Bone wax

At postoperative week 8, bone tissue samples were removed and fixed with paraformaldehyde. Then, bone samples were completely decalcified in 15% EDTA solution. After decalcification, bone tissue samples were embedded in paraffin wax and cut into tissue sections. The tissue sections were stained with Masson trichrome, H&E and sirius red in accordance with standard protocols, and samples were observed using a light microscope and polarization microscope.

Study design
The primary objective of this prospective (May 2022-April 2023), parallel-group, two-arm, randomized, single-blind, multicenter study was the comparison of sternal dehiscence rate within 26 weeks of the median sternotomy closure after applying Truwax® or Ethicon® bone wax. Secondary objectives were to elucidate the effect of two hemostats on average time to sternal hemostasis, overall intraoperative handling assessment of bone wax, effect of two hemostats on surgical site bleeding, postoperative wound healing, incisional pain post-sternotomy, and other postoperative complications of median sternotomy closure, viz., amount of postoperative drainage, bleeding, blood transfusion requirement, superficial and deep sternal wound infection, re-exploration (re-sternotomy) and all-cause mortality rates, subject satisfaction and health-related quality of life (QoL) post-sternotomy, time taken to return back to work and to normal day-to-day activities, material problems, and other adverse events (AEs) in both groups.
Ethics
The study was registered in the Clinical Trial Registry of India (CTRI) (registration number: CTRI/2022/03/041192 (registered on 17/03/2022)) and approved by the Ethics Committees of Mathikere Sampangi (MS) Ramaiah Medical College and Hospitals (date: 28/02/2022) and Ganesh Shankar Vidyarthi Memorial (GSVM) Medical College (approval number: EC/53/March/2022; date: 16/03/2022). Ethical principles of the Declaration of Helsinki Agreement, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline for Good Clinical Practice (GCP) E6 R2, European Standard (EN) International Organisation for Standardisation (ISO) 14155:2020, Indian Medical Devices Rules (MDR) 2017, MDR (EU) 2017/745, New Drugs and CT Rules 2019, and Consolidated Standards of Reporting Trials (CONSORT) were followed for conducting and reporting the study.
Study participants
Male or female adults (18-70 years) scheduled for sternotomy for surgical procedures on the heart, great vessels, and mediastinal lesions were included in the study after receiving written informed consent in the vernacular language prior to performing any study-specific procedures.
Participants with American Society of Anesthesiologists (ASA) grade 5 or hemoglobin (Hb) A1C of >10 or not requiring usage of bone wax to stop bleeding from the bone exposed by the sternotomy were excluded. Moreover, participants with a history of median sternotomy or bleeding disorders, or allergy to bee wax or similar products, or active infection at or around the skin incision site were excluded. Participants with a mental disorder/learning disability/language barrier or who received an experimental drug/device within 30 days prior to the study or pregnant or lactating female participants were also excluded. In addition to these, other criteria for exclusion were participation in another cardiovascular or similar study, or unlikely to comply with study procedure, or unlikely to complete the scheduled visits, or direct involvement in this study or other studies of the investigators or the institutions.
Study setting
The study was conducted at the Department of Cardiothoracic Surgery in MS Ramaiah Medical College and GSVM Medical College of India.
Intervention
Truwax® and Ethicon® bone waxes are a sterile mixture of bee wax, paraffin wax, and isopropyl palmitate. Both bone waxes are indicated to be used for the control of bleeding from bone surfaces; however, they are contraindicated to be used where rapid osseous regeneration and fusion are desired.
Study procedure
The pre-surgery screening was performed (week 26 to day -1), and the subjects were enrolled to undergo median sternotomy on day 0 for surgical procedures on the heart, great vessels, and mediastinal lesions. The surgery was performed in theatre by the investigators with recognized expertise in sternotomy along with their team members using institutional and standard surgical protocol. Immediately after sternotomy, Truwax® or Ethicon® bone wax was spread by digital pressure on the sternal surfaces to prevent sternal bleeding. Chest drains were connected to underwater seal drainage systems and left with suction to collect drainage from the pleural cavity. The presternal fascia, muscle, and skin were closed as per the standard institutional protocol. Subjects were transferred to the intensive care unit (ICU) for observation and continued their medical flow as per the standard of care. Subjects having a good postoperative recovery, no discharge from the sternal wound, and a stable sternum were discharged. Post-surgical follow-ups include day 3, days 4-15 (discharge), weeks 4-6, week 12, and week 26.
Baseline characteristics
Subject demographics, lab investigations regarding Hb, platelet count, prothrombin time, activated partial thromboplastin time (aPTT), and international normalized ratio (INR), classifications of ASA and NYHA, the reason for undergoing sternotomy, and history of smoking, alcohol consumption, treatment/surgery, and cardiovascular disease in the family were recorded. In addition, a physical examination for any anomaly was performed.
Study outcomes
Primary Outcome
A proportion of subjects having sternal dehiscence within 26 weeks of the median sternotomy closure was evaluated based on clinical findings of a sternal click or evidence of sternal instability during coughing or respiration. Chest radiography was performed as per investigator's discretion.
Secondary Outcomes
During the surgery, the surgeon watched both sides of the sternum for hemostasis after the application of bone wax, and the time from the start of the application to hemostasis was recorded. In addition, bone wax properties such as easy applicability, adhesiveness, easy to be shaped, satisfaction with sternal bleeding control, and packaging integrity were rated by the surgeons on a Likert scale (1 point for "very unsatisfied" and 5 points for "very satisfied"). Bleeding during surgery was based on the universal definition for perioperative bleeding (UDPB). Blood and its products like packed red blood cells (PRBCs), fresh frozen plasma (FFP), concentrated erythrocytes, platelet concentrates (PLT), and electrolyte solutions used were also captured. Other standard perioperative details such as type of surgery (full sternotomy/mini-sternotomy), any abnormality in the sternum, whether osteoporotic sternum, number of grafts, length of surgery, sternotomy incision length, cardiopulmonary bypass time, amount of bone wax used, quantity of initially used bone wax removed from the area of application before approximating the sternum, description of the surgery, type of suture used for sternal closure, number of blood transfusion, antibiotic and thrombosis prophylaxis, and perioperative complications were also recorded.
Postoperative bleeding/oozing from the surgical site was clinically assessed, and the number of dressing changes was noted. The Sternal Instability Scale (0 is "clinically stable sternum" and 3 is "completely separated sternum") was used to assess the stability of the sternum [11 ]. Sternal bone instability was only confirmed by chest radiography at the investigator's discretion. Findings such as a mid-sternum line of lucency of >2 mm indicating sternum diastasis, any sternum wire displacement, and any obvious interruption/dislocation were recorded in case a chest radiograph was performed. Postoperative experience of pain at rest, during coughing, and on movement was assessed using the Visual Analogue Scale (VAS) with 0 corresponding to "no pain" and 100 to "worst pain." Other complications after sternotomy, viz., amount of postoperative drainage through mediastinal drains in the first 24 hours and till discharge, superficial and deep sternal wound infection, reoperations, mediastinitis, hemothorax, steel wire fracture, all-cause mortality rates, other complications of sternal closure, other bone wax-related complications, and blood and blood products used postoperatively, were recorded. Moreover, the duration of stay at the ICU and hospital and time taken to return back to work and to normal day-to-day activities were noted. Subject satisfaction and QoL were measured using the EuroQoL Five-Dimensional Three-Level (EQ-5D-3L) Questionnaire. The five dimensions of EQ-5D-3L comprised mobility, self-care, usual activities, pain or discomfort, and anxiety or depression, which were marked on three levels: no, some, and extreme problems. Similarly, EuroQoL-VAS (EQ-VAS) was used for the assessment of the subject's health on a 0-100 scale, where 0 is the worst imaginable health and 100 is the best imaginable health.
Any abnormal medical occurrence/disease/injury/clinical sign was considered as an AE. Serious AE was marked when a subject had any life-threatening condition, leading to hospitalization or death. Concomitant or prescribed medications were also documented.
Sample size
The postoperative sternal dehiscence rate was reported as 1.4% in patients without bone wax and 2.5% in patients with bone wax [12 (link)]. Therefore, the proportion of subjects with sternal dehiscence in the Ethicon® bone wax arm was assumed as 2.5%, i.e., π1=0.025. Further assuming a type I error as 5%, power as 80%, and a difference to be detected as 0.2% for the proportion of subjects having sternal dehiscence in the Truwax® arm (π₂=2.7%, 0.027), with 10% margin of non-inferiority, the sample size of 38 in each arm was required. The total sample size requirement was 76. The required sample size was increased to 92 (46 subjects in each arm) after considering 20% dropout and post-randomization exclusion. Two centers were involved in the study and enrolled a proposed sample of up to 46 subjects, with 23 subjects block randomized to the Truwax® arm and 23 subjects to the Ethicon® bone wax arm. The sample size calculation formula was as follows: π₁-π₂>δ n_i=(Z_α+Z_β)²(π₁(1-π₂)+π₂(1-π₂))/(π₁-π₂ -δ)² (two-sample parallel non-inferiority). Here, n_i is the sample size required in each group; Z_α, the conventional multiplier for alpha; Z_β, the conventional multiplier for power; π₁, the proportion of patients having sternal dehiscence in the Ethicon® bone wax arm; π₂, the proportion of patients having sternal dehiscence in the Truwax® arm; δ, the margin of non-inferiority difference; and π₁-π₂, the size of difference of clinical importance.
Randomization and blinding
Two random lists of size n=46 (23 vs. 23) were generated by Random Allocation Software (V 1.0) using block sizes of 4, 6, or 8. Block randomization of variable block length as per study sites was performed, and the randomization codes were issued to the sites in sequentially numbered opaque sealed envelopes. Subjects were allocated randomly to one of the two treatment arms (Truwax® or Ethicon® bone wax) just before the surgical procedure. The detail of the treatment arm was blinded to the subjects only.
Statistical analysis
Statistical analysis of primary and secondary outcomes was performed using IBM SPSS Statistics for Windows, Version 28.0 (Released 2021; IBM Corp., Armonk, New York, United States) in a per-protocol (PP) analysis set, consisting of all randomized subjects who completed the study till 26 weeks. Continuous variables were represented as mean±SD and categorical variables as numbers (n) and percentages (%). For comparisons of continuous variables, the t-test or Mann-Whitney U test (depending on data distribution) was used, and for categorical variables, the chi-squared test was used. A probability of less than 0.05 was considered significant.

(1) Pre-operative alginate is taken from the conventional model and photographs of the patient are taken inside and outside the mouth.

(2) The surgeon designs a diagnostic wax-up and makes a pressure film guide on the diagnostic wax-up.

The periodontist performs the periodontal crown lengthening surgery under the guidance of the pressure film guide: The incision in the affected area is positioned according to the ideal gingival margin position on the guide and the excess gingiva is excised; the guide is removed, the flap is incised and the alveolar bone is exposed, and the position of the bone apex in the operation requires a combination of the indication of the periodontal probe [11 , 12 (link)] and a combination of the use of the shu two-armed ruler to indicate the position of the bone crest apex [13 (link)], and the alveolar bone is removed using a bone chisel/bullet drill so that the gingival margin is at least 3 mm from the crest of the alveolar ridge to match the biological width and is displaced towards the crest of the adjacent teeth.

(4) Fabricate the temporary restoration: Remove the sutures after 1 week and refer to a prosthodontist for tooth preparation and chairside fabrication of DMG resin temporaries concerning the diagnostic wax-up.

(5) Follow-up observation: Wear the temporary crown after surgery and record the amount of gingival change, red and white aesthetic index and patient satisfaction at 4 weeks, 3 months and 6 months to evaluate the clinical restoration effect. Since the gingiva will change over time after crown extension surgery, the prosthodontist should adjust the shape of the temporary crown according to the patient's intraoral situation when the patient wears it for inspection, so that the gingiva is better guided and shaped.