A total of 30 systemically healthy adult patients, male and female between the age group of 16-35 years were selected for this double blind randomized clinical trial. This study was conducted in the Department of Oral & Maxillofacial Surgery, KLES Institute of Dental Sciences, Belgaum for a period of six months, from May 2008 to October 2008. The patients were selected randomly from the outpatient department based on the following criteria.

1. Patients requiring extraction of bilaterally impacted lower third molars with similar degree of impactions (moderate to difficult).

2. Male and female patients, age ranging from 16-35 years.

3. Class II position B impaction based on radiographs (Pell and Gregory’s classification).

4. Patients without any infection, pain and inflammation at the site of operation one week before the procedure.

5. Patients with any symptoms of pre-op infection were treated with antibiotics and then taken up for surgery once the infection was under control.

1. Patients with systemic diseases, pregnancy and breast-feeding.

2. Patients taking any medications that could interfere with the healing process.

3. Patients under any antibiotic regimen or taking anti inflammatory drugs within the one week period before the procedure.

4. Patients who exhibited any hypersensitivity to the drug aprotinin.

All patients were explained the details about the surgical procedure and the possible complications associated with the same. A written consent accordingly was obtained from all the patients before the surgery was carried out. The study was approved by the institutional ethical committee board.

The selected 30 patients were undertaken for surgical removal of impacted third molar on both the sides simultaneously.

A general medical history was recorded following which the clinical examination was done. Following which measurements for pain and swelling were carried out.

Postoperative pain was assessed using the (Verbal Pain Intensity Scale) [25], which measures pain verbally and a numeric value was attached to each, 0 = No pain; 1= Mild pain; 2 = Moderate pain; 3 = Severe pain; 4 = Very severe pain; 5 = Worst possible pain. Mean readings were taken at each 1^st, 2^nd, and 7^th Postoperative days [Table/Fig-1].

[Table/Fig-1]:

Clinical assessment of facial swelling on aprotinin and control side. Right side was control and left was Aprotinin: a) Aprotinin injection; b) Swelling measurement; c) Pre operative; d) Swelling 1^st day Postoperatively; e) Swelling 2^nd day Postoperative; f) Swelling 7^th day post op.

Patients were asked to keep the neck straight and sit in a relaxed position on the dental chair and look straight. Swelling was measured by drawing lines from angle of the mandible to 5 various points viz: (1) Tragus of ear; (2) Lateral canthus of eye; (3) Alae of nose; (4) Corner of mouth; (5) Menton on both sides of the patient on the 1^st, 2^nd, and 7^th Postoperative days [26] [Table/Fig-1b]. The preoperative readings for each patient were taken as the standard values. The statistical significance was analysed using Student t-test.

All patients in the study routinely received a prophylactic antibiotic (1 g Amoxicillin), 1 hour prior to the surgery. Multi vial bottle containing 50ml of the solution, each milliliter containing 10,000 KIU of aprotinin in saline was used for the study [Table/Fig-1a]. A test dose for aprotinin was first administered subcutaneously on the ventral surface of right forearm, marked and looked for any hypersensitivity reaction. Once no signs and symptoms were ascertained, the patients were asked to pick one out of two chits which decided the study side for that individual.

On the basis of this random selection, five minutes before commencement of the surgical procedure the study side was submucosally infiltrated with 1ml of 10,000 KIU/ml injection aprotinin in isotonic saline around the surgical site as follows. 0.3 ml submucosally each in lingual and buccal area followed by 0.4 ml in the retromolar area in systematic divided doses. The contra lateral side was infiltrated with 1ml of isotonic saline in the same manner. This was done before obtaining local anaesthesia [Table/Fig-2].

[Table/Fig-2]:

Depicting the intra oral peri apical radiographs and clinical site of injection of aprotinin: a) Periapical radiograph of lower right impacted third molar(48); b) Periapical radiograph of lower left impacted third molar (38); c) Submucousal injection of saline on right side; d) Submucosal injection of aprotinin on left side.

All the surgeries were performed by the same operator with identical surgical technique using the standard armamentarium. The patient’s face was prepared with 10% povidone-iodine solution (BetadineTM) and then draped following which inferior alveolar nerve, lingual nerve, and long buccal nerve block were administered using 2% Lignocaine Hydrochloride (with 1:80,000 Adrenaline) in 2 ml syringe with 26 gauge needle to achieve desired local anaesthetic effect. A standard incision (Ward’s incision) [27] was made in all cases and a mucoperiosteal flap was reflected using a Howarth’s periosteal elevator. The surrounding bone was removed with stainless steel burs (no.8). Wound closure was achieved using 3-0 black braided silk. After the surgery details of operation and duration of surgery (from incision to last suture) were recorded. Immediate postoperative instructions were given and no postoperative antibiotics were prescribed. However, all patients were prescribed paracetamol 500 mg (1 tablet every 6 h for two days) postoperatively and advised to avoid any medications but those prescribed. Patients were asked to take diclofenac sodium 50 mg 1 tablet if they experienced intolerable pain, only after rating their daily pain score as severe pain.

The patients were recalled for follow up on the first, second and seventh postoperative day by the same examiner. The patients were assessed for swelling, pain, wound healing and global pain on day 7 after removal of sutures. At the end of investigation, patients were asked to choose the least painful side. The mean value and standard deviation for each of the parameters was considered and checked for statistical significance using the Mann-Whitney test, which is used for non-parametric values.

All 30 patients completed the study.

When global pain was assessed, 24 (80%) of the patients chose aprotinin side to be less painful than the control. Two (7%) patients chose control side less painful and four (13%) patients found no difference between study and control side [Table/Fig-3]. Mean readings were taken on each 1^st, 2^nd, and 7^th postoperative days according to the degree of pain the patients described verbally as mild, moderate, severe, very severe and worst pain (0, 1, 2, 3, 4 and 5). No patients complained of very severe pain and worst possible pain. Mann-Whitney U-test was used to calculate the difference in pain between aprotinin and control side. Subjects experienced less pain on the aprotinin side compared to control side postoperatively. On the 1^st and 2^nd Postoperative days the mean pain scores on the aprotinin side was less compared to the control side [Table/Fig-4] with p-values of 0.000 and 0.000 respectively which was highly significant. On the 7^th Postoperative day the mean pain score was 0.03 on the aprotinin side, whereas it was 0.20 on the control side (p-value of 0.046) which was also significant.

[Table/Fig-3]:

Global pain assessment between study and control side.

The pain distribution was assessed on 1^st, 2^nd and 7^th day postoperatively where patients complained of no pain, mild pain, moderate pain and severe pain on both study and control side [Table/Fig-5]. The difference in pain distribution between aprotinin and control side on all days was not significant. The p-values on 1^st, 2^nd and 7^th day were 0.73, 0.14 and 0.57 respectively. Zero percent of patients complained of pain on the aprotinin side on 1^st Postoperative day whereas on test side 4% complained of severe pain. On the 7^th day 96.6 % patients complained of no pain [Table/Fig-6].

[Table/Fig-5]:

Pain distribution on 1^st, 2^nd and 7^th day Postoperatively on both study and control side.

The total time for surgery was noted (from the time of incision was placed till the last suture was given). It was found that on aprotinin side the minimum time taken for surgery was 51.3±10.3 minutes. In the saline group the mean duration of surgery was 59.4±8.9 min. Hence there was no significant difference between both groups.

The swelling was measured on pre operative, 1^st, 2^nd and 7^th postoperative days. On the 1^st postoperative day, the mean swelling was 1.07±0.64 mm on the aprotinin side, whereas on the control side the mean was 1.80±0.66 mm. On 2^nd Postoperative day the mean was 0.33±0.48 mm on the aprotinin side, whereas on the control side the mean was 1.20±0.48 mm. On the 7^th Postoperative day the mean was 0.03±0.18 mm on the aprotinin side, whereas on the control side the mean was 0.20 ± 0.41mm. The difference in swelling was not statistically significant. The average swelling in millimeters on each day is depicted in [Table/Fig-7].

[Table/Fig-7]:

Comparison of mean swelling size between study and control side Postoperatively.

However, clinically it was evident that swelling on the side with aprotinin was less as compared to the control side on the 1^st, 2^nd, and 7^th postoperative days [Table/Fig-1d-f]. The comparison of mean swelling size between test and control side is depicted in [Table/Fig-7]. It can be observed that swelling size is evidently lesser in the aprotinin side compared to control. The total time for surgery was also noted and found that on aprotinin side the mean time taken for surgery was 51 minutes whereas on control side was 50 minutes respectively.

The adverse effects associated with the drug were also recorded. No intraoperative or postoperative complications were encountered in any of the patients included in the study.

In the present study the efficacy of aprotinin in management of postoperative pain and swelling after mandibular third molar surgery was assessed. It has been observed that use of aprotinin clinically rarely causes hypersensitivity reaction [19]. Surgical removal of impacted third molar causes moderate to severe pain and forms a useful model for assessing the efficacy of analgesics [6,28]. Therefore, the mandibular impacted third molar was chosen as a clinical model in the present study.

The interpretation of pain, experienced by the patients has always been a challenge since it is based on individual perception. Based on studied methods of pain assessment the Verbal Pain Intensity Scale was used to evaluate pain in the present study [25]. Evaluation of facial swelling resulting from surgical procedure was difficult as swelling involves a three dimensional volumetric change at the tissue and cellular level. Methods used to evaluate swelling include photographic analysis [29], modified face-bow [30], linear measurements [26] and subjective assessment [31]. It has been observed that linear measurements are a practical and reliable technique for measuring swelling [26]. In agreement to this observation, linear measurement for assessing postoperative facial size was incorporated in the present study. All patients were given antibiotics one hour prior to surgery and anti inflammatory drugs following the surgery. The surgical trauma between the aprotinin and control sides was equalized so that patient’s responses would be meaningful.

The postoperative pain assessment reflected that pain was considerably reduced on aprotinin side of the mouth following extraction of third molars on all follow up days. The pain reduction by aprotinin was highly significant on the 1^st and 2^nd postoperative days and significantly lesser on the 7^th postoperative day [Table/Fig-3,4].

The interrelation between complaints after surgical removal of mandibular molar and time taken for surgery has been studied and it has been concluded that longer the operation takes more the postoperative pain [32]. In the present study, surgical time for all cases was noted and mean time taken for both the aprotinin and control side was equal.

Linear measurements of swelling on the 1^st, 2^nd and the 7^th postoperative days were comparatively lesser on the side of the face in which aprotinin was injected than the control side but not statistically significant on either side [Table/Fig-7]. This may be due to the smaller sample size included in the study. The facial size clinically was seen to reach normalcy on the aprotinin side by the 7^th postoperative day but not on the control side [Table/Fig-1].

The results of the present study was similar to a previous study conducted to evaluate the value of aprotinin in third molar surgery and which concluded that aprotinin reduced pain and swelling postoperatively [11].

A major concern was healing of the sockets on the side where aprotinin was given which might be compromised because of inhibition of the initial acute inflammatory reaction. However, when sockets were checked for adequate healing after one week, both the sides showed satisfactory healing in all the patients. When the patients were reviewed a week following extractions, most confirmed that aprotinin side of the mouth had been less painful and the discomfort was lesser than the control side. Results from this investigation may be correlated with the unique property of aprotinin in inhibiting the mediators of acute inflammation. These mediators, which cause pain when applied to nerve endings and increase vascular permeability, were not activated and therefore the tissue reaction to trauma was reduced. In view of this observation injection of 1ml aprotinin, submucousally around the surgical site 5 minutes before the surgical procedure markedly reduces postoperative pain and swelling clinically, thereby helping the patient resume normalcy at the earliest.

The study had a few limitations. Firstly, aprotinin could have been used in a larger number of patients undergoing surgical removal of mandibular third molar. Secondly, comparative studies with other drugs like corticosteroids to ascertain its efficacy conclusively would have provided a better result.

Within the limitations of the present study it may be concluded that aprotinin proved to have definite benefits for relief of postoperative pain and swelling clinically. A local infiltration of aprotinin is less traumatic than corticosterois which have to be administered through a more invasive parenteral route. Injection of aprotinin noticeably reduces pain and swelling postoperatively. Although the present study was standardized to maximum possible extent more studies should be encouraged in long term therapy of aprotinin in terms of safety, its use in other surgical procedures.