percentage of bone fill in experiment group was 45.54%, which is 21% more than control group [Table 1].

DISCUSSION

The ultimate goal of periodontal therapy is creation of an environment that is conducive to maintain dentition in state of optimum health, comfort and function. Schwartz et al. have stated that current widespread use of DFDBA is based on its purported osteoinductive ability.[13] Histologically, DFDBA has been shown to be more effective in inducing regeneration of periodontium.[141516] Garret has stated that growth factors can provide the additional stimulus.[17] Growth factors have pleiotropic effects, are potent modulators of cells residing within the periodontium and have been shown to exert favorable effect on periodontium.[18] A simple way to obtain these growth factors is PRP, a rich source of platelet-derived growth factors. PRP combined with hydroxyapatite, bovine porous bone mineral and barrier membranes has been tested in periodontal defects.[192021]

Human DFDBA with particle size 500-1000 μm was used in present study, as these sizes are recommended. The retention of blood clots is difficult when the between-particle distance is too large, whereas blood vessels cannot readily enter the material when the distance is too small.[2223]

Our study showed a statistically highly significant change in plaque index of both test and control groups from baseline to 3 months and 6 months. The comparison within the groups showed the change to be significant at 3 months, but not significant at 6 months. A statistically highly significant change in the gingival index of the test and control groups from baseline to 3 months and 6 months was noted. Comparison within the groups showed change to be not significant at 3 months and 6 months. Cortellini et al. have stated that such change in index scores may be attributed to good maintenance of oral hygiene by patients throughout the study period.[24] Piemontese et al., Yukna et al. and Srikanth et al. have shown similar changes in plaque index and gingival index in their study.[12526]

In the present study, probing pocket depth showed a highly significant reduction in both test and control groups from baseline to 3 months and 6 months. However, comparison within the groups showed change to be not significant at 3 months (P = 0.47) but significant at 6 months (P = 0.02), which is in accordance with studies by Piemontese et al., Richardson et al., Bender et al. and Hanna et al.[1272829] Greater reduction in Probing Depth observed in test group may be explained by additional biologic effects of PRP. Wikesjo et al. have stated that because of its high fibrin content, PRP preparation has a ‘sticky’ characteristic that works as a hemostatic and stabilizing agent.[30] It may aid in immobilizing the blood clot and bone graft in defect area, an important event in early phases of wound healing in periodontal regeneration.

In the present study, both test and control groups showed a statistically highly significant change in clinical attachment level from baseline to 3 months and 6 months. Comparison within the groups showed change to be not significant at 3 months and 6 months. These results are consistent with studies of Hanna et al. and Camargo et al.[2931]

In the present study, test and control groups did not show a statistically significant change in gingival marginal position from baseline to 3 months and 6 months. The comparison within the groups showed change to be not significant at 3 months (P = 0.79) and 6 months (P = 0.79). Piemontese et al. have reported contrasting results.[1] However, Park et al. and Lovelace et al. showed similar recession values, probably due to the fact that these studies were of equal duration i.e., 6 months.[3233] Primary reason for this change after treatment can be attributed to reduction in gingival inflammation and shrinkage of pocket wall.[21]

Radiographic evaluation of experimental and control sites showed progressive changes in appearance of graft material from time of placement to 3 and 6 months. Comparison across the groups also showed results to be not statistically significant at the end of 3 months but highly significant at 6 months. These results are in accordance with a study by Piemontese et al.[1] However, studies by Okuda et al., Aghaloo et al. and Wiltfang et al. show contrasting results.[193435] This may be explained by the fact that growth factors present in high concentrations at inappropriate times or for an extended duration can adversely affect cell behavior.[3536]

A combination of platelet-rich plasma (PRP), bovine porous bone mineral (BPBM) and guided tissue regeneration (GTR) has been shown to be effective in promoting clinical signs of periodontal regeneration in intrabony defects.[5] Camargo et al. have evaluated the additional benefits provided by the incorporation of platelet-rich plasma (PRP) into a regenerative protocol consisting of bovine porous bone mineral (BPBM) and guided tissue regeneration (GTR) in the treatment of intrabony defects in humans. They have concluded that PRP did not significantly augment the effects of BPBM and GTR in promoting the clinical resolution of intrabony defects.[37] However, recent studies by Lekovic et al. indicate that platelet-rich fibrin (PRF) can improve clinical parameters associated with human intrabony periodontal defects, and BPBM has the ability to augment the effects of PRF in reducing pocket depth, improving clinical attachment levels and promoting defect fill.[38] On the contrary, Dori et al. have shown that at one year after regenerative surgery with PRP + anorganic bovine bone material (ABBM) and ABBM alone, significant probing depth reductions and clinical attachment level gains were found, and the use of PRP failed to improve the results obtained with ABBM alone.[39]

The present study demonstrated good soft and hard tissue response after treatment of intrabony defects with either DFDBA alone or in combination with PRP. Both treatment groups showed a significant PD reduction, CAL gain, hard-tissue fill and bone-depth reduction at 6 months after surgery compared to baseline. Addition of PRP to DFDBA did not show any significant improvement in clinical parameters, except plaque index at 3 months and probing pocket depth at 6 months. However, radiographic defect fill showed a significant improvement in PRP + DFDBA group than in DFDBA group at 6 months, postoperatively. The limitations of the present study were a small sample size, shorter follow-up period, absence of re-entry and non-digital radiographic evaluation.

CONCLUSION

Within limits of the present study, combination of DFDBA + PRP, though effective in improving radiologic parameters did not enhance clinical outcome of therapy compared to DFDBA alone. Long-term trials, large samples and histological examination are needed to evaluate regenerative potential of this combination. Further studies using PRP are necessary to examine the individual role played by PRP in achieving such results.