Clinical and histological evaluation on application of platelet concentrates on depigmented gingival epithelium.

BACKGROUND: The platelet concentrate is a windfall in the field of regenerative therapy in periodontology. It accelerates wound healing by excellent neovascularization and promoting fast cicatricle tissue remodeling. AIM: This study aims to evaluate clinically and histologically accelerated effect of platelet-rich fibrin (PRF) membrane and PRF matrix (PRFM) following depigmentation procedure.
MATERIALS AND METHODS: Eleven individuals were divided into three groups after depigmentation procedure. PRF membrane and PRFM gel were prepared as per standard protocol. Group A and B received PRF membrane and PRFM gel followed by periodontal dressing, respectively, and the only periodontal dressing was placed in Group C. The individuals were evaluated for visual analog scale (VAS) and healing index (HI) on 3rd and 5th day. Epithelization test and histologic analysis from punch biopsy were done on the 5th day. At 3rd month, reevaluation was performed.
RESULTS: The intergroup statistical analysis in respect to VAS, HI, epithelization test, and histologic analysis showed a statistically significant results with P < 0.001 in Groups A and B compared to Group C. Clinical evaluation of epithelization test and histologic analysis revealed better-wound healing and moderate to no inflammatory cell infiltrate in Groups A and B, respectively, as compared to Group C, which appeared more erythematous with dense inflammatory cells.
CONCLUSION: Thus, the application of PRF membrane and PRFM gel has shown a successful approach to protect the raw wound area of depigmented sites with better patient comfort and faster healing.

INTRODUCTION

In today's era, facial cosmetic concerns, as well as increased intraoral awareness, have created a demand for esthetics in the periodontal practice. The gingiva is considered to be the most frequently pigmented region of the oral cavity. The color variation in the gingiva mainly depends on the thickness of the gingival epithelium, blood supply, amount of the keratinization, and number of the pigmented cells present.[1] Melanin, a nonhemoglobin endogenous-derived pigment, is responsible for normal pigmentation of the skin, gingiva, and remainder of the oral mucous membrane. The pigmentation appears as a diffuse purplish discoloration or light-brown patches which were irregularly arranged. Clinically, it is manifested as multifocal or diffuse melanin pigmentation. The distribution of the pigmentation is variable with different ethnic groups worldwide and is prevalent in all races.[23] The physiologic melanin deposition takes place by melanocytes present between basal and suprabasal cell layers of epithelium. The melanin pigmentation was mostly seen at a higher degree in the anterior region.[45] The size and degree of melanin pigmentation are directly proportional to the degree of melanin pigmentation. The brown or dark pigmentation or discoloration could be considered due to various multifactorial etiology and also due to genetic factors.[67891011]

Clinical melanin pigmentation is benign and is not a medical problem, but pigmented gingiva may be a cause of an esthetic concern particularly, especially during speech and smile. The demand for the cosmetic surgery was especially more seen in the fair-skinned individual with high smile line. Gingival depigmentation can be performed by various techniques including scalpel, bur, laser, and chemicals whereby the gingival hyperpigmentation is removed or reduced. The procedure aims in the removal of gingival epithelium along with a layer of the underlying connective tissue and allowing the denuded connective tissue to heal by secondary intention.[12] Coverage of exposed connective tissue minimizes the likelihood of postoperative bleeding and facilitates healing by preventing surface trauma. The use of platelet concentrates known for its immense reparative potential [13] could be a boon for rapid epithelization on the depigmented gingival surface.

Thus, the present research aimed to evaluate clinically and histologically the effect of platelet concentrates (platelet-rich fibrin [PRF] membrane and PRF matrix [PRFM] gel) after depigmentation.

MATERIALS AND METHODS

Study design

The nonrandomized split-mouth interventional study was conducted in Bengaluru, India. 11 healthy controls were recruited who had an esthetic concern due to gingival hyperpigmentation and demanded a need for depigmentation procedure. Ethical clearance was obtained from the local ethical committee of the institution.

The inclusion criteria included systemically healthy controls with an age range of 20–30 years. The individuals presented moderate clinical pigmentation which appeared to be medium brown or mixed pink and brown color as seen in Figure 1.[14] The plaque index score of <1[15] and gingival index score <1,[16] thick biotype, >2 mm of attached gingiva, and absence of endodontically involved teeth. The exclusion criteria included the presence of gingival recession, thin gingival biotype, individuals on antibiotic or anti-inflammatory drugs for past 3 months, underwent periodontal surgery in past 6 months, individuals with a habit of smoking/tobacco chewing and pregnant and lactating mothers.

Figure 1

Preoperative view of pigmentation

On fulfilling the inclusion as mentioned above and exclusion criteria, the individuals were informed about the surgical procedure along with the application of platelet concentrates and the benefit that can be obtained from it. A signed informed consent was obtained from all the 11 individuals.

The individuals were divided into 3 groups based on the application of platelet concentrates after the depigmentation procedure.

The depigmentation procedure was carried out by abrasion technique as observed in Figure 2.[17] Following the depigmentation procedure, the groups were divided in the following way as noted in Figure 3:

Figure 2

Depigmentation by bur abrasion method

Figure 3

Immediate postoperative view following depigmentation

Group A: Right maxillary quadrant received the PRF membrane

Group B: Left maxillary quadrant received the PRFM gel

Group C: The mandibular anterior sextant received only the periodontal pack placement.

The individuals were refrained from brushing for 3 days following the procedure and were instructed to rinse mouth with 0.2% chlorhexidine Digluconate mouthwash. On the 3rd day, the individuals were recalled, and the periodontal pack was removed.

The clinical and histological parameters were evaluated following the surgical procedure. The visual analog scale (VAS)[18] and healing index (HI)[19] were evaluated at 3rd and 5th day [18] and epithelialization test at 5th day.[20] The histological analysis was carried out on the 5th day through punch biopsy from the depigmented gingival epithelium.

Preparation of platelet concentrates

10 ml of blood samples was collected from individuals participating in the clinical trial. The samples were further processed for preparation of PRF membrane and PRFM gel as per the conventional centrifugation protocol.

Preparation of platelet-rich fibrin membrane

5 ml of blood present in glass vacutainer was centrifuged in REMI4 (*R-4C, REMI Laboratory Instruments, Mumbai, India) centrifugation machine for 2800 rpm for 12 min. The gel obtained following the centrifugation had three layers. The top layer being platelet poor plasma and bottom layer consisted of concentrated red blood cells. The middle layer harboured fibrin network gel as observed in Figure 4.

Figure 4

Preparation of platelet-rich fibrin membrane (a) Blood withdrawn from antecubital vein; (b) centrifuged at 2700 rpm for 12 minutes; (c) the Platelet rich fibrin (PRF) obtained; (d) PRF gel was squeezed between sterile gauze to form Platelet rich fibrin membrane

The gel was then compressed in between sterile gauze pieces, and the supernatant was discarded to obtain the PRF membrane.[21]

Preparation of platelet-rich fibrin matrix

Five milliliter of blood was transferred to Meresis PRFM kit (Meresis, laboratory, Bangalore, India) using a single spin centrifugation method. It was centrifuged at rpm of 3000 for 10 min. The supernatant obtained at the top of gel was removed through syringe, and activator containing 0.1% gluconate was added and mixed for 9–10 times to achieve the PRFM gel as seen in Figure 5.[22]

Figure 5

Preparation of platelet-rich fibrin matrix (a) blood was withdrawn fromantecubital vein; (b) transferred to Meresis PRFM kit; (c) centrifuged at rpm of 3000 for 10 min; (d) The supernatant obtained at the top of gel was removed through syringe; (e) activator containing 0.1% gluconate was added and mixed for 9–10 times; (f) PRFM gel was obtained

Surgical procedure

The depigmentation procedure was carried out by abrasion technique with the use of a medium grit football-shaped diamond bur with feather-like brushing strokes under copious water lavage. The individuals underwent depigmentation procedure with abrasion technique in maxillary right and left and mandibular anterior teeth region. On the maxillary upper quadrant, PRF membrane was placed as contemplated in Figure 6. The membrane was stabilized with 5- 0 absorbable suture followed by periodontal pack placement. On left maxillary quadrant, PRFM gel was uniformly spread over the depigmented epithelium as observed in Figure 7 and was covered with the periodontal pack. The mandibular anterior sextant directly received periodontal pack following depigmentation procedure as seen in Figure 8. The postoperative view at 3rd and 5th day was contemplated in Figures 9 and 10.

Figure 6

Application of platelet-rich fibrin membrane on thefirst quadrant following depigmentation (a) The de-epitheliliazed 1st quadrant;(b) platelet rich fibrin (PRF) membrane placed; (c) periodontal pack placed

Figure 7

Application of platelet-rich fibrin matrix on second quadrant following depigmentation (a) The de-epitheliliazed 2nd quadrant site; (b) platelet rich fibrin matrix gel (PRFM) placed; (c) periodontal pack placed

Figure 8

Application of periodontal pack on lower anterior teeth region following depigmentation (a) The de- epithelialized site in mandibular anterior site; (b) periodontal pack was placed

Figure 9

Post operative view at 3rd day (a) The 1st, 2nd quadrant; (b) mandibular anterior teeth region lower anterior teeth region

Figure 10

Postoperative view at 5th day

Statistical analysis

The results were statistically evaluated using SPSS Inc., (Released 2009 PASW Statistics for Windows, Version 18.0. Chicago). The power of the study was 90%, and a P < 0.05 was considered statistically significant. The intergroup comparison of the clinical parameters was evaluated with Fischer's extract test.

RESULTS

The application of platelet concentrates on the maxillary right and left quadrant has shown promising results concerning the clinical parameters as compared to Group C which received periodontal pack placement [Figure 11].

Figure 11

(a) Pre-operative view and (b) Post operative at 3rd month interval

Visual analog scale

The VAS in the individuals at 3rd day showed 5 individuals receiving platelet concentrate had moderate pain with 45.5% whereas 9 individuals with 81.09% in periodontal pack placement in mandibular anterior teeth region have demonstrated severe pain. Statistically, the percentage evaluation in Group A and B was statistically significant as compared to Group C with the P < 0.001.

On the 5th day, all the 11 individuals with 100% VAS score did not have pain in the platelet concentrate depigmented area whereas 11 individuals in Group C had moderate pain which when evaluated statistically showed significant results for Group A and B as compared to Group C with the P < 0.001. The same observation is tabulated in Table 1 and Figure 12.

Table 1

Percentage of healing index score obtained in Groups A, B, and C on 3rd and 5th day postsurgery

Figure 12

Percentage of visual analog scale (VAS)perceived by individuals at 3rd and 5th day in Platelet rich fibrin (PRF), Platelet rich fibrin matrix (PRFM) and Periodontal pack (PACK) groups

Healing index

The various scores present in the healing index as per Landry et al.19 have shown that on the 3rd day following depigmentation, all the individuals had good healing in Groups A and B as compared to Group C where 7 individuals with 63.6% had the poor healing score and 4 individuals had good healing. Statistical tabulation observed a statistically significant difference with the P < 0.001 in Groups A and B as compared to Group C as observed in Figure 13.

Figure 13

Percentage of healing observed in Groups A, B, and C on 3rd and 5th day as per healing index in Platelet rich fibrin (PRF), Platelet rich fibrin matrix (PRFM) and Periodontal pack( PACK) groups

On the 5th day, all the 11 individuals in the Group C had shown good healing, and the sites that received platelet concentrates had a very good healing score. The same has been observed in Table 2 and Figure 13.

Table 2

Percentage of visual analog scale rating sore obtained on 3rd and 5th day in Groups A, B, and C

Epithelialization test

On the 5th day, the maxillary right and left quadrant and mandibular anterior sextant was substantially stained with toluidine blue. 10 individuals with 90.90% in the Group B (PRFM gel) quadrant and 6 individuals 54.50% in Group A had taken the mild staining which was statistically significant with P < 0.001 as compared to mandibular anterior sextant of Group C where 5 individuals had severe stain uptake with 45.5%. It denoted the presence of less inflammatory cells in the platelet concentrates group as compared to periodontal pack group with uneventful healing. The toluidine blue uptake was seen in Figure 14. The statistical tabulation is observed in Table 3 and Figure 15.

Figure 14

Epithelialization test performed at 5th day

Table 3

Percentage of epithelialization test score obtained on 5th day for Groups A, B, and C

Figure 15

Percentage of epithelialization observed at 5th day with toluidine blue in Platelet rich fibrin (PRF), Platelet rich fibrin matrix (PRFM) and Periodontal pack (PACK) groups.

Histological analysis

At 5th day after the surgical procedure, a section of tissue from the lateral incisor region in maxillary arch and mandibular arch was taken through punch biopsy from all the treated sites. Hematoxylin and eosin stained slides was prepared from all the tissues. At ×40 magnification, the inflammatory cell infiltrates was observed in Figure 16. The 7 individuals in Group B, i.e., PRFM group had shown a distinct parakeratinized stratified squamous epithelium with fibrous connective tissue with nil inflammatory cell infiltrates and 8 individuals in Group A, i.e., PRF group had demonstrated moderate inflammatory cell infiltrate with 70% and 9 individuals in Group C had severe inflammatory cell infiltrate with 81.8%. Statistically, when tabulated, Group B was statistically significant as compared to Groups A and C. These data can be contemplated in Table 4 and Figure 17.

Figure 16

Histological analysis at 5th day with toluidine blue in Platelet rich fibrin (PRF), Platelet rich fibrin matrix (PRFM) and Periodontal pack (PACK) groups

Table 4

Percentage of histological analysis for the presence of inflammatory cells on 5th day in Groups A, B, and C

Figure 17

Presence of inflammatory cell infiltrate through histological analysis at 5th day in Platelet rich fibrin (PRF), Platelet rich fibrin matrix (PRFM), Periodontal pack (PACK) groups

DISCUSSION

The hyperpigmentation of the gingiva is an esthetic concern for the individual; though not a medical condition, it requires the removal of the hyperpigmented gingival epithelium. The depigmentation leaves behind an exposed connective tissue on the gingival surface for the healing to take place by secondary intention.[23] An attempt to cover the surgical site with platelet concentrates has been evaluated. The results obtained from the study using the platelet concentrates could be elaborated due to the immense potential of the biomaterial in terms of VAS, HI, epithelialization test, and histological analysis as compared to periodontal pack.

The 2nd generation PRF has been utilized and investigated in various forms in the field of periodontology. A measurable result was obtained when used in the treatment of intrabony defects,[24] gingival recession,[25] and furcation defects.[26] The PRF membrane has been used as palatal bandage following free gingival graft procedure where satisfactory patient comfort was achieved.[27] The distinguishable results from various treatment modalities could be attributed to its fibrin structure and the release of various growth factors for a prolong period. The PRF blood clot contains >97% of platelets that was sufficient to accelerate soft- and hard-tissue healing. The fibrin matrix being in a tetramolecular structure incorporates platelets, leukocytes, cytokines, and circulating stem cells.

The fibrin matrix also contains glycol aminoglycans which have a strong affinity for circulating peptides and can support cell migration and healing process. PRF is the activated form of a plasmatic molecule called fibrinogen.[28] The fibrin formed after the centrifugation is transformed into a kind of biologic glue which consolidates the initial platelet cluster, thus constituting a protective wall along vascular breaches during coagulation. The fibrin architecture entraps various numbers of leukocytes in the fibrin matrix allowing intense slow release of growth factors.[29] It favors the sealing of wound borders and allows an uneventful proliferative phase by the steady release of growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin growth factor (IGF), and transforming growth factor (TGF) which are known to play pivotal role in wound healing process.[2130313233]

The first-generation platelet concentrate introduced by Marx et al., in 1998 is known to act on healing inducible cells to increase their numbers through mitogenesis and perform angiogenesis by stimulating vascular ingrowth.[34] The PRF matrix PRFM gel used in the study was a first-generation platelet concentrate in which addition of calcium gluconate as an activator was known to enhance the release of various growth factors in a more substantial quantity for a shorter period. Kobayashi et al., in 2016 evaluated the growth factor release from PRF, PRP, and APRF advanced platelet rich fibrin for 10 days which showed a robust release of PRP for a shorter period as compared to other platelet concentrates.[31]

In the present study, a statistical significant difference was observed in HI, epithelialization test and VAS score in the test groups i.e. Group A (PRF) and Group B (PRFM) as compared to Group C (Periodontal pack). The result could be attributed to the release of PDGF, VEGF, EGF, FGF, IGF, and from both the platelet concentrates.

The histological analysis done at 5th day has observed a statistically significant difference in PRFM group as compared to PRF and periodontal pack group. The reason behind the absence of inflammatory cell infiltrates in Group B was due to its robust and rapid release of growth factors for a shorter period of time. Studies done by Gassling et al., in 2009[21] and Lucarelli et al.[32] in 2010 has evaluated the release of PDGF, EGF, TGF1, EGF, and FGF for 10 days and observed a maximum release of growth factors were seen on the 1st day of PRP group and a constant release of growth factors was seen in PRF group suggesting a rapid release of growth factors from first-generation platelet concentrates in the initial stage of healing period which enhanced the wound healing process. Carroll et al. in 2005[33] evaluated the PRFM group for 10 days and observed 5 times more platelet entrapment in the fibrin mesh leading to a faster and increased concentration of growth factors (PDGF, VEGF, and EGF).

Severe inflammatory infiltrates remained in Group C as the periodontal pack does not have any curative properties and is a dimensionally unstable material which shows contraction during the first minutes after completion of their setting, resulting in delayed healing.[3536]

The present study is in accordance with the only study conducted by Bansal et al. in 2016[37] where PRF membrane along with periodontal pack was placed after depigmentation procedure in 5 individuals. However, the present study had compared two platelet concentrates, i.e., PRF and PRFM and had recruited a larger sample size comprising of 11 individuals. All the results were clinically and statistically tabulated. The use of platelet concentrates is necessary to cover the exposed lamina propria as healing occurs with secondary intention, which is delayed if the surgical site is left exposed. The connective tissue repair would establish and maintain the adherence of fibrin clot which is a natural phenomenon of wound healing process. Thus, with the addition of platelet concentrates, a better patient comfort and rapid wound healing was viewed. As per our thorough literature research, the present research is the first of its own kind where the application of PRFM gel along with PRF membrane has been evaluated in gingival epithelialization and yielded an enhanced response in wound healing process.

CONCLUSION

Thus, the application of PRF membrane and PRFM gel in the present study has shown a successful approach to protect the raw wound area of depigmented sites with a better patient comfort and faster healing than the use of periodontal pack alone.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.