Platelet-rich fibrin-a cost-effective, donor less and indigenous therapy for obtaining root coverage-a case report

Among the periodontal infections, gingival recession is a separate clinical entity which demands a permanent solution plan. Treatment therapies are based on either elimination (as in case of Miller’s class I/II recession) or increasing the width of keratinized gingiva for prevention of further progression (as in case of Miller’s class III/IV recession). From the centuries, graft surgeries like free gingival graft, lateral pedicle and Coronally advanced flaps have been successful in the treatment of gingival recession. As every technique has its own merits and demerits, clinicians have tried to stress upon addition of certain adjuncts or biomaterials to ensure rapid healing and less postoperative discomfort. Platelet concentrates are an excellent biomaterials for increasing width of attached gingiva and enhancing wound healing. They are cost effective, do not require donor tissue for harvesting and contain variety of growth factors for initiating regeneration. In this paper, Platelet-rich fibrin a 2nd generation concentrates has been used as an adjunct to Coronally advanced flap with a purpose of increasing width of keratinized gingiva, obtaining complete root coverage and achieving gingival harmony.


Introduction
Oral diseases comprise of vast array of destructive processes, which are chiefly concerned with either pulpal or periodontal pathology. Pulpal infections, majorly; manifest themselves as either reversible or irreversible pulpitis while periodontic infections initiate in the form of gingivitis or periodontitis. Pulpitis is easily treated by root canal treatment or ultimately by extraction, but periodontal infections have a different strategy for cure & prevention. Often patients present with dentinal hypersensitivity which upon clinical observation demands a permanent treatment therapy. This condition is commonly referred to as 'Gingival recession" which is characterized by exposure of root surface by an apical shift in position of gingiva (Newman et al.2014, p.735). Gingival recession is a challenging task for clinicians because in some cases, esthetic concerns are the primary treatment modality besides elimination of dentinal sensitivity. Probably, there can be chances of even root caries (Raetzke1985, p.397).Hence, Hentreatmentrbecomes essentialsfortpreservingeoralihealth. Whilehcompletecrootecoverageccanrbeeachievedainiboth Class,hI Cands III Miller'sMgingivalgrecessionedefects, donlytpartial cov-eragecmayrbeeexpectedeineClassi IIIl and ClassnIVCMiller'sMgingivalgrecessionedefects (Newman eteala.2014(Newman eteala. ,l p.1931 p.1931) An ideal outcome of a root coverage procedure can be achieved only if the environment is plaque free (on tooth surface) and adequate periodontal support is present (Raetzke PB1985, p.397-402). Another important clinical entity is the amount of keratinized gingivae, which prevents further progression of gingival recession (Newman et al.2014(Newman et al. , p.1948) Hence, all these components must be considered for restoring periodontal health.
The therapeutic modalitiesinclude pedicle soft tissue graft procedures (advanced or rotational), or free soft-tissue graft procedures (epithelialized graft or sub-epithelial connective tissue graft) (Lindheet al.2008,p.972-977)Further, rotational flap procedures are differentiated into the laterally sliding flap, double papilla flap and oblique rotated flap while the advanced flap procedures are subdivided intocoronally advanced flap and semilunar coronally advanced flap. As every surgical procedure has both advantages and disadvantages, selection of cases remains the key for elimination of gingival recession. The free gingival graft is always associated with painful post-operative palatal wound and unpleasant aesthetics (due to harvesting of graft from a palatal mucosa) while lateral pedicle graft results in occurrence of recession at the donor site (Miller 1988, p.674-681;Sato 2000,p.342).On the other hand, Semilunar coronally advanced flap ,though suture-less, is successful in maxillary arch only while Coronally advanced flap (CAF) is associated with shortening of vestibule (Miller 1988, p.674-681). Furthermore, sub-epithelial connective tissue (SCTG), which remains the gold standard for root coverage (mean root coverage between 52 and 98 %),is a technique sensitive procedure and requires a second site (donor) for harvesting of graft, which is not always possible (Sato 2000 The initial concept of these autologous preparations was to concentrate platelets, growth factors in a plasma solution and lastly, activate it into a fibrin gel on a surgical site so that local healing is improved (Zhao et al.2013, p.3).Platelet-rich fibrin (PRF), which was first discovered by Choukroun et al (2001)of France, is a second generation platelet concentrate,and it has been used significantly in the field of periodontal plastic surgery (Dohanet al.2013,p.38-40) Many clinicians have performed Coronally advanced flap along with platelet-rich fibrin (PRF) for the treatment of gingival recession and found that PRF can be used effectively for root coverage as well asa healing biomaterial(Urazet al.

Case report
A 43 years-old male came to the Department of Periodontology, Bhojia dental college &Hospital, Baddi , with a chief complaint of unpleasant looks in right upper front tooth region. On clinical examination, it was noticed that there was attrition present along with Miller's class, I gingival recession with respect to right maxillary canine (tooth no.-13). Pre-operative recession width (RW),width width of keratinized tissue (KT) &Recession depth (RD) were recorded with William's probe (University of Michi-gan1,2,3,5,7,8,9 & 10 mm) while gingival thickness (GT) was recorded with reamer and rubber stopper as the protocol mentioned by Arocaet al. 2009,p.245-48. (Fig.-3).Recession depth (RD) was 3 mm pre-operatively while recession width (RW) was calculated to be about 4mm ( Fig.-1 & 2). Width of keratinized tissue came to be around 4 mm (Fig.-2) while gingival thickness (GT) was 1 mm, pre-operatively (Fig.-3).Full mouth scaling and root planing was done by hand and ultrasonic instruments, at least4-5 weeks prior to the surgery and oral hygiene instructions were given and reinforced at each visit. After completion of phase I therapy, patients were scheduled for surgical phase.

Surgical procedure
After proper blood investigations, the patient was given written and verbal information on the nature, risks and benefits of the surgical procedure and a signed, informed consent was obtained prior to the treatment .  (Fig.-4). Then, a split-thickness flap was prepared by sharp dissection at mesial and distal portions of the recession defect. These portions were interconnected by an intra-crevicular incision (Fig.-4). Apical to the receded soft tissue margin on the facial aspect of the tooth, a fullthickness flap was elevated by using Molt's Periosteal elevator (No. 9) to achieve maximum thickness for root coverage (Fig.-5).
Approximately 3mm apical to the marginal bone, a horizontal incision was made which was followed by blunt dissection into the vestibular lining mucosa so that muscle tension was relieved. This blunt dissection was extended buccally and laterally to make the mucosal graft tension free while positioning it coronally at the level of CEJ. Then, the facial portion (mesial and distal portion of interdental papilla coronal to flap) was de-epithelialized using currette/ cuminescaler to allow for the final placement of the flap margin. Proper isolation was done by using cotton rolls and an orthodontic button was cemented/fixed at the central most area of the crown of the involved tooth (Fig.-4). After that, platelet-rich fibrin membrane (PRF-M) was prepared.

Prf preparation
The PRF was prepared following the protocol developed by Choukroun et al (2001). Just prior to the surgery, intravenous blood (approx.10 ml) was collected without anticoagulant, by venepuncturing the anticubital vein with the help of 10 ml syringe and was immediately transferred to a glass test tube. The test tube was immediately centrifuged at 3000 rpm for 10 minutes at room temperature. After centrifugation, blood settles into 3 layers (Fig.6): 1) Red lower fraction containing RBCs.
2) PRF clot in the middle.
3) Upper straw colouredAcellularPlasma. The PRF clot was removed from the test tube using sterile tweezers, separating it from the RBC base and placed in a sterile dappen dish (Fig.-7, 8). Excess plasma was squeezed out with the gauze and then it was compressed with gauze so that it can be converted into a membrane (Fig.-9) The membrane was placed on the denuded root and sutured (sling suture) by using ½ round cutting needle with 5-0 absorbable chromic gut sutures (Fig.-10).After that, the flap was coronally advanced,adjusted foroptimal fit to the prepared recipient bed andsecured at the level of the CEJ by suturing(interrupted & sling sutures) the flapto the connective tissue bed in the papilla with 4-0 non-absorbable braided silk sutures (Fig.11). The central area of the flap was stabilized by suturing (sling suture) it to an orthodontic button/bracket and a periodontal dressing (coepack) was given to avoid any direct trauma to the working/operative site. Patient was prescribed the necessary antibiotics, analgesics, antiinflammatory drugs and chlorhexidine mouthwash (0.2 %). Routine verbal and written post-surgical instructions were given to the patient and the patient was asked to report after one week, for suture removal and at any time, in case of an adverse event. Thepatient was placed on a soft diet for a period of 10 days and packing was changed after a week and further, placed for another 5 to 7 days for stability. He was told to apply minimal pressure during brushing and use a soft nylon bristle brush during then extend upto 2 to 3 weeks following pack removal. Recall was done after 3 and 6 months.    As mentioned earlier, recalling was done after 7 days, 3 and 6 months and all clinical parameters were recoded (Fig.-12,13,14).
There was complete root coverage and healing was uneventful. Also, there were no post-operative complications.

Results
The amount of root coverage was determined by calculation of recession depth /width reduction. It was calculated in percentage (%) (Zucchelli&Sanctis2000, p.1510): i.e. Initial recession depth or width-Final recession depth or widthX100 Final recession or width Recession depth (RD) reduced from 3mm pre-operativelyto 0 mm post-operatively while Recession width (RW) decreased preoperatively from 4 mm pre-operatively to 0 mm post-operatively demonstrating 100% root coverage. Width of keratinized tissue (KT) also increased to 5 mm from 4 mm post-operatively. Gingival thickness (GT) increased to 1.5 mm from 1mm postoperatively.

Discussion
Healing is a crucial phenomenon after the completion of any surgical procedure is over. It is associated with a complex process comprising of both intracellular and extracellular events that are regulated by some signaling proteins. It is not completely understood, but it is quite obvious that, platelets play a major role in formation of blood clot ( [1][2][3][4][5][6].Directinteraction between fibrin and the osseous tissue has no supportive evidence. But, bone morphogenic proteins (BMP) enmeshed in fibrin matrix have the ability to be released consistently highlighting the angiogenic, hemostatic and osteo conductive properties (Khiste&Tari2013; p.1-6).Fibrin has the credit of acting as a supportive matrix for BMP. BMPs enmeshed in fibrin are progressively released and are able to induce boneformation. Consistent release of vacular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) helps in angiogenesis. Hemostasis is achieved through the ability of fibrin clot to trap circulating stem cells, allowing vascular and tissue restoration (Khiste&Tari2013; p.1-6). Ross et al(1974) was among the pioneers who first described growth factors from platelets (Khiste&Tari,2013,p.1-6).Plateletrich plasma (PRP) was introduced for the first time by Marx et al in 1998(Khiste&Tari2013;p.1-6).This biomaterial was advantageous as it was easy to handle and did not provoke chances of infectious diseases (Kauret al.2011, p.86-89).But, it was suggested that there was release of growth factors for a shorter period of time and antibodies to bovine factor Va might cross react with human factor Va and might produce coagulopathies and rare bleeding episodes.(Kauret al. 2011 p.86-89; Harmon et al.2011,p.8-9).It is contraindicated in patients having Platelet dysfunction syndrome or Critical thrombocytopenia and who are sensitive to bovine antigen (Harmon et al.2011, p.8-9) This led to the development of platelet-rich fibrin (PRF), a 2 nd generation platelet concentrates (natural concentrate is produced without any anticoagulants or jellifying agents.) having various components like fibrin matrix polymer, blood aggregates like platelet derived growth factor (PDGF) and transforming growth factors (TGF-β), cytokines, leucocytes and circulating stem cells (Zhao et al.2013, p.3-5).It was found that the use of platelet-rich fibrin eliminates the need for donor tissue grafting along with fewer amounts of post-operative discomfort (Huang et al.2005(Huang et al. , 1770(Huang et al. -1774.It is produced by a simple method, safer than PRP, does not require any anticoagulant or bovine thrombin, is costeffective and readily available due to which it has been utilized in various treatment modalities like orthopaedic sand plastic surger-ies (Zhao et al. 2013, p.3-5).The presence of leukocytes and various cytokines enables the self-regulation of the infectious and inflammatory processe (Kauret al. 2011, p.86-89).Various studies have concluded that PRF has a proliferatives effect on different types of cells such as dental pulp cells, human osteoblasts, human gingival and periodontal ligament fibroblasts, dermal prekeratinocytes and preadipcytes (Tsai et al.2009  But, this biomaterial too have certain limitations. It can't be used in uncooperative patients, not willing to give blood and who are suffering from Platelet dysfunction syndrome or Critical thrombocytopenia (Harmon et al.2011, p.8-9).Also, the success of PRF preparation depends on the speed of blood collection and transfer to the centrifuge machine. Since there is absence of anticoagulant, the blood samples still start to coagulate immediately on contacting the tube glass .Another limitation is that the dehydration and inadequate volume of the membrane can result in failure of the PRF membrane (Arocaet al.2009, p.247-250). Quick handling is the only way to obtain clinically usable PRF clot (Zhao QM et al.2013p.4-5).In my case, the technique of PRF preparation was followed carefully, and it was prepared immediately before placement into the recession defect. Also, it was observed that the applications of orthodontic/button and sling sutures were quite effective in maintaining the coronal displacement of

Conclusion
Application of PRF as an adjunct to the Coronally advanced flap is a beneficial process for increasing the width of keratinized gingiva and achieve uneventful healing. Coronal stabilization during first two weeks of Coronally advanced flap adds to its long-term stability. However, dehydration and inadequate volume of the membrane can be responsible for causing failure of the PRF membrane and therefore, there is a need to quickly handle is the PRF clot.