Platelet Rich Fibrin Membrane Grafting After Laser Excision for Oral Mucosal Lesions

Abstract

Platelet rich fibrin (PRF) is a novel surgical biomaterial which has shown immense healing and regenerative potential with diverse clinical applications. Surgical excision is a routinely employed treatment modality for mucosal oral lesions with or without grafting or repair of the base of the wound. We proposed a hypothesis that covering of the base of excised lesion with this platelet rich fibrin membrane can accelerate the rate of physiological healing process and regeneration. To evaluate the role, efficacy, advantages and adverse effects if any, of placement of PRF membrane grafts over surgical excision sites of oral mucosal lesions. Platelet rich fibrin membrane graft was prepared as per recommendation and established protocols. Patients were selected for surgical excision of localized, superficial oral mucosal lesions after meticulous clinical and radiological considerations and informed consent was taken. After laser excision with safe margins, the base of wound was covered with PRF membrane graft, stitched in place by 3-0 vicryl sutures. The operated site was clinically evaluated at regular intervals and a healing score was calculated and statistically tabulated on the basis of various parameters of healing. A total of 34 patients were included in the study with a male preponderance (21 males and 13 females). Satisfactory and clinically acceptable wound healing was observed in most of the patient with minimal morbidities. Surgical site demonstrated good healing score and clinically complete healing with good epithelialisation was achieved in all patients. Platelet rich fibrin membrane is an effective grafting biomaterial after excision of oral mucosal lesions as it enhances the rate of healing with minimal complications. We recommend further multicentre studies with higher sample size to explore its utility and clinical applications in different avenues of oral and head neck surgery.

Introduction

Wound healing is a highly coordinated sequence of biochemical, physiological, cellular and molecular responses involving a cascade of events to restore tissue integrity and functional capacity after injury. Advancements in the clinical understanding of pathophysiology of wounds have commanded significant biomedical innovations. Various biomaterials, skin substitutes, biomembranes and scaffolds have been developed to facilitate wound healing through various mechanisms [1]. Platelet rich fibrin (PRF) developed by Choukrou et al. [2] is a second generation platelet concentrate that has shown promising results in enhancing wound healing as an autologous biomaterial. It is essentially a fibrin matrix in which platelet concentrates, cytokines and growth factors are embedded which is favorable for healing and regeneration. Review of literature also reveals clinical studies supporting the usage of this biomaterial in promoting wound healing, wound sealing, and hemostasis [3].

Oral cavity is a common site of occurrence for variety of lesions, ranging from benign pathologies to potentially malignant and life threatening diseases. Surgical excision of such lesions is a preferred treatment modality for many of these lesions with or without covering of the base of the wound. It was hypothesized that usage of a PRF membrane graft as a biomaterial to cover the base of carefully selected oral mucosal lesion after laser excision could accelerate the rate and quality of wound healing and regeneration. This study was thus undertaken to evaluate the efficacy, usability and benefits of PRF membrane graft as a covering biomaterial over the wound base of surgically excised oral mucosal lesions.

Materials and Methods

This prospective interventional study on utility of PRF membrane graft in treatment protocol for oral mucosal lesions was conducted at an urban health care centre in India over a period of 1 year. Patients presenting with mucosal lesions in the oral cavity were clinically evaluated and suitable diagnostic tests were performed to confirm the diagnosis, ascertain the margins of the lesions, lymph node status and fitness of surgery. The diagnostic test battery involved radiological tests such as contrast enhanced Computed Tomography (CT)/Magnetic Resonance Imaging (MRI), detailed hematological work up and punch biopsy if required for histological confirmation. Based on this comprehensive evaluation, an individualized treatment plan was charted out for each patient in a multidisciplinary team approach. Patients in whom conservative approach was adopted were kept in an observational phase with regular follow up and explanations regarding warning signs for future intervention.

Surgical excision was planned for carefully selected cases. The procedure of laser excision and PRF membrane grafting was explained in detail and informed documented consent was taken as per guidelines of Declaration of Helsinki. The inclusion criteria included in this study were as following:

• Oral mucosal lesion less than 3 cm in maximum dimension.

• No involvement of underlying structures and neighboring bones.

• Absence of nodal involvement.

• Possibility of safe surgical margins without need of reconstruction.

• No contraindication for general anesthesia for surgery.

• Patient willing for regular follow up.

Platelet Rich Fibrin (PRF) membrane graft production protocol

10 mL of blood was drawn from the antecubital vein of the patient into a 21 gauze disposable 10 cc syringe. The blood was immediately filled into a disposable 10 mL glass coated plastic test tube (Becton–Dickinson Vacutainer, Franklin Lakes, NJ, USA) without any anticoagulants. The vacutainer with blood was balanced with an identical vacutainer filled with 10 mL of Normal Saline and kept in the opposite slot in the centrifuge machine. The tubes were centrifuged straight away at 3000 rpm for 10 min in the centrifugation machine (REMI Laboratories) at room temperature.

After centrifugation, the blood in the tube settled in 3 layers:

• Red colored lower fraction containing red blood cells (RBC).

• Middle fraction containing the PRF clot.

• Straw colored upper fraction containing Platelet Poor Plasma (PPP).

The PRF clot was removed from the test tube using sterile tweezers after discarding the PPP and was separated from the RBC base using scissors. The PRF clot was gently pressed by squeezing it between sterile dry gauze or glass slab to drain the extra fluid and obtain a membrane like graft of Platelet Rich Fibrin.

Surgical Procedure

The oral mucosal lesion was excised using CO₂ laser after local anesthetic infiltration under strict aseptic precautions. A partial thickness wound was created and adequate safe margin was taken around the lesion as per the clinical diagnosis of every case. The PRF membrane was placed on the base of the wound and sutured into place using absorbable 3-0 sutures. Hemostasis was secured and moist pressure dressing was applied to the surgical site.

Routine precautions after oral surgery was explained to the patient and post operative antibiotics and analgesics were prescribed. Sutures were removed between 7 and 10 days after surgery. The patients were followed up meticulously on day 1, 3, 7, 15 and 30 days after surgery and a detailed documentation of every case was maintained. The success of the graft placement and wound healing was measured on parameters of epithelialisation, status of wound margins, appearance, bleeding, edema and infection at the site of graft placement. Patients were kept for a longer follow up if required and postoperative histopathology report dictated future course of treatment in these patients. The records were organized, tabulated and statistically interpreted for records and academic interest (Figs. 1, 2, 3, 4, 5, 6).

Fig. 1.

Leucoplakia on the lateral surface of tongue

Fig. 2.

Platelet Rich Fibrin graft prepared from patient blood

Fig. 3.

Laser excision of oral mucosal lesion

Fig. 4.

Grafting of Platelet rich Fibrin membrane over the defect

Fig. 5.

Site of wound on the day of suture removal

Fig. 6.

Well healed wound site on 2 weeks after PRF grafting

Evaluation of Healing

The healing of the surgical wound was statistically analyzed on the basis of a healing score on set criteria evaluated on 7, 15 and 30th postoperative day. The criteria observed were colour of the tissue, granulation tissue, bleeding from the site of wound, suppuration, pain on palpation, contour irregularity and cosmetic appearance of the surgical site. This 7 point scoring system was utilized to put in order a scoring system to assess the status of wound after PRF grafting (Table 1).

Table 1.

Scoring system for evaluation of healing after PRF grafting

Criteria	Yes	No
Is redness present at surgical site?	0	1
Is Granulation tissue present at surgical site?	0	1
Is there bleeding from the surgical site?	0	1
Is there suppuration at the surgical site?	0	1
Is there contour irregularity at the surgical site?	0	1
Is there pain on palpation at the surgical site?	0	1
Is the surgical site appearance unacceptable?	0	1

The sum of these scores for individual criteria was taken to be as the healing score. Lower the score, poorer was the healing after PRF grafting. A higher score signified better postoperative wound healing.

Results

A total of 34 patients were included in this study that underwent PRF membrane grafting after laser excision of the oral mucosal lesion. Out of 33, there were 23 male and 11 female patients, showing a male preponderance (male: female ratio of 2.09:1). The age group varied from 22 to 59 years with an average age of presentation equal to 37.4 years. The most common pathology was leucoplakia (13 cases) followed by erythroleucoplakia (7 cases), suspicious ulcers (7 cases), papilloma (4 cases), lichen planus (4 cases) and pigmented melanotic macules (2 cases). The clinical diagnoses were later collaborated with histopathological findings for confirmation (Fig. 7). 9 patients were reported as malignant lesions of the oral cavity after biopsy report and were accordingly treated further. Tongue and buccal mucosa (12 cases each) were the most frequent sites of involvement. Mucosa of the lower lip (4 cases), vestibule (2 cases), retromolar trigone (2 cases), soft palate (1 case) and alveolar mucosa (1 case) were the other sites of involvement (Fig. 8).

Fig. 7.

Frequency of oral mucosal lesions

Fig. 8.

Site of involvement of oral lesions

Healing score of every patient was calculated as described in Table 1. There was a significant improvement in healing score across all parameters over a period of 3 assessments. Redness was present in all patients at first post operative visit and was absent at the 30 days for all patients. Granulation tissue was observed in 29 patients at 7 days, 7 patients at 15 days and none at 30 days. Bleeding was observed in just 2 patients on the day of surgery which was managed conservatively. There was presence of suppuration in 1 patient at 7 days and 1 patient at 2nd post operative visit. Both these patients had poor oral cavity hygiene and infection was managed successfully under antibiotic coverage. Patients were prescribed post operative analgesics for 5 days. None of the patients complained of pain after completing the course of post operative medications. Contour irregularity was noted in 4 patients at 15 days and 1 patient after 1 month. These patients had comparatively larger lesion requiring a greater area and depth of dissection. All patients were satisfied with appearance of surgical site at the end of post operative visits.

On a long term follow up, 1 patient complained of pain at surgical site 3 months after surgery. Clinical assessment revealed no pathology and pain subsided after assurance from the surgeon. 2 patients presented with hypertrophy around the edges of surgical excision 6 months after surgery without any other complaints. Clinical and radiological examinations were not significant. Histopathological examination revealed no abnormality or recurrence. Patients on a longer follow up have shown complete epithelialisation without any known case of recurrence of disease.

Discussion

Wound healing is a complex and dynamic cascade of cellular and molecular events mediated by various signaling events to establish tissue integrity and functional normalcy after tissue injury. There has been a continuous effort to accelerate and regulate the process of healing and regeneration through newer therapies, biomaterials and surgical additives. The role of platelets and its derivatives attains paramount importance in this scenario as upon activation, platelets release various growth factors that stimulate cellular growth, proliferation, healing, and cellular differentiation [4]. There has been a better understanding of physiological properties of platelets in wound healing in last few decades that has led to increased therapeutic application in various forms with varying results [5]. Introduction of the concept of Platelet Rich Plasma (PRP) introduced by Whitman et al. [6] in 1997 was of such a platelet derivative as an autologous concentration of platelets in a small volume of plasma containing wound healing proteins that had utility in oral surgeries. However, the potential risk of developing antibodies leading to life threatening coagulopathies due to the presence of bovine thrombin in PRP restricted their further usage [7]. This led to further exploration into platelet derived biomaterials and research into platelet gel therapeutic concept with simplified processing without artificial biochemical modifications. Choukroun et al. in 2001 [2] introduced a new biomaterial called Platelet Rich Fibrin (PRF), a second generation platelet derivative that attempted to collect platelet derivatives like growth factors and cytokines in a fibrin clot. Today, PRF is recognized as a promising biomaterial with several potential usages that have revolutionized the field of reconstructive biology [3].

PRF is increasingly used its potentials are being explored all over the world for applications in different fields of medicine. Structurally, soluble fibrinogen in plasma transforms to fibrin which polymerizes to form a three dimensional matrix [8] into which platelets activated by centrifugation are trapped and forms a storage pool of growth factors. Thus, the PRF membrane is essentially a three dimensional, biocompatible, biodegradable scaffold which can slowly and sustainably release bioactive molecules which facilitate cell adhesion and proliferation [9]. The 3D architecture of the fibrin matrix provides the PRF membrane with great density, elasticity, flexibility and strength that are excellently suited for handling, manipulation and suturing [10]. The various growth factors that are released by the activated platelets include platelet derived growth factors (PDGF), vascular endothelial growth factors (VEGF), transforming growth factor β, insulin growth factors, fibroblast growth factors, epidermal growth factors [11]. There are various other feature of PRF that have captured the attention of researchers worldwide:

• Autologous PRF is derived from patients own blood hence it is an allogenic biomaterial without possibility of foreign body reaction [12].

• Simplified preparation Preparing PRF is easy, fast and user-friendly to use within the daily clinical routine without along waiting time [13].

• Efficient usage PRF membrane, as mentioned above due to its polymeric structure of fibrin matrix provides great freedom maneuverability and handling.

• Cost effective PRF is an inexpensive and affordable biomaterial with immensely favorable cost to benefit ratio.

• No contraindications PRF can be used in all kinds of patients, especially in systemic conditions where healing is compromised or surgically compromised situations [14].

Excision of lesions of the oral cavity has been a routinely employed method of intervention for various types of lesions. Therapeutic excision for localized unresponsive benign pathologies as well as suspicious or premalignant conditions has been the preferred treatment modality in number of cases. However, surgically difficult anatomy, difficulty to maintain immobility and oral flora often prevents adequate reconstruction of excised wound site. Primary wound closure is restricted to small accessible lesions and it is not unusual for post surgical wounds to heal by secondary intention in the oral cavity. This leads to granulations tissue formation causing bleeding, scarring, trismus and other morbidities. There have been attempts to cover the wound site with a local flaps and grafts, both autologous and alloplastic [15, 16]. However, these methods have their own limitation such as donor site morbidity and have never been practiced regularly or universally. We decided to utilize the PRF membrane taking into consideration its benefits, regenerative potential and reports of successful utilization as a healing booster at other grafted sites [13]. PRF membranes is a highly stimulating matrix that permits cells to migrate through it, permitting angiogenesis that assists regenerative and healing interactions between the tissues below and above the PRF membrane. We hypothesized that slowly releasing growth factors from a stable fibrin matrix over a wound site would accelerate the rate and improve the quality of healing in carefully selected lesion of the oral mucosa. CO₂ laser was utilized to excise the lesions as it has been shown to supersede conventional scalpel in terms of better intra operative performance, hemostasis and less scarring [17]. Covering of the wound site after excision with the PRF membrane resulted in good post operative results in our study. There were no significant adverse effects or long term morbidity after PRF grafting.

The clinical application of Platelet Rich Fibrin has expanded multifold in the past decade and newer avenues of its uses are being added with outstanding outcomes. PRF has become a commonly used biomaterial in advanced surgical dentistry with multiple applications. Periodental regeneration, regenerative endodontics, impaction surgeries, ridge augmentation, socket preservation, sinus lift procedure, gingival recession, transalveolar extraction procedures, tissue volumerization and fat transfer procedures are some of the numerous dental procedures which utilize the properties of PRF [18–20]. The field of dermatology and cosmetic has successfully applied PRF in areas such as facial rejuvenation [21], facial aesthetics, facial lipostructure correction [22], hair fall treatment [23], scars, contour defects and striae distansea. The field of otorhinolaryngology has applied platelet rich fibrin membrane for the reconstruction of tympanic membrane [24]; however, the scope for utilization in several subspecialties of is still unexplored. The use of PRF is healing of chronic non healing ulcers and tendon and cartilage repair are the newer avenues of PRF usage that have shown promising results [25, 26]. There are aspects of PRF preparation, handling, quality control and effects that need a better understanding. As we enhance our current knowledge regarding PRF, an enhancement in the list of therapeutic application is inevitable. There has been an attempt to improve the characteristics of PRF to make it sterile, uniform, homogenous suturable biomaterials through interventions that may lead further application of PRF in different fields of medicine [27].

Platelet rich fibrin graft; however has its limitation at present that must be kept in mind while advocating its application across different fields. PRF graft manufacture requires quick handling and experienced manipulation during preparation. The volume that is available is limited owing to its autologous nature and it is non transferrable to other patient. Long term storage is improbable as tissue banks are not feasible for storage. In spite of these limitations, we look towards a future where newer innovation in molecular research can alter and resolve the shortcomings of Platelet rich fibrin.

Limitations

The efficacy of Platelet rich fibrin in healing of oral lesions has been demonstrated but comparison with other grafting biomaterials in a larger sample size is called for a better understanding of grafting mechanism. The role of other oral pathology on the status of graft efficacy is unclear.

Conclusion

We conclude that Platelet rich fibrin membrane grafting after laser excision or superficial oral mucosal lesion is an effective, safe, simple and convenient approach to management. PRF membrane has several biological peculiarities that can be utilized as a useful surgical biomaterial. We recommend further multicentre studies to analyze and establish its effectiveness in surgical applications.

Funding

None.

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have no conflict of interest.

Informed Consent

Obtained.