Clinicoradiographic evaluation of advanced-platelet rich fibrin block (A PRF + i PRF + nanohydroxyapatite) compared to nanohydroxyapatite alone in the management of periodontal intrabony defects.

Background: Several bone grafting formulations have been given clinically acceptable outcomes in treating intrabony defects. Platelet rich fibrin (PRF), an autologous platelet concentrate holds potential to be used for regenerative treatment. The purpose of this study was to evaluate clinical and radiographic outcomes in periodontal intrabony defects treated with advanced-PRF block (A PRF + i PRF + nanohydroxyapatite [nHA]) compared to nHA alone.
Methods: Twenty-eight sites in chronic periodontitis patients having probing pocket depth (PPD) ≥6 mm and 3 walled intrabony defects (depth of ≥3 mm) were selected, randomly allotted into two groups: Group A was treated with A-PRF block and Group B with nHA (Sybograf™). Clinical parameters including plaque index (PI), gingival index (GI), PPD, relative attachment level (RAL) and radiographically linear and volumetric defect fill were assessed using cone beam computed tomography at baseline and 6 months postoperatively.
Results: Intragroup comparison using paired t-test and intergroup comparison using unpaired t-test was done. Group A demonstrated significantly higher reduction in PPD and gain in RAL when compared to Group B (P ≤ 0.05) at the end of 6 months. Similarly gain in bone volume was greater in Group A (0.1 ± 0.05) as compared to Group B (0.04 ± 0.02) (P ≤ 0.05).
Conclusion: Advanced-PRF block showed significant clinical and radiographic improvement as compared to nHA alone which depicts that, it may be an ideal graft to be used for the treatment of periodontal intrabony defects. Copyright:

INTRODUCTION

Periodontitis is a polymicrobial disease where the inflammatory process results in progressive tissue destruction. The ultimate aim of periodontal therapy is to arrest the disease progression and to regenerate the lost tissues.[1] Till date, several procedures have been developed in an attempt to regenerate the lost bone with the use of various bone graft materials, guided tissue regeneration, and/or the combination therapy with growth factors.[234] Nanosized ceramics is a new class of material which demonstrates superior properties over its microscale counterpart. Use of nanocrystalline hydroxyapatite (nHA) (Sybograf™) promotes osteoblast cell differentiation, proliferation, adhesion, and deposition of calcium containing minerals on bone surface better than microcrystalline HA; thus enhancing new tissue formation within a short period.[5]

Platelet rich fibrin (PRF) is the second generation autologous platelet concentrate introduced by Choukroun et al. in 2001. Various modifications of PRF clots have been introduced by reducing the speed and increasing time which exhibited promising outcome for the clinicians to perform research. Recently, Ghanaati et al. in 2014 proposed a new modification, Advanced-PRF (A-PRF) based on low speed centrifugation protocol which has an added benefit of evenly distributed cells facilitating regeneration.[67] Further, Miron et al. demonstrated a new protocol of injectable-PRF (i PRF), a liquid formulation which clots outside the PRF tube to accelerate the number of growth factors responsible for tissue regeneration.[8] Recently, a novel method of “PRF block” consisting of pieces of L PRF mixed with i-PRF and bone graft formed into a cohesive block was proposed.[9] This block forms a volumetric matrix which can be used to fill various osseous defects. Cortellini et al. used PRF block for ridge augmentation procedures and observed good results with superior bone formation.[10] It is also observed that the presence of platelets in the PRF block bio-activates the bone grafts over a period of time within the defect.[11]

This clinical study emphasizes the importance of combined use of different formulations of PRF (A PRF + i PRF) with bone graft, making it a user friendly matrix which can occupy 3 dimensional osseous defects. On thorough research, there was no available study where A-PRF block was used in the management of intrabony defects. Hence, the present study was aimed to evaluate clinically and radiographically, the regenerative potential of A-PRF block over nHA (Sybograf™) in the management of periodontal intrabony defects.

METHODS

A total of 28 sites from 20 chronic periodontitis patients, in the age group of 30–50 years were selected from the outpatient section, Department of Periodontics. Calculation of sample size (n) was done using the G power 3.1.9.4, alpha was fixed at 0.05 and beta at 0.80, effect size = 1.125. To calculate the sample size mean difference of bone density at the end of 6 months was considered.[12] Minimum sample size obtained was 14 per group and the total sample size was 28. The sites were divided into 2 groups with an allocation ratio of 1:1 i.e., group A treated with A-PRF block and group B treated with nHA (Sybograf™) alone.

Ethical clearance was obtained from the Institutional ethical Review board. Written consent was obtained from all the participants and the study was carried out in accordance with the declaration of Helsinki.

Chronic periodontitis patients were selected based on 1999 AAP classification. Inclusion criteria were: Patients of either sex with good oral hygiene[13] having platelet count within normal limits (1.5–3 lakhs/μL). Site specific criteria were, presence of atleast one/two sites with probing pocket depth (PPD) of ≥6 mm and 3 walled defect of depth of ≥3 mm as confirmed by cone beam computed tomography (CBCT).[12] The study excluded chronic smokers, patients with hypertension, diabetes or any other systemic disorders which affect the outcome of the treatment. Patients who were unwilling to participate in the study, pregnant and lactating mothers were also excluded.

Clinical parameters recorded at baseline and 6 months after surgery included PI,[14] Gingival index (GI),[14] PPD, relative attachment level (RAL) [Figure 1b and 2b].[1214] RAL was measured by making vertical grooves on the stent to guide the placement of UNC 15 probe (Hu-Friedy, Chicago, IL) and [Figures 1a and 2a]. All the clinical parameters were assessed by JM and referee was TMG who were both blinded to the study groups.

Figure 1

Experimental site A: (a) Preoperative clinical measurement of relative attachment level and probing pocket depth. (b) Intrasurgical measurement of the defect. (c) Preparation of A-platelet rich fibrin block (A platelet rich fibrin + i platelet rich fibrin + Sybograf™). (d) A-platelet rich fibrin block prepared. (e) Prepared graft placed in the defect secured with the suture. (f) 6-month postoperative view

Figure 2

Experimental site B. (a) Preoperative clinical measurement of relative attachment level and probing pocket depth. (b) Intrasurgical measurement of the defect. (c) Graft placement in the defect. (d) 6-month postoperative view

For the radiographic assessment of the intrabony defect, CBCT scans were taken at baseline and 6 months postoperatively. The slice thickness was standardized to 1.0 mm in sagittal section. Base of the defect (BOD), alveolar crest (AC) and CEJ of the adjacent tooth was located. Defects fill (change in distance from cement enamel junction to BOD from baseline to 6 months) and defect resolution (change in distance from AC to base of defect from baseline to 6 months) was recorded [Figures 3a and 4a].

Figure 3

Experimental site A (a) Preoperative linear measurement of the defect of 3.2 mm. (b) 6 month postoperative linear measurement of the defect of 2.85 mm. (c) Coronal view of preoperative volumetric assessment of the defect area of 143 mm3. (d) Sagittal sectional view of preoperative volumetric assessment of the defect of 80 mm3. (e) Coronal view of postoperative volumetric assessment of the defect of 92 mm3. (f) Sagittal sectional view of postoperative volumetric assessment of the defect of 71mm3

Figure 4

Experimental site B. (a) Preoperative linear measurement of the defect of 7.5mm. (b) 6 month postoperative linear measurement of the defect of 6.45mm. (c) Coronal view of preoperative volumetric assessment of the defect of 148mm3. (d) Sagittal sectional view of preoperative volumetric assessment of the defect of 65mm3. (e) Coronal view of postoperative volumetric assessment of the defect of 131mm3. (f) Sagittal sectional view of postoperative volumetric assessment of the defect of 71mm3

Routine blood investigations were performed prior to the commencement of the study. After 6–8 weeks of phase I therapy the sites were divided randomly into Group A and Group B. Following open flap debridement (OFD), Group A was treated with A-PRF block (APRF + i PRF + nHA) and Group B with nHA alone. All the surgical procedures were carried out by DV.

Following aseptic precautions, an adequate local anesthesia was administered. A full thickness mucoperiosteal flap was reflected, thorough debridement of the osseous defect was done using Gracey curettes and Columbia universal curettes (Hu-Friedy, Chicago, IL).

A-PRF was prepared according to the protocol given by Ghanaati et al.[1516] Ten ml of venous blood was drawn from the patient's antecubital vein in the Choukroun A-PRF tubes and was centrifuged (Choukroun A-PRF™ 12) immediately at 1500 rpm for 14 min. The fibrin clot was then placed in the expression box to obtain A-PRF membrane.

I-PRF was prepared as per the protocol by Miron.[8] Nine ml of venous blood was collected in Choukroun i-PRF tube and centrifuged immediately at 700 rpm for 3 min. Upon termination of centrifugation, liquid i-PRF was aspirated from the top layer of the tube using a 2 ml syringe.

Alloplast nHA graft (0.5cc Sybograf™ Particle size: 600-700 microns, Eucare Pharmaceuticals Private Limited, Chennai, India) was taken in a bone well, to which i-PRF was mixed and the cut pieces of the A-PRF membrane was incorporated during the polymerization phase [Figure 1c]. The resulted cohesive, packable graft was “A-PRF block” [Figure 1d] which was then placed into the defect [Figure 1e].

In Group B, nHA (Sybograf™) was mixed with normal saline and placed into the defect with light incremental pressure till the level of adjacent bone crest [Figure 2c]. Care was taken not to overfill the defect. After grafting, in both the groups, the flaps were repositioned, ethicon nonresorbable interrupted and vertical mattress sutures were given to obtain primary closure [Figure 2].

The patients were prescribed Amoxicillin 500 mg thrice daily for 5 days and analgesic (Diclofenac sodium 50 mg) were prescribed as and when required. Chlorhexidine 0.2% (Rexidin®, Indoco Remedies Ltd, Mumbai, India) was advised for 2 weeks. Patients were recalled 2 weeks after surgery for suture removal. At the end of 6 months, all the clinical and radiographic parameters [Figures 1e and f, 3e and f, 4e and f] were recorded.

All the patients completed their follow up and there were no drop outs from the study. Intragroup comparison was done using paired t-test and intergroup comparison using unpaired t-test. P ≤ 0.05 was considered statistically significant and P ≤ 0.001 was considered statistically highly significant.

The two investigators (DV and MJ) underwent training for the assessment of clinical parameters and radiological defect fill, defect resolution before the commencement of the study. The gold standard (TMG) examiner conducted demonstration and examination of clinical cases to the investigators. The inter examiner Correlation Coefficient value for radiographic parameters was 0.8 and for clinical parameter was 0.9.

RESULTS

The mean age range of the participants in group A was 39.6 ± 4.33 years and for group B it was 40.2 ± 5.70 years (P > 0.05). There was no statistically significant difference in intra and inter group scores in the PI and GI at baseline and 6 months in both the groups [P > 0.05, Table 1]. Whereas Group A demonstrated significantly higher reduction in PPD (baseline 7.1 ± 1.38 mm to 6 months 3.6 ± 0.74 mm, P ≤ 0.001) when compared to group B (baseline 6.5 ± 0.74 mm to 6 months 4.3 ± 0.82 mm). Also, gain in RAL in group A (baseline 9.8 ± 1.72 mm to 6 months 6.5 ± 1.34 mm) was significantly better when compared to group B (baseline 10 ± 1.93 mm to 6 months 7.9 ± 2.10 mm) [P ≤ 0.05, Table 1].

Table 1

Comparison of clinical parameters between the experimental site A and site B at baseline and 6 months

Parameters	Mean±SD
	Group A		Group B
	Baseline	6 months	Baseline	6 months
PI	0.7±0.16	0.6±0.20	0.8±0.17	0.7±0.11
GI	0.7±0.17	0.6±0.16	0.7±0.30	0.7±0.19
PPD (mm)	7.1±1.38	3.6±0.74**	6.5±0.74	4.3±0.82*
RAL (mm)	9.8±1.72	6.5±1.34**	10±1.93	7.9±2.10*

*Statistically significant intragroup difference from baseline to 6 months in site A and site B (P<0.05), **Statistically highly significant difference in site A when compared to site B from baseline to 6 months (P<0.001), ‘P’ (calculated probability) value <0.05 is considered as statistically significant. PI – Plaque index; GI – Gingival index; PPD – Probing pocket depth; RAL – Relative attachment level; SD – Standard deviation

Radiographically, mean change in AC height and mesio-distal width from baseline to 6 months was statistically highly significant for both the groups (P ≤ 0.001). However, the difference between the groups was not significant at 6 months [P > 0.05, Table 2]. The mean defect fill for Group A was 3.2 ± 0.72 mm where as for Group B it was 2.3 ± 0.67 mm and the difference between them was found to be statistically highly significant [P ≤ 0.001, Table 2]. Similarly, the mean defect resolution was 4.0 ± 0.7 mm for group A whereas it was 2.8 ± 0.73 mm for group B and the difference between them was statistically highly significant [P ≤ 0.001, Table 2]. Also, mean volume of bone gain from baseline to 6 months was found to be 0.1 ± 0.05 mm3 and 0.04 ± 0.02 mm3 for group A and group B respectively and the difference between them was found to be statistically significant [P < 0.05; Table 2].

Table 2

Comparison of radiographic parameters between the experimental site A and site B at baseline and 6 months

Parameters	Mean±SD
	Group A		Group B
	Baseline	6 months	Baseline	6 months
Defect fill (mm)	8.3±1.19	5.0±0.85**	8.4±1.46	6.2±1.34*
Alveolar crest height (mm)	3.1±0.98	3.8±0.95**	3.7±1.35	4.2±1.33*
Mesio-distal width (mm)	3.3±0.88	2.8±0.74**	3.6±0.54	3.1±0.43*
Defect resolution (mm)	5.2±0.99	1.3±0.91**	4.7±0.38	2.0±0.88*
Mean volume of bone gain (mm3)	0.1±0.05**		0.04±0.02

*Statistically significant intragroup difference from baseline to 6 months in site A and site B (P<0.05), **Statistically highly significant difference in site A when compared to site B from baseline to 6 months (P<0.001) (P<0.001 is statistically highly significant), ‘P’ (calculated probability) value <0.05 is considered as statistically significant. SD – Standard deviation

DISCUSSION

Attempts to achieve periodontal regeneration have evolved from root debridement, soft tissue curettage to the advanced approaches in aiding guided regeneration by the utilization of growth factors. PRF is one such formulation having the capacity for stimulating tissue regeneration and acts as a immune modulating node and trap for stem cells.[15] In a recent systematic review it was concluded that, PRF gives superior results in the augmentation of intrabony defects as compared to OFD.[17] A novel formulation of PRF is the PRF block, it is a combination of liquid fibrinogen and a bone graft (LPRF + iPRF + bone graft), which was used in horizontal ridge augmentation procedure with superior bone formation.[910] Similarly in the present study, APRF block was prepared using APRF + iPRF + nHA (Sybograf™) which may provide beneficial results as compared to nHA (Sybograf™) alone in the management of periodontal intrabony defects.

In this study the mean reduction in probing depth and gain in RAL was statistically highly significant in group A (P ≤ 0.001) over Group B at 6 months postoperatively. Results of the present study is in agreement from the previous studies, where PRF and nHA demonstrated statistically significant advantages with respect to PPD, gain in RAL and radiographic osseous fill.[18] Singh et al. observed clinical benefit with nHA (Sybograf™) with collagen membrane compared to OFD alone.[19] In another study Pradeep et al. compared PRF membrane combined with nHA (Sybograf™) to OFD and observed increase in the regenerative effect of nHA in 3 wall intrabony defects. Kasaj et al. in their clinical study observed that nHA provides increased surface area for its attachment to the bone, thereby improves the bone healing by stimulating the osteoblastic activity.[12]

In the present study, the radiographic parameters were assessed through CBCT, as it allows three-dimensional assessment and volumetric analysis which may depict a better representation of an intrabony defect than linear measurements, and could be an effective alternative for the surgical re-entry [Figure 3b-d and 4b-d].[20212223] A significant gain in the bone volume (P ≤ 0.05) and a highly significant defect fill (P ≤ 0.001), defect resolution (P ≤ 0.001) was observed in group A as compared to group B at 6 months post operatively. Our radiographic results are in agreement with the studies done by Elegandy et al.,[18] Kasaj et al.[12] and Pradeep et al.[20]

Superior clinical and radiographic results obtained in our study can be attributed to the porous nature of A-PRF which is based on low speed centrifugation concept (LSCC). LSCC incorporates even distribution of platelet cytokines and neutrophillic granulocytes which are released gradually over a period of 7–10 days as the matrix degradation occurs.[15] Further, Chatterjee and coworker observed, PRF releases steady and consistent growth factors over a period of 23 days,[24] such distinctive property of PRF may have contributed to the better results in our study. Invitro studies revealed, the interconnected fibrin network of PRF block showed marked growth factor release, osteoblastic activity and accelerated wound closure with increased tissue remodeling.[2526] In A-PRF block, the cut membrane of the A-PRF acts as a biological matrix and promotes migration of osteoprogenitor cells to the centre of the graft.[10] A study by Clark and co-workers, assessed ridge augmentation using PRF block, the histomorphometric analysis revealed significant vital bone formation in the A-PRF with FDBA (freeze dried bone allograft) compared to the FDBA alone. They attributed this to the space maintenance characteristics of the PRF block which was the key success of their study.[27] This could be another reason for improvement of results of this current study.

Injectable-PRF polymerizes along with the graft and A-PRF to form a packable cohesive mass which can be easily compacted into the defect site.[2829] The benefit of adding i-PRF is bifold. One, it provides osteoinductive properties to the graft material by virtue of the sustained delivery of platelet growth factors like bone morphogenetic protein-2 and platelet-derived growth factor (PDGF).[92930] Another being, the addition of A-PRF and i-PRF to the bone graft acts as a biological connector,[153132] this advantage was utilized in our study. Invitro studies on i-PRF have demonstrated superior fibroblast migration with expression of PDGF, transforming growth factor-β, and collagen.[3033] It has higher antimicrobial efficacy compared to PRP and PRF, thereby reducing the chances of postoperative infection.[34] This could be the reason for good postoperative healing during the follow up of our patients.

Nanohydroxyapatite bone graft (Sybograf™) has a longer resorption time and provided space maintenance for new bone formation. Also an invitro study has proven that nHA has an increased expression of bone morphogenetic proteins on its surface leading to increased recruitment of osteoprogenitor cells thereby enhancing bone formation. It also promoted the migration and differentiation of human alveolar osteoblasts and encouraged bone regeneration.[35] This benefit of Sybograf™ was utilized as a choice of material in this study.

A study was done by Cortellini et al., where PRF block (L-PRF, DBBM, Liquid fibrinogen) was used for augmentation of alveolar bone. Significant gain in alveolar ridge width with 61% increase in the bone volume as compared to 39% of particulate bone graft material alone was observed.[10] Another study by Abdallah et al. used PRF block (L-PRF mixed with Easy-Graft Crystal) for alveolar ridge splitting procedure presented superior marginal bone level and bone density.[9] Similarly, in our study A PRF block (A PRF + i-PRF + nHA) showed superior clinical and radiographic parameters.

This is the first study to analyze the efficacy of A PRF block with nHA (Sybograf™) for the treatment of intrabony defects. Further studies with larger sample size with histologic analysis may validate the results obtained from the present study.

CONCLUSION

The concomitant use of A PRF, i PRF with nHA (Sybograf™) in periodontal intrabony defect hastened the bone formation and significantly increased the bone volume in comparison to nHA (Sybograf™) alone. The above study gives a new insight into its application in wide variety of osseous defects including intrabony defects, furcation defects, ridge augmentation and for sinus lift procedures.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.