Oral Maxillofac Surg DOI 10.1007/s10006-013-0435-0

ORIGINAL ARTICLE

Effect of platelet-rich plasma on bone regeneration after removal of cysts and benign tumours of the jaws Arvind Ramanathan & K. M. Cariappa

Received: 6 June 2013 / Accepted: 11 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Introduction Platelets are involved in regeneration at sites of pathology, apart from their role in clotting. A preparation composed mainly of platelets (platelet-rich plasma gel) applied to sites of bony pathology, after surgical treatment of lesions, may hasten bone regeneration. Materials and methods An autologous platelet-rich plasma (PRP) gel was prepared using a standardized technique, without using thrombin clot accelerator, and applied to surgical site in six patients of study group. Five patients were enrolled as controls, in whom PRP gel was not used. The differences in the occurrence of radiographic changes between the study and control group at 6, 12, 18 and 24 weeks after surgery were analysed with chi-square test. Intragroup radiographic changes, i.e. within the study and control groups occurring over the 24 weeks of follow-up, were analysed with Friedman test. Results A trend towards more rapid healing was observed in the study group at 6, 12, 18 and 24 weeks. However, these differences between the study and control group were not statistically significant. Both the study and control group demonstrated significant healing changes over the 24 weeks of follow-up. Conclusions It is possible to prepare platelet-rich plasma gel without using thrombin clot accelerator. PRP, as prepared and applied to surgical sites in this study, was not observed to significantly enhance bone regeneration. All surgical sites, both in the PRP and control group, showed significant healing changes over 6 months. A. Ramanathan (*) Manipal College of Dental Sciences, Manipal University Mangalore, Mangalore, Karnataka, India e-mail: [email protected] K. M. Cariappa Manipal College of Dental Sciences, Manipal University Manipal, Manipal, Karnataka, India

Keywords Platelet-rich plasma . Bone regeneration . Growth factors

Introduction Platelets contribute not only to the haemostatic process but also to wound healing through the release of growth factors (cytokines). These growth factors initiate and sustain wound repair. An interesting idea developed that the addition of a concentrate of platelets, to wounded tissues or surgical sites, may enhance the body’s natural wound healing mechanisms [1]. Platelet-rich plasma (PRP) is the accepted term to describe the preparation made out of autologous platelets suspended in autologous plasma, in concentration greater than normally found in peripheral blood [2]. PRP has been used in various surgical fields such as otolaryngology, head and neck surgery, neurosurgery, general surgery, oral and maxillofacial surgery, and periodontics. PRP has been called ‘an autologous alternative to fibrin glue’ [3]. Because it is autologous, PRP avoids the risk of transmissible diseases such as HIV, Hepatitis B, C or D and other blood-borne pathogens [2]. A clot accelerator such as bovine thrombin is added to gel the PRP or PRP is added to other materials such as collagen, gelfoam, etc. In the present study, the effect of PRP on bone regeneration when applied to the site of removal of cyst and benign tumours of the jaws is evaluated. PRP was delivered to the site combined with gelfoam. Thrombin clot accelerator was not used.

Materials and methods This study was conducted on adult patients diagnosed to have cysts or benign tumours of the jaws, after institutional approval of study.

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Inclusion criteria are the following:

Fig. 1 Separation of whole blood after first spin

1. Patients with cyst or benign tumour of the maxilla or mandible 2. Cysts that had become infected were also included 3. The lesion could be visualized radiographically 4. Patients within the age range of 20 to 50 years 5. Patients who were non-smokers and non-alcoholics Exclusion criteria are the following: 1. 2. 3. 4.

Patients with systemic diseases History of bleeding tendencies, blood dyscrasia Low platelet counts Patients unable or unwilling to return for follow-up

Patients who reported between October 2005 and March 2006, satisfying the above criteria were included in the study. Patient consent was obtained. They were assigned to the study or control group. Patients who had been operated on prior to this time interval but were on regular follow-up were included as controls. The size or specific locations of lesions was not used as criteria for patient selection. Lesions were divided into small (3 cm) for descriptive purposes.

transferred into Vacutainer tubes and again centrifuged for 10 min at 2,000 rpm. This results in an upper portion of clear yellow supernatant serum and the bottom red-tinged layer consisting of highly concentrated PRP (Fig. 2). The upper clear layer is aspirated until 1 ml of serum is left. The contents of the tube are mixed well (Fig. 3). Platelet counts were obtained before and after platelet enrichment using Haematology Analyzer (ABX Pentra).

Preparation of PRP Surgical procedure Preoperatively, platelet-rich plasma was prepared in the Haematology Laboratory from 20 ml of the patient’s blood using an R8-C Laboratory Centrifuge (Remi Motors Ltd., Mumbai) using an aseptic technique. Armamentarium

The enucleation of cyst or tumour and any further treatment to eliminate pathology was performed under local anesthesia, or in case of large lesions, treatment was performed under general anesthesia. Affected teeth were either removed or treated endodontically.

Anticoagulant citrate phosphate dextrose, sterile 10 ml test tubes (2 nos.), sterile pipette, Vacutainer tubes 5 ml (2 nos.), 10 ml syringe and 21 gauge needle were used. Procedure Twenty millilitres of venous blood is collected from antecubital region with a 20-ml syringe and transferred to two test tubes containing 1.4 ml anticoagulant (citrate phosphate dextrose solution) each. (A ratio of 1.4 ml of anticoagulant to 10 ml of blood) It is then centrifuged for 10 min at 1,300 rpm. The result is a separation of whole blood into a lower red blood cell region and an upper straw coloured plasma region (Fig. 1). The upper straw coloured plasma layer (platelet poor plasma) and 1 to 2 mm of top part of RBC layer is aspirated and

Fig. 2 After second spin highly concentrated PRP at the bottom

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Fig. 5 PRP gel

Follow-up Fig. 3 Platelet-rich plasma (PRP)

In the study group, for filling the bony defect PRP gel was used. At the time of application, 1 ml of 10 % calcium gluconate was added to the PRP and mixed well. A small cube of Gelfoam was added to the test tube to soak load PRP (Fig. 4). The test tube was left standing for 5 min. A gel formed that was placed inside the defect (Figs. 5 and 6). Primary wound closure was performed. In the control group, PRP gel had not been placed into the bone defect that resulted after treatment of pathologic lesion. In one case of a unicystic ameloblastoma involving a large part of one half of the body of the mandible, a Bismuth Iodoform Paraffin Paste pack had been placed after elimination of the lesion. Sutures were removed after 7 days. At this time, the wounds were observed for primary wound healing, visible dehiscence or wound infection with pus.

Bone healing in the defect after treatment of lesions was evaluated radiographically. Patients were recalled at intervals of 6 weeks for follow-up of 6 months. A single IOPA radiograph or standard occlusal radiograph was obtained during follow-up appointments. A panoramic radiograph was made if the dimension of osseous defect was very large. Standardized radiographic techniques were used. Making of multiple radiographs was strictly avoided. The IOPA or occlusal radiograph was manually processed while the panoramic radiograph was processed in an automatic dental film processor. Assessment of bone healing The radiographs were assessed by a single examiner, the first author. Bone healing was assessed according to the radiographic changes that occurred in the site margins and site interior [4]. Post-surgical radiographic changes in the site margins were categorized as the following: 1. Unchanged —Original lesion margin completely preserved

Fig. 4 Adding gelfoam to PRP

Fig. 6 PRP gel

Oral Maxillofac Surg Table 1 Specific diagnosis of lesions, location and size

Study group

Age

Sex

Diagnosis

Site

Size

Case 1 Case II Case III Case IV Case V

29 39 24 36 28

M M M F F

Central giant cell granuloma Periapical cyst with infection Periapical cyst with infection Periapical cyst Complex odontoma

Anterior maxilla Anterior maxilla Anterior maxilla Posterior maxilla Mandible molar region

Large Small Small Small Large

Case VI Control I Control II Control III Control IV Control V

24 32 31 50 24 34

M M F F M M

Odontogenic keratocyst Unicystic ameloblastoma Periapical cyst with infection OKC OKC Periapical cyst with infection

Mandible canine region Mandible symphysis and body Anterior maxilla Mandible third molar region Mandible ramus Anterior mandible

Large Very large Small Large Large Small

2. Partly reduced —Original lesion margin is partly decreased in width and clarity 3. Entirely absent—Original lesion margin entirely absent The post-surgical radiographic appearance of the internal portion of the surgical site was classified [5] as follows: 1. No closure: The bone defect remained radiolucent with the exception of limited new bone apposition at the defect margins 2. Partial closure: The bone defect was partially radiopaque, exhibiting regions of radiolucency and radiopacity without establishing bone continuity 3. Complete closure: The bone defect showed radiopacity/ osseous continuity between the defect margins This was symbolically denoted as follows: No closure ‘−’ Partial closure ‘+ +’ Complete closure ‘+ + +’

females and four males, mean age 30.00 years. The control group had three males and two females, mean age 34.20 years. With regard to size of lesion, the study group had three ‘small’ (3 cm in diameter) lesions. In the control group, there were three large and two small lesions. The specific diagnosis of lesions in each group, location and size are summarized in Table 1. The technique used to prepare platelet-rich plasma yielded a platelet enrichment of more than 300 % (mean 318 %) (Table 2). In one case, clumping of platelets was observed during enrichment. The patient was included in the study as control. In one patient in the study group, wound dehiscence was observed at the time of suture removal (case no. 5). The wound was without any signs of acute infection and was managed during follow-up applying local rinses. The radiographs of the 11 patients were assessed. Observations of bone healing over the 6 months of follow-up are presented in Tables 3 and 4 and photographic illustrative in Fig. 7.

Statistical analysis The differences in the occurrence of radiographic changes between the study and control group at 6, 12, 18 and 24 weeks after surgery were analysed with chi-square test. Intragroup radiographic changes, i.e. within the study and control groups occurring over the 24 weeks of follow-up, were analysed with Friedman test.

Results The study included 11 subjects—six in the study group and five in the control group. The study group had two

Table 2 Platelet enrichment in PRP

Case 1 Case 2 Case 3 Case 4 Case 5 Case 6

Pre-enrichment platelet counts (1,000/cumm)

Platelet counts in PRP (1,000/cumm)

Percent enrichment (%)

198 144 207 373 214 150

618 447 690 1,189 671 500

312 310 333 318 313 333

Oral Maxillofac Surg Table 3 Radiographic changes in lesion margin. ‘X’—data unavailable

Serial number

6 weeks

12 weeks

18 weeks

24 weeks

Case I Case II Case III Case IV Case V

Unchanged Partly reduced Partly reduced Unchanged Partly reduced

X Partly reduced Entirely absent Partly reduced Partly reduced

Entirely absent Partly reduced Entirely absent Partly reduced Partly reduced

Entirely absent Entirely absent Entirely absent Entirely absent Entirely absent

Case VI Control I Control II Control III Control IV Control V

Unchanged Unchanged Unchanged X Unchanged Unchanged

Partly reduced Partly reduced Unchanged Entirely absent Partly reduced Unchanged

Entirely absent Partly reduced Partly reduced Entirely absent Partly reduced Partly reduced

Entirely absent Entirely absent Entirely absent Entirely absent Entirely absent Entirely absent

On comparison of site margins at 6 weeks, the ‘unchanged’ status persisted in all the subjects of the control group and in half the subjects of the study group. The other half of the subjects in the study group had progressed to ‘partly reduced’ margin. By 12 weeks, all the patients in the study group had progressed to ‘partly reduced’ (66.7 %) or ‘completely absent’ margins. These numbers were less in the control group, 40 % partly reduced and 20 % completely absent. In 40 % (two patients), the margins remained unchanged. By 18 weeks, there were no subjects with unchanged margins. In 50 % of the subjects in the study group and 20 % in the control group, the lesion margins were entirely absent. By 24 weeks, lesion margins in both the groups were completely absent. Although healing changes in the margins of the lesion appeared to progress more rapidly in the PRP group, these differences were not statistically significant (Chi-square test; p >0.05). Bone regeneration into the site interior was evident in one subject (16.7 %) in the study group by 6 weeks. In all the other subjects, the lesion interior remained radiolucent. By

12 weeks, 60 % of the subjects in the study and 40 % of the subjects in the control group showed partial closure of defect by bone. By 18 weeks, two cases (33.3 %) in the study group showed osseous continuity/complete closure between the defect margins. Two cases in the control group had a persistent radiolucent interior. This was attributed to very large size of the lesion in one case and persistence of periapical infection in one case. By 24 weeks, 80 % of the subjects in the study and 60 % of the subjects in the control group showed complete closure of the defect. The differences were not statistically significant (Chi-square test; p >0.05). Using the Friedman test, radiographic changes in the margins of the lesions observed over 24 weeks in both the study group and control group were significant. Similarly, significant changes occurred in the interior of the lesion in both the groups (Tables 5 and 6).

Table 4 Radiographic changes in lesion interior no closure (−), partial closure (++), complete closure (+++), date unavailable ‘X’

There have been conflicting results from a variety of studies on the ability of PRP to enhance healing. A possible explanation for this could be that the techniques used to prepare PRP are varied and this would influence success [6, 7]. The technique used to prepare PRP, in this study, yielded a mean concentration of 318 %. Bovine thrombin was not used to accelerate gel formation. Apart from any potential risks associated with its use, it was difficult to procure. Instead, PRP was soak-loaded onto a piece of Gelfoam. Gelfoam is an absorbable gelatin sponge. It is a haemostatic agent that acts by initiation of clotting cascade through contact activation [8]. When implanted in tissues, it is completely absorbed within 4 to 6 weeks. Various studies on osseous regeneration in the presence of Gelfoam have demonstrated that this material initially provokes an inflammatory reaction but has no long-term deleterious effects on bone formation

Serial number

6 weeks

12 weeks

18 weeks

24 weeks

Case I Case II Case III Case IV

− − − ++

X ++ ++ ++

+++ ++ +++ ++

+++ +++ +++ +++

Case V Case VI Control I Control II Control III Control IV Control V

− − − − X − −

− − − − ++ ++ −

− ++ − ++ X ++ −

++ X ++ +++ +++ +++ ++

Discussion

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Fig. 7 Photographic illustrative table

[9–12]. Further, as explained by Schulte [13], insertion of a blood coagulum stabilized with gelation sponge may prevent

wound infection in cyst cavities of the jaws (>2 cm) on account of clot retraction.

Oral Maxillofac Surg Table 5 Radiographic changes in site margin over 24 weeks (Friedman test)

Case Control

Chi-square p Chi-square p

8.143 0.043 sig 11.000 0.012 sig

Another difference from ‘traditional’ PRP preparations is the choice of 10 % calcium gluconate, rather than calcium chloride, added to counter the effects of anticoagulant citrate. Ten percent calcium gluconate ampoule was readily available. The treatment rendered to eliminate cyst or tumour is not the same for all lesions. As the site, size, histopathologic diagnoses, biologic behaviour of lesions, operating surgeons and patient-related factors have varied, so have the individual steps of treatment. Hence, arguably, the study and control groups are not comparable in many respects. However, the end result at surgical site is a bony defect and observations have been made about status of bone healing. For assessment of bone changes, healing in the site margin was designated ‘Unchanged’, ‘Partly reduced’ and ‘Entirely absent’. Bone regeneration in site interior was recorded as ‘no closure’, ‘partial closure’ or ‘complete closure’. These designations were applied to simplify radiographic assessment. Lesions in both the study and control groups showed progressive bone regeneration in the 6 months of follow-up. In the control group, two cases did not show complete bone regeneration at 6 months (control I and control V). This can be attributed to the very large size of the lesion in control I and persistent periapical infection in control V. One case in the study group (case V) did not show complete bone regeneration at 6 months due to failure of primary closure with a resultant delay in bone healing. Both the study and control group demonstrated significant healing changes over the 24 weeks of followup. A trend towards more rapid healing is observed in the study group at 6, 12, 18 and 24 weeks. However, these differences between the study and control group were not statistically significant. Table 6 Radiographic changes in site interior over 24 weeks (Friedman test)

Case

Control

Chi-square df Asymp. Sig. Chi-square df Asymp. Sig.

9.700 3 0.021 sig 10.800 3 0.013 sig

Many studies on PRP have assessed its effects on soft tissue healing, pain and swelling [6, 14, 15]. Due to variations in the location, size of the lesions and type of anesthesia used (local or general), these factors could not have been assessed meaningfully. The PRP being a sticky gel with haemostatic properties was an aid to flap adaptation and primary closure, and PRP gel foam can be filler for empty bone cavity. This study could not demonstrate a significant enhancement of bone regeneration with PRP after removal of cysts and benign tumours of the jaws. The study is limited by small sample size, by the observational nature of radiographic evaluation. As previously stated, the technique of PRP preparation and gelation is different from other studies and the amount and type of growth factors released is not established. The amount of bone formation is assessed in the study, but evaluation of the density of bone being formed would have provided valuable insight. Histologic and histomorphometric techniques may have brought out differences not apparent on radiographs.

Summary and conclusion An attempt has been made to evaluate the effect of plateletrich plasma on bone regeneration after removal of cysts and benign tumours of the jaws. Eleven subjects were included in the study, six in the PRP and five in the control group. Subjects were followed up for 6 months at six weekly intervals. PRP was prepared in a laboratory centrifuge and platelet enrichment of 318 % was obtained. To deliver the PRP to the surgical site, it was combined with Gelfoam. Hence, it was possible to prepare platelet-rich plasma gel without using thrombin clot accelerator. However, the amount and type of growth factor released with this preparation is not established. When applied to the surgical site, PRP being a sticky gel was an aid to flap adaptation and primary closure, and PRP– gelfoam can be filler for empty bone cavity. Bone regeneration assessed on radiographs at the site margins and interior at 6, 12, 18 and 24 weeks was not found to be significantly different from that at control sites. By 24 weeks (6 months), all the surgical sites showed significant bone healing. Some of the limitations of the study are its small sample size and observational nature of radiographic evaluation. Histologic examination of sites and assessment of density of bone formed using histomorphometric techniques may have brought out differences not apparent on radiographs. To conclude, PRP as prepared and applied to surgical sites in this study was not observed to significantly enhance bone regeneration. All surgical sites, both in the PRP and control group, showed significant healing changes over 6 months.

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Alternatives to thrombin to activate PRP and the benefits of PRP when used alone require further research. Acknowledgments I gratefully acknowledge the surgeons, haematologist, statistician and laboratory technicians who provided their valuable guidance in the conduct of this study. Conflict of interest None

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Effect of platelet-rich plasma on bone regeneration after removal of cysts and benign tumours of the jaws.

Platelets are involved in regeneration at sites of pathology, apart from their role in clotting. A preparation composed mainly of platelets (platelet-...
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