Clinical and histological comparison of platelet-rich fibrin versus non-eugenol periodontal dressing in the treatment of gingival hyperpigmentation.

BACKGROUND: The platelet-rich fibrin (PRF) has proven an immense role in angiogenesis and epithelization in a wound healing process. The present study aims to ascertain PRF's beneficial role in wound healing after depigmentation surgery.
MATERIALS AND METHODS: A total of 12 systemically healthy controls included were divided into two groups after scalpel depigmentation procedure. PRF was prepared according to Choukroun's standard protocol. Using split-mouth design after depigmentation, one group received PRF membrane, and in second group non-eugenol periodontal dressing was placed. The participants were evaluated for visual analog scale (VAS), healing index (HI) on 3rd and 5th day. Epithelization test using toluidine blue and histological examination employing punch biopsy was done on the 5th day.
RESULTS: On statistical scale, VAS, HI, epithelization test, and histological findings were statistically significant in the two study groups. PRF group proved better epithelization test and inflammatory cell infiltration was less in PRF group which confirmed superior wound healing in the group.
CONCLUSION: PRF membrane postdepigmentation provided satisfactory patient comfort and enhanced the wound healing cascade.

INTRODUCTION

In the contemporary world, esthetic demands and concerns have increased the number of patients receiving gingival depigmentation surgeries for creating the pleasing appearance of gingiva. Among various endogenous factors implicated, namely thickness of the gingival epithelium, blood supply and amount of keratinization, melanin pigmentation reigns as most instrumental in determining the overall color of gingiva even though other pigments such as carotene, oxyhemoglobin, and reduced hemoglobin also contribute to the normal color of the integument, are also found in the masticatory mucosa.[12] Exogenous factors namely various drugs, heavy metals, endocrinal disturbances, genetics, inflammation, and various syndromes may exaggerate its expression further.

Melanocytes of dark-skinned and black individuals are uniformly highly reactive than in light-skinned individuals thereby amplifying phenotypic expression.[3] In spite of being a physiologic condition, it is the patient's esthetic need and concerns that have increased the number of patients receiving gingival depigmentation surgeries for creating pleasing appearance of gingiva. Various periodontal plastic surgical procedures have been employed for gingival depigmentation, specifically by chemicals, abrasion employing diamond bur, gingivectomy, soft-tissue autografts, partial thickness flap, cryosurgery, and lasers with each technique offering advantages and limitations.[456789]

One of the first and still popular techniques to be employed is the surgical removal of undesirable pigmentation using scalpels.[10] The procedure essentially involves surgical removal of gingival epithelium along with a layer of underlying connective tissue, thereby allowing the denuded connective tissue to heal by secondary intention. The new epithelium that forms is devoid of melanin pigmentation thereby providing the patient desirable results.[10]

The coverage of exposed connective tissue during the initial postsurgical phase minimizes the likelihood of postoperative bleeding and facilitates unperturbed healing by preventing surface trauma. Conventionally, periodontal dressings have been the material of choice for the clinicians owing to its inert mechanical advantages.[11] However, a feeling of foreignness in the mouth and abundant plaque accumulation along the undersurface during the healing phase has led the clinicians to explore more viable options.[12] The use of platelet-rich fibrin (PRF), a second-generation platelet concentrate (PC) first developed by Choukroun et al. as a membrane nonetheless could provide its innate wound healing properties and thus aid in faster epithelization.[13]

Thus, the present study was aimed to compare and evaluate clinically and histologically the wound healing with PRF membrane and non-eugenol periodontal dressing (NEPD) after depigmentation by use of scalpel technique.

MATERIALS AND METHODS

A total number of 12 systemically healthy controls falling in the age group of 16–33 (mean age 23.5) years were selected for this prospective, parallel arm, nonrandomized split mouth interventional study conducted in Haryana, India, between May and July 2018. The experiments were undertaken in accordance with the Helsinki Declaration of 1975, as revised in 2000. Ethical clearance was obtained from the Ethical Committee of the Institute. All participants were verbally informed about the study and written informed consent was obtained before participation.

All the patients reported of “blackish gums” which esthetically interfered with their smile. The patients' history revealed blackish discoloration of gingiva, which was present since birth suggestive of physiologic melanin pigmentation.[14] Exclusion criteria were patients with a history of severe acute or chronic systemic disease affecting periodontal condition, patients who had received periodontal therapy in the past 6 months and/or are taking antibiotics for any chronic inflammatory condition, use of tobacco in any form, pregnant and/or lactating females, patients having teeth with inadequate endodontic treatments, gingival recessions. Intraoral examination of the selected patients exhibited generalized diffuse blackish pigmentation of gingiva, on the labial surface of both maxillary and mandibular arches; however, it was healthy and free of any inflammation. The patients selected for the study had thick biotype and ≥2 mm of attached gingiva [Figure 1].

Figure 1

Pre-operative picture showing melanin pigmentation

On fulfilling the criteria, the selected patients were made aware of the surgical procedure, the possible benefits of the use of PRF and all participants were also told about the follow-up visits before treatment.

Surgical procedure

After the administration of infiltration anesthesia with 2% lignocaine with adrenaline 1;200,000, a partial thickness flap was raised with Bard Parker handle with No. 15 surgical blade where a thin layer of gingival epithelium along with a layer of connective tissue was removed. This procedure was carried out from the first premolar of the right side maxillary/mandibular arch to the first premolar of the maxillary/mandibular arch in all the patients. Hemorrhage during the surgical procedure was controlled by direct pressure applied with sterile saline soaked gauge. The entire visible pigmentation was removed, exposing the underlying connective tissue. The PRF was prepared according to the recommended protocol by Choukroun [Figure 2].[13] In all the patients, the gingiva of 2nd quadrant was covered with PRF membrane, and simple interrupted sutures were given with 3-0 nonabsorbable silk suture, and that of 1st quadrant was covered with a non-eugenol periodontal dressing [Figure 3]. All the patients were prescribed analgesic (Ibuprofen 400 mg three times a day for 3 days) and were refrained from brushing for 3 days following the procedure and were instructed to rinse mouth with 0.2% chlorhexidine digluconate mouth wash. On the 3rd postoperative day, patients were recalled, and the sutures were removed, and non-eugenol periodontal dressing was also removed [Figure 4].

Figure 2

PRF isolated following Choukroun's protocol

Figure 3

Non-eugenol periodontal pack applied in 1st quadrant while PRF membrane sutured in 2nd quadrant

Figure 4

3rd post-operative day view after non-eugenol periodontal pack removal and suture removal

The clinical and histological parameters were evaluated following the surgical procedure. The visual analog scale (VAS) and healing index (HI) were assessed at 3rd and 5th day.[1516] Epithelization test by toluidine blue was carried out 5th day [Figure 5].[17] The histological analysis was done on the 5th day by punch biopsy at mid of 11 and 21 region of both quadrants [Figures 6 and 7].

Figure 5

Epithelization test on 5th post-operative day

Figure 6

H&E stained histomicrograph at non-eugenol periodontal pack site at 10X magnification

Figure 7

H&E stained histomicrograph at PRF site at 10X magnification

Statistical analysis

The results were statistically evaluated using Statistical package for the social sciences (SPSS) version 21 (IBM Corp., Chicago, Illinois, USA). All the variables were summarized as mean and standard deviation. Inferential statistics were performed using independent t-test, post hoc pairwise comparison was done using post hoc Turkey's test. The level of statistical significance was set at 0.05.

RESULTS

The employment of PRF on the scalpel treated depigmented sites showed encouraging results with respect to clinical parameters and histological analysis compared to sites which received the application of periodontal dressing after 2 weeks [Figure 8].

Figure 8

Final post-operative view after two weeks

Visual analog scale

The VAS was checked at 3rd and 5th day postoperatively. On the 3rd day, 5 (41.7%) patients reported of moderate pain in PRF group whereas in non-eugenol periodontal dressing group 10 (83.3%) patients complained of severe pain. On the 5th day, all 12 (100%) patients in PRF group reported of no pain, while on the other hand, moderate pain was seen in all 12 (100%) patients in non-eugenol periodontal dressing group. Intergroup results were statistically significant on both 3rd and 5th day (P<0.0001) [Table 1 and Graph 1].

Table 1

Visual analog scale rating obtained on 3rd and 5th postoperative day

Pain (VAS scores)	At 3rd day		At 5th day
	PRF, n (%)	NEPD, n (%)	PRF, n (%)	NEPD, n (%)
No pain	7 (58.3)	0	12 (100)	0
Mild pain	0	0	0	0
Moderate pain	5 (41.7)	2 (16.7)	0	12 (100)
Severe pain	0	10 (83.3)	0	0
Pa	<0.0001		<0.0001

aChi-square test; P<0.05 implicating statistically significant difference. PRF – Platelet-Rich Fibrin; n – Number of individuals;P<0.0001; NEPD – Non-eugenol periodontal dressing; VAS – Visual Analog Scale

Graph 1

Percentage of visual analog scale perceived by patients at 3rd and 5th postoperative day

Healing index

HI was evaluated according to Landry et al.[16] On the 3rd postoperative day, all the individuals of PRF group showed good healing, and it was very good on 5th postoperative day in the same group. In non-eugenol periodontal dressing group, 7 (58.3%) patients had poor healing score on the 3rd day after surgery. Intergroup comparison of HI score was statistically significant (P<0.0001) as can be seen in Table 2 and Graph 2.

Table 2

Healing index score obtained on 3rd and 5th postoperative day

Healing index	At 3rd day		At 5th day
	PRF, n (%)	NEPD, n (%)	PRF, n (%)	NEPD, n (%)
Very good	0	0	12 (100)	0
Good	12 (100)	5 (41.7)	0	12 (100)
Fair	0	0	0	0
Poor	0	7 (58.3)	0	0
Pa	<0.0001		<0.0001

aChi-square test; P<0.05 implicating statistically significant difference. PRF – Platelet-Rich Fibrin; n – Number of individuals;P<0.0001; NEPD – Non-eugenol periodontal dressing

Graph 2

Percentage of healing index score at 3rd and 5th postoperative day

Epithelization test

All surgically depigmented sites were stained with toluidine blue on 5th day postoperatively. The amount of staining a site picks up tells about the inflammatory concentrate underneath. 7 (58.3%) individuals in PRF group picked up mild staining, and it was statistically significant (P<0.0001) as compared to non-eugenol periodontal dressing group where 8 (66.7%) patients picked up moderate and 4 (33.3%) patients reported of severe staining. The toluidine blue score clearly indicates the presence of more inflammatory cells in non-eugenol periodontal dressing group which was further confirmed through histological analysis [Table 3 and Graph 3].

Table 3

Epithelization test score on 5th postoperative day

Epithelization test	At 5th day
	PRF, n (%)	NEPD, n (%)
Mild	7 (58.3)	0
Moderate	5 (41.7)	8 (66.7)
Severe	0	4 (33.3)
Pa	<0.0001

aChi-square test; P<0.05 implicating statistically significant difference, PRF – Platelet-Rich Fibrin; n – Number of individuals; P<0.0001; NEPD – Non-eugenol periodontal dressing

Graph 3

Percentage of epithelization at 5th postoperative day

Histological evaluation

Histological analysis was carried out by means of punch biopsy in which a section of tissue was taken out from mid of 11 and 21 region of both quadrants. Hematological and eosin stained slides were prepared and evaluated at ×40 magnification. Eight (66.7%) individuals in PRF treated sites showed negligible inflammatory infiltrate in the connective tissue whereas in non-eugenol periodontal dressing sites, 9 (75%) of individuals demonstrated chronic inflammatory cell infiltrate consisting of lymphocytes in the connective tissues. The results of the histological analysis were statistically significant between the two groups (P<0.0001) [Table 4 and Graph 4].

Table 4

Histological analysis for inflammatory cells on the 5th postoperative day

Percentage of histological analysis	At 5th day
	PRF, n (%)	NEPD, n (%)
Nil	0	0
Mild	4 (33.3)	0
Moderate	8 (66.7)	3 (25.0)
Severe	0	9 (75.0)
Pa	<0.0001

aChi-square test; P<0.05 implicating statistically significant difference. PRF – Platelet-Rich Fibrin; n – Number of individuals; P<0.0001; NEPD – Non-eugenol periodontal dressing

Graph 4

Percentage of histological findings at 5th postoperative day

DISCUSSION

When gingival hyperpigmentation becomes an esthetic concern, it compels the patients to undergo depigmentation surgery despite it is not a medical condition. The scalpel surgery though may cause unpleasant bleeding during and after the procedure and leaves behind an open connective tissue wound to heal by second intention, however, the vested advantage of cost: benefit ratio with scalpel's use and faster postoperative healing makes it a treatment of choice among others.

The gingival depigmentation surgery leaves behind an open connective tissue wound, and periodontal dressings have long been used to cover the exposed lamina propria at surgical sites.[18] PRF, on the other hand, has been found to deliver growth factors (GFs) in high concentration to the surgical sites and thus promote wound healing, maturation, wound sealing, and hemostasis.[19] Thereby, the present study was undertaken to evaluate the possible augmented healing effects of PRF as a membrane as visualized through VAS, HI, epithelization test, and histological evaluation as compared to non-eugenol periodontal dressing following depigmentation procedure.

A split-mouth design was chosen to compare the two treatment modalities which is an excellent method to eliminate the influence of numerous inter-subject factors, such as age, facial complexion, genetics, and environmental risk factors.

Literature amply documents the fact that non-eugenol periodontal dressing is a dimensionally unstable material which shows contraction during the 1st min after completion of their setting, resulting in delayed healing.[20] Although biocompatible, yet there are chances of foreign body reaction if non-eugenol periodontal dressing becomes embedded in the tissues or underneath the flap.[21] A number of clinical trials have proposed that the use of this non-eugenol periodontal dressing accumulates plaque-causing inflammation, irritates the healing tissue, and produces transient bacteremia postoperatively.[2223]

PRF, an activated fibrinogen, a second generation PCs, has properties superior to first-generation PCs, i.e., platelet-rich plasma.[13] The fundamental advantage offered in PRF is because of elimination of the addition of bovine-derived thrombin step, and thereby the conversion of fibrinogen to fibrin takes place slowly with small amounts of physiologically available thrombin present in the blood sample itself. Consequently, the physiological architecture of fibrin structure attained is due to the slow polymerization process which is favorable to the healing process as it causes release of various GFs for a prolonged period.[19]

The intrinsic incorporation of cytokines within the fibrin mesh allows for their progressive release over time (7–10 days) as the network of fibrin disintegrates.[24] Corso MD et al. reported that fibrin network allows for cellular migration, particularly for endothelial cells necessary for neoangiogenesis and vascularization.[19] Furthermore, it causes steady release of platelet-derived GF, transforming GF, vascular endothelial GF (EGF), EGF, fibroblast GF, and insulin GF as the fibrin matrix gets resorbed, thus creating a perpetual process of healing.[25]

Fibrin and fibrinogen degradation products increase the membrane's expression of CD11c/CD18 receptor which permits the adhesion of neutrophils to endothelium and fibrinogen as well as transmigration of neutrophils that is crucial to initiate repair. Concomitantly around the wound margins, epithelial cells lose their basal and apical polarity and produce basal and lateral extensions towards the wound side thereby instituting re-epithelization. The remodeling of fibrin matrix hence produces a more resistant connective tissue thereby reigning PRF membranes superior for all types of superficial cutaneous and mucous wound healing.[26] This truly justifies the term “healing biomaterial” attributed to PRF.[13] In coherence with the aforementioned facts, a statistically significant difference was observed in VAS score, HI, and epithelization test in PRF Group compared to non-eugenol periodontal dressing group in the present study.

The histological examination also revealed a statistically significant difference in PRF applied sites compared to periodontal pack group. The lack of inflammatory infiltrate and signs of parakeratinized epithelium in PRF group may be due to slow polymerization process and entrapment of leukocytes and cytokines in PRF scaffold which play an important role in GF release.[2728] It is the high concentration of platelets in PRF which are considered to be the main precursor for the regeneration of epithelium and connective tissue in the PRF-treated sites. Platelets are reservoir of GFs that lead to cascade of events in series, i.e., neovascularization, collagen synthesis, cell division, cell differentiation induction, and migration of other cells to the injured/treated site.[29]

Conversely, periodontal pack shows contraction during the first few minutes after completion of their setting resulting in delayed healing.[30] Similar results were obtained by Bansal et al. in 2016 where the PRF membrane and non-eugenol periodontal dressing were applied in five patients.[26] Debnath and Chatterjee in 2018, evaluated use of PCs in depigmented gingival epithelium. They used PRF, PRF matrix (PRFM), and periodontal pack and the use of PRF was found to be superior as compared to PRFM and periodontal pack.[31] PRF group produced better results as release of GFs occurs in a steady manner over a period of 10 days whereas in first-generation PCs (PRFM), there is a rapid release of GFs as observed in our study group.

The results of the present research study are in favor of PRF as a postsurgical dressing but shorter sample size and shorter duration kept for the evaluation of results were its limitations. Future studies should be directed to enroll a larger number of participants, and the results should be assessed on a long-term basis so that the results can be extrapolated for PRF's use as a dressing in diverse surgical techniques.

CONCLUSION

In recent times, the beneficial role of PRF in augmenting periodontal wound healing has been researched on a lot. The synergistic effect of three-dimensional structure of fibrin and entrapment of leukocytes augment the healing process. A better understanding of this interesting bioactive material's inflammatory features will further enhance its therapeutic application in various periodontal surgical procedures.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.