Use of autologous platelet rich plasma to treat gingival recession in esthetic periodontal surgery.

BACKGROUND: Multiple approaches have been used to replace lost, damaged or diseased gingival tissues. Coronally advanced flap (CAF) and the use of guided tissue regeneration are among the successfully used surgical techniques to treat gingival recession. Platelet rich plasma (PRP), containing autologous growth factors, has been shown to promote soft-tissue healing. Therefore, the purpose of this study was to evaluate the efficacy of PRP in combination with CAF in the treatment of gingival recession.
MATERIALS AND METHODS: A total of 15 systemically healthy patients with buccal Miller's class I and class II gingival recession in cuspids or premolars participated in the study. CAF procedure was performed and PRP with collagen sponge was placed over the defect. Clinical parameters such as recession depth, recession width, surface area, width of keratinized gingival (KG), clinical attachment level (CAL), probing depth, plaque index and gingival index were evaluated at 3, 6 and 9 months post-surgery. The percentage of root coverage was calculated.
RESULTS: The results of this study suggest that the CAF procedure provides a predictable and simple technique in the treatment of localized Class I and Class II gingival recession. The additional application of PRP does significantly increase the width of KG and gain in clinical attachment.
CONCLUSION: CAF procedure is a predictable and simple technique in the treatment of gingival recession and the additional application of PRP does significantly increase the width of KG and gain in CAL. The long-term benefits following surgical treatment of such defects needs to be determined further.

INTRODUCTION

The primary objective of periodontal therapy is to improve the periodontal health and to preserve the dentition. However, esthetics has become an inseparable part of oral therapy recently. With the increasing popularity of cosmetic dentistry, root coverage procedures to enhance the level of keratinized tissue around exposed tooth surfaces have gained popularity over the last few years.

Gingival recession can be localized or generalized and can cause major functional and esthetic problems as it may result in a higher incidence of root caries, periodontal attachment loss and hypersensitivity;[1] more importantly gingival recession needs to be treated because of esthetic concerns.[2] Therefore, root coverage procedures have been developed to treat these conditions. Various modalities of root coverage procedures exist. These include laterally sliding flap,[3] double papilla flap,[4] subepithelial connective tissue graft,[5] coronally advanced flap (CAF),[6] free gingival autograft[7] and guided tissue regeneration techniques.[8] The most recent advances in root coverage include the use of various adjunctive agents such as acellular dermal matrix graft,[9] enamel matrix derivative,[10] recombinant human growth factor and platelet rich plasma (PRP). PRP derived from concentrated platelets is identified as one mediator, which has many growth factors. These growth factors are important at all stages of cell cycle and may stimulate chemotaxis and production of extracellular matrix proteins.[11] Administration of these growth factors may be combined with tissue regeneration techniques in repair of intrabony defects,[12] furcation[13] and sinus augmentation.[14] All these procedures have demonstrated new bone formation and bone healing. However, there is limited evidence regarding its effects on soft -tissue healing. There are only few case reports wherein PRP is used to treat gingival recession.[1516] Hence, the present study was taken up by doing a series of cases to assess the outcome of CAF along with PRP in the treatment of gingival recession. The objective of this study was to evaluate the effectiveness of PRP with collagen sponge as an adjunct to CAF procedure and to assess the soft-tissue healing with the use of PRP.

MATERIALS AND METHODS

The study was approved by the ethical committee of Oxford Dental College. Patients who visited the out-patient department of Periodontics, Oxford Dental College, Bangalore, India, for either dentin hypersensitivity or esthetic concerns were evaluated to be included in the study. The clinical trial included 15 male patients ranging in age from 21 years to 38 years (mean age 27 years).

Patient selection criteria for this clinical study included the following:

Age of 18 years or more

Miller's class I or II buccal recession defect with recession depth (RD) of ≥2 mm

Radiographic evidence of sufficient interdental bone

Probing sulcus depth (PD) of ≤3 mm

A minimum width of keratinized gingiva (KG) of 1 mm.

Exclusion criteria were

Medically compromised patients

Smokers

Those on corticosteroids and other immune modulators

Tooth with the endo-perio problem

Aggressive periodontitis.

Each participant completed initial therapy consisting of oral hygiene instructions, scaling, root planing of the required teeth, polishing and occlusal adjustment if necessary. After 4 weeks, patient's oral hygiene was evaluated. Study models of the teeth were prepared.

Clinical measurements

Data were collected on the standard case history proforma. All clinical measures were made to the nearest 0.5 mm with a standard University Of North Carolina (UNC-15) manual probe. For the tooth selected for surgery, the following measurements were recorded: RD, recession width (RW), surface area (SA) of the defect, width of KG, clinical attachment level (CAL) and PD.

RD was measured from the midfacial point of the cemento-enamel junction (CEJ) to the free gingival margin (FGM). The recession width (RW) was recorded at a level of 1 mm apical to the CEJ. The width of KG was determined by subtracting the RD measure from the CEJ- mucogingival junction (MGJ) distance. The SA of the defect was measured by placing tin foil over the defect, conform it to the shape of the defect, placing this foil over a graph paper and then counting the number of squares in the area of the foil. The SA was calculated in square millimetres. Two half squares were considered as one square. All parameters were again measured at 3, 6 and 9 months. Wound healing index (WHI)[17] was recorded 1 week after surgery using the following criteria: Score 1 = uneventful healing with no gingival edema, erythema, suppuration, patient discomfort or flap dehiscence; Score 2 = uneventful healing with slight gingival edema, erythema, patient discomfort or flap dehiscence, but no suppuration; and Score 3 = poor wound healing with significant gingival edema, erythema, patient discomfort, flap dehiscence or any suppuration. Additional clinical data were obtained by taking pre-operative photographs and at each post-operative visit.

The percentage of root coverage (PRC) was calculated according to the following formula:[18]

To measure oral hygiene, plaque index was recorded according to the criteria for the PI (Silness and Loe 1964).[19] Recordings for gingival status was made according to the criteria for GI by Loe and Silness.[20]

Preparation of PRP

One hour before the surgery, 8 ml of blood was drawn from the antecubital vein into the vacutainers (VacuetteR) containing 3.2% anticoagulant sodium citrate. To separate and concentrate platelets, two separate centrifugations (referred to as spin) were done. In the first spin, the blood was centrifuged at 2000 rpm for 2 min. This separates the red blood cells from the rest of the whole blood (white blood cells, Platelets and Plasma) with a thin white line in between (called as buffy coat), which has maximum concentration of platelets. The plasma and the buffy coat were pipetted out in a separate test tube and centrifuged (second spin) at 4000 rpm for 8 min. The second spin results in two separate fragments. The bottom layer is the PRP which is overlaid by supernatant fluid platelet poor plasma (PPP). PPP is pipetted out in a separate test tube. The PRP is then used for the procedure.[21] For the purpose of this study, PRP was obtained by the modified method of Curasan.[22]

Surgical procedure

The surgical procedure was performed under local infiltration with 2% lignocaine containing adrenaline at a concentration of 1:200,000. After obtaining adequate anesthesia, two vertically divergent incisions (i.e., one mesial and one distal, immediately adjacent to the defect) extending beyond the MGJ was made on the buccal aspect of the involved tooth. Sulcular incision was then placed up to the end of vertical incisions [Figures 1 and 2]. Care was taken to see that the papilla was intact. A trapezoidal mucoperiosteal flap was elevated by blunt dissection to the level of the MGJ. In the area apical to the MGJ, partial thickness dissection was employed [Figure 3]. The vertical incision was extended apically, well beyond the MGJ and into the mucobuccal fold, to a point where the flap could be positioned over the original defect without tension. In the adjoining interdental papilla, de-epithelialization was carried out with a B.P blade no. 11 to provide a bleeding connective tissue bed for the future CAF.

Figure 1

Case 1: Pre-operative view of recession in relation to 23

Figure 2

Case 1: Vertical and sulcular incisions given

Figure 3

Case 1: Full and partial thickness flap reflected

The exposed root surface was thoroughly planed and contoured by no. 3-4 Gracey curette as a part of mechanical root biomodification. A measurement of the appropriate width necessary for the graft was obtained with a periodontal probe. Here, collagen sponge (CollacoteR Integra LifeSciences Corporation.) was used as a carrier for PRP. The collagen sponge was properly trimmed according to the measured defect size. The PRP was poured over the membrane until it was fully soaked. Then two drops of 10% calcium chloride and autologous blood was used for activation of PRP to initiate coagulation and formation of PRP gel. The platelet concentrate graft was placed over the denuded root extending from 2 mm apical to the crest of the alveolar bone up to the CEJ below and to the incision lines at the sides [Figure 4]. The flap was then coronally positioned to completely cover the graft and secured by sling suture using no. 4-0 bioabsorbable suture (VICRYL™, Johnson and Johnson). The vertical incisions were then sutured with two direct interrupted sutures on either side [Figure 5]. A tin foil of suitable size was placed on the buccal aspect and a non-eugenol periodontal dressing (Coe-Pak, GC) was adapted [Figure 6]. Post-operative instructions were given and patient was recalled for follow-up regimen (1 week, 3, 6 and 9 months). Similar surgical protocol was adopted for all patients.

Figure 4

Case 1: Platelet rich plasma with collagen sponge placed over the defect

Figure 5

Case 1: Flap coronally advanced and sutured

Figure 6

Case 1: Periodontal pack in place

Post-operative care

Analgesics (non-steroidal anti-inflammatory drug Ibuprofen 200 mg) were prescribed to control post-operative discomfort. No antibiotics were prescribed. Patients were asked to refrain from oral hygiene measures on the treated areas for 10-12 days and instructed to rinse the mouth with warm salt water twice daily for 2 weeks. From the third week of surgery, patients were instructed to resume mechanical tooth cleaning using a soft brush with a careful roll technique.

Patients were recalled after one week for post-operative check up to evaluate the healing [Figure 7]. Periodontal pack was removed followed by irrigation with saline.

Figure 7

Case 1: 1 week post-operative view

The follow-up was carried out at three, six and nine months. All clinical measurements were recorded during the follow-up visit.

Statistical analysis

Quantitative data were summarized as mean ± standard deviation. Repeated Measures Analysis of variance (ANOVA) has been used to find the significance of study parameters between baseline, at 3 months, at 6 months and at 9 months

Student t-test with Bonferroni correction has been used to investigate the significance of the difference between two population means. No assumption is made about the population variances.[23]

Statistical software

The Statistical software namely SPSS 15.0, Stata 8.0, MedCalc 9.0.1 and Systat 11.0 were used for the analysis of the data and Microsoft Word and Excel have been used to generate graphs and tables.

RESULTS

Fifteen patients aged between 21 years and 38 years (mean age 27 years) were enrolled in the study of which thirteen patients completed all the follow-up protocol and were considered for the study. A total of 15 gingival recessions comprising of 9 canines and 6 premolars were treated with CAF and PRP.

Healing was uneventful in all patients with very minimal pain post-operatively. The mean WHI was assessed by examining for erythema and edema, was 1.33 ± 0.59, which was more towards normal and favorable with uneventful healing. However, in one case, flap perforation occurred inadvertently, which was managed by coronally advancing the flap and suturing it has done in the rest of the cases.

Platelet count

PRP was prepared one hour prior to surgery by the modified Curasan method[21] as per the technique described in the methodology. PRP samples of two cases were randomly selected for platelet count using cell counter. The count was in the range of 4.8 lakhs to 7 lakhs/μl.

Plaque index and gingival index

The post-surgery defect specific plaque index and gingival index did not increase beyond 0.5 at any time during the post-operative period. There was no statistically significant difference between the plaque index and gingival index at baseline and that observed post operatively (P = 0.584 and P = 0.274, respectively) [Tables 1 and 2].

Table 1

Plaque index

Table 2

Gingival index

Probing depth

There was a gradual decline of probing depth from baseline to 3 and 6 months and at the end of 9 months it was moderately significant with a P value of 0.019. The mean probing depth at 3 months was 1.57 mm (reduced by 0.13 mm compared to baseline) while it was 1.07 mm at 6 months (reduced by 0.63 mm) and 0.81 mm at the end of 9 months (reduced by 0.89 mm) [Table 3, Graph 1].

Table 3

Summary of change from baseline to 3, 6 and 9 month interval with p value of the following clinical parameters

Graph 1

Probing depth in mm

Clinical attachment level

The mean baseline CAL was 3.97 mm. At 3 months, the mean CAL was 2.63 mm reflecting an attachment gain of 1.34 mm. The mean CAL at 6 and 9 months were 2.10 mm and 1.43 mm reflecting an attachment gain of 1.87 mm respectively. At 6 months, the mean CAL was 2.10 mm showing an attachment gain of 1.87 mm. At 9 months, the mean CAL was 1.43 mm again showing an attachment gain of 2.54 mm. The change was highly significant with P < 0.001 [Table 3, Graph 2].

Graph 2

Clinical attachment level

Recession width

The mean RW was 2.63 mm at baseline, which decreased to 1.47 mm at 3 months, 1.57 mm at 6 months and to 0.94 mm at 9 months. The decrease in RW was highly significant (P < 0.001) with the percentage of decrease in RW being 61.3% at the end of 9 months [Table 3, Graph 3].

Graph 3

Recession width

Recession depth

The mean recession depth was 2.33 mm at baseline, which decreased to 0.87 mm at 3 months and 0.80 mm at 6 months. The recession depth significantly reduced to 0.5 mm at 9 months. The decrease in recession depth was highly significant (P < 0.001) with an improvement of 78.5% at 9 month [Table 3, Graph 4].

Graph 4

Recession depth

SA of the defect

The mean baseline SA was 5.27 mm2, which decreased to 2.20 mm2 at 3 months and 2.63 mm2 at 6 months postoperatively. Further, the mean SA significantly reduced to 1.63 mm2 at 9 months (69.1% decrease) P < 0.001; [Table 3, Graph 5].

Graph 5

Surface area of the defect

Width of KG

The mean width of KG at baseline was 2.63 mm. At 3 months, the width increased to a mean of 3.27 mm and 3.43 mm at 6 months. At the end of 9 months, the width increased to 3.50 mm. The change was highly significant with P < 0.001 amounting to an increase in gain of KG by 33.1% [Table 3, Graph 6].

Graph 6

Width of keratinized gingiva

PRC

PRC is the result of decrease in RW, recession depth and SA of the defect. All treated sites gained root coverage. At 3 months, the mean root coverage was 62.55% while it was 65.33% and 78.75% at 6 months and 9 months, respectively [Table 3, Graph 7].

Graph 7

Percentage of root coverage at 3 months, 6 months and 9 months

Among the study group, three of the 15 defects showed complete root coverage [Figures 8-12]. The site with buccal perforation showed the least, with 20% root coverage.

Figure 8

Case 1: 3 month post-operative view

Figure 12

Case 2: 9 month post-operative view with complete root coverage

Case 1: 6 month post-operative view with complete root coverage

Case 1: 9 month post-operative view with 100% root coverage

Case 2: Pre-operative recession depth of 3 mm in relation to 44

DISCUSSION

Although root coverage is a desirable outcome, it is necessary to achieve an increased zone of KG, reduced probing depth as well as gain in CAL. PRP, rich in growth factors has been shown to enhance soft-tissue healing, promote initial clot stabilization and revascularization of the flaps and grafts in root coverage procedures. However, limited evidence is currently available verifying these claims. Therefore, this study was conducted to study the effects of PRP on CAF procedure.

In general, PRP, when used along with bone graft, acts as a scaffold and enables to be placed easily in an intrabony defect or a furcation defect. Since placement of PRP on a recession defect is difficult, a collagen sponge (CollacoteR Integra LifeSciences Corporation), a wound dressing material, was used as a carrier for PRP. The benefits of this collagen sponge include controlling the bleeding and stabilization of blood clot, protection of the wound bed, provides a matrix for tissue ingrowth and gets fully absorbed in 10-14 days.

The results of the present study demonstrated that use of PRP in CAF procedure may lead to consistent RD reduction and considerable improvement in root coverage.

In the present study, PRP was prepared one hour prior to surgery and when the platelet count was randomly checked, the number of platelets were 4 times greater than the baseline value of 1.5 lakh/μl. This was similar to the value obtained by Marx[24] who has shown that this concentration was enough to produce clinical benefits. This method is preferred because it requires only 8 ml of blood, less time for preparation and can be used in routine dental set up.

In the present study, during one week post-operative examination, the gingival appearance was evaluated by examining for erythema and edema. In all cases, gingival appearance was nearly normal with appearance varying between “no gingival erythema or edema to a slight erythema and edema” (as evident in Figure 7). Patient discomfort was present only on the first day and with very minimal post-operative pain. This shows an accelerated soft-tissue healing. PRP is known to contain higher levels of platelet derived growth factor (PDGF), transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) and these growth factors may reportedly enhance soft-tissue healing by hastening the angiogenesis and matrix biosynthesis during early wound healing.

Plaque and gingival indices remained relatively constant at all-time intervals during the experimental period. Probing depth was included as an assessment parameter to detect if there was a negative effect of therapy in terms of increasing buccal probing depth. There was a reduction in probing depth of 0.63 mm, which was moderately significant from pre-operative to post-operative examination.

There was an average gain in CAL of 1.87 mm at 6 months and 2.54 mm at 9 months. This gain in CAL was accompanied by reduction in probing depth and recession depth. Considering that root coverage was achieved in these situations, the gain in CAL suggests the formation of new attachment to the root surface, although, in the absence of histological evidence, it is not possible to determine the type of gain in attachment. The gain in CAL might be attributed to the growth factors secreted by PRP would have attached to the membrane in the cells in the flaps and to the underlying root surface and prevented shrinkage.

The presence of an “adequate” zone of gingiva has been considered critical for the maintenance of gingival health.[25] There was a gain of 0.8 mm from baseline to 6 months while Huang et al.[17] achieved 0.4 mm gain of KG with the use of PRP on CAF. Since the stimulation for keratinization of surface epithelium is provided by underlying connective tissue, it can be construed that the newly formed connective tissue had the ability to induce keratinization. The newly formed connective tissue, helped by PRP, possessed the ability to induce keratinization of overlying epithelium.

However, the mean recession depth reduction was less as compared to Pini Prato study, where a mean recession depth reduction of 4.12 mm was seen for CAF technique alone and 2.3 mm mean recession depth reduction by Huang et al. for CAF procedure along with PRP and whereas, in our study, the mean depth reduction was 1.53 mm. This difference in the mean reduction can be explained based on the selection of the size of the defect sites. In the present study, a baseline value of 2-3 mm of recession depth was selected, whereas in the group selected by Pini Prato, the recession depth ranged from 3-8 mm. As reported by Pini Prato[26] sites with deeper recession defects tend to respond more favourably than shallower sites.

The 6 month and 9 month data indicates that a CAF operation with PRP is as efficient as the other procedures in terms of root coverage. The mean root coverage at 6 months was 65.33% and 78.75% at 9 months. Studies show mean defect coverage ranging from 50% to 98% with a mean for all studies of 78%. Predictability data indicated that 90% or greater defect coverage was achieved 39% of the time.[27] The percentage root coverage obtained in the present study is within the range of reported results of 64.2%,[28] 75%,[29] 78%,[30] 97.1%,[31] 97.8%[32] and 98.8%.[33]

The difference in the outcomes of treatment in the present study and other studies may be attributed to technical and anatomic factors. Technical factors such as operator experience/surgical skills and the occurrence of a learning curve during the study may also account for the difference between the studies. The measurement techniques employed also differ in some studies. Other anatomical factors such as root prominence, depth of the vestibule, soft-tissue quality – all these variables were not considered and might also have accounted for a less favourable outcome.

When keeping in mind the various limitations, the degree of root coverage obtained in the present study compares well with other reports. The additional benefits obtained with the use of PRP are:

It decreased the incidence of both intraoperative and post-operative bleeding at the receptor sites because of its inherent hemostatic properties

Decreased the incidence of postoperative pain

Also, the thickness of the KG was increased.

In addition, the use of PRP promotes a more rapid vascularization delivering growth factors specific for capillary formation. Hence in the present study, one week post-operative cases showed less erythema and the tissues appeared normal and comparable with the adjacent area. PRP is known to contain higher levels of PDGF, TGF-β and VEGF. These growth factors might enhance soft-tissue healing by increasing the angiogenesis and matrix biosynthesis during early wound healing.

However, this short term benefit did not significantly influence the final clinical outcomes, e.g., PRC. But a significant increase in CAL and width of KG was definitely observed. Nevertheless, histological examination needs to be performed to determine the type of attachment.

CONCLUSION

Keeping in mind the ample information available about the properties of PRP and the improvements seen in the various clinical parameters, it can be assumed that PRP along with collagen sponge is beneficial in the treatment of gingival recession. However, a clinical trial with larger sample size supported by histologic study is needed to provide evidence of PRP's impact on wound healing and the clinician's quest for periodontal regeneration.