In vitro cytotoxicity
Primary gingival fibroblasts were obtained from the gingival tissue of a healthy patient, after the study was approved by the Ethics Committee of Universidade Federal do Rio Grande do Sul. The primary cells were cultivated in Dubellco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, and 100 IU/mL penicillin, 100 μg/mL streptomycin (Thermo Fischer Scientific, Waltham, Massachusets, USA) at 37ºC and 5% CO2, until the cells could be used for culture. To test the effect of adhesives on cell viability, three independent samples were poured into 24-well plates and kept at 37ºC for 72h to allow polymerization of adhesives. After this, DMEM at 37ºC was added on top of adhesives for 24h. The media in contact with the adhesives were used to treat the cells during the test. To perform the test, gingival fibroblast cells were seeded in 96-well plates (5x103) and after 24h the subconfluent cell monolayer was treated with conditioned media for 72h. Cells were cultivated with pure DMEM as a positive control. All conditions were tested in triplicate for each independent sample. After treatment, cells were fixed with 50% trichloroacetic acid (Sigma Aldrich) and left at 4ºC for one hour. Cells were stained with 0.4% SRB solution to identify the cells that had viable proteins after the treatment. The stained monolayer was suspended in 10% Trisma and the quantification was performed at 560nm in a Microplate Spectrophotometer (Multiskan GO, Thermo Fisher Scientific, USA). The absorbance values of cells treated with pure DMEM were used to normalize the viability of cells in contact with the conditioned medium, and thus, the percentage of viable cells was calculated.
Clinical Evaluation
A case series was performed by evaluating the post-operative healing patients, in a total of 10 post-extraction alveoli of single-rooted teeth. The study protocol was carried out according to the guidelines of the 1975 Declaration of Helsinki, (revised in 2013) and approved by the ethics committee of the Universidad Cientifica del Sur, by registration 064-2018-PRE8. The study included male and female patients aged 20-70 years, in need of single-tooth extraction with mobility degree III (Miller, 1938), who had been referred to the University Cientifica del Sur for clinic treatment. The patients were excluded in case of smoking, systematic diseases, use of medications and when acute infection in teeth was identified. The corresponding medical records of the patients who met the inclusion and exclusion criteria were compiled, in which their important medical history was detailed. All patients included in the study signed the term of free and informed consent and were notified about the possible complications of the treatments.
A trained operator performed all the tooth extraction and socket preservation procedures. Periapical radiographs and cone beam computed tomography (CBCT) scans were used to record the position, angulation, remaining bone plates and anatomical repairs of the teeth to be extracted, to avoid complications at the time of tooth extraction. Prior to the extraction, asepsis of the oral cavity was performed with a 0.12% chlorhexidine-based antiseptic (Perioaid®, treatment) for 30 seconds; and the patient’s face was decontaminated with a 10% iodopovidone solution. The teeth were anesthetized by applying topical anesthesia with 20% benzocaine gel (Benzotop®), and then the infiltrative anesthesia was administered, using lidocaine 2%, with epinephrine 1:80 000 (New Stetic®). Sulcular incision and vertical liberating incisions were made with a 15C blade, and full-thickness flap elevation with the use of a Molt curette, with the purpose of obtaining direct visualization of the vestibular bone plate. The teeth were excised with the use of a peristome, and once the tooth had been dislocated, an anterior forceps or elevator was used to perform avulsion, depending on the teeth to be extracted and the surrounding structures. On conclusion of the extraction, the walls of the alveolus were cleaned using a Lucas 48 curette, and washed with a 0.9% Sodium Chloride solution. After socket preparation, the alveolar flange preservation technique was performed, in which 0.5 grams of 300-500 μm mineralized cortical allograft (Puros®, Zimmer Biomet dental, Miami, USA) was applied, compacted into the alveolus and covered with a collagen membrane (OSSIX ® PLUS). The vestibular flap was re-positioned without traction and an internal cross-type suture stitch and single stitches made with monofilament non-absorbable e-PTFE suture thread with a 16mm 3 / 8c cutting needle ( GORE-TEX®) (Figure 1) were used on the proximal surfaces to close the wound, but intentionally leaving the membrane exposed.
After conclusion of the alveolar flange preservation technique, the cyanoacrylate-based tissue adhesive (PeriAcryl® 90 HV) was applied both on the perialveolar stitches and on the intentionally exposed membrane, creating a protective film, by using a plastic dosing pipette, in accordance with the manufacturer's instructions. The amount of adhesive to be used depended on the size of the exposed membrane to be covered in each case as shown in (Figure 1). The patients were provided with post-surgical recommendations with regard to restriction on brushing in the area, and were instructed to use 0.12% chlorhexidine-based mouthwashes every 12 hours for 2 weeks. All patients received postoperative antibiotic and anti-inflammatory therapy based on 500 mg Amoxicillin taken every 8 hours for 10 days, and 500 mg Paracetamol, depending on pain experienced.
Post-surgical controls were performed at time intervals of 12, 30 and 60 days after surgery, in which the presence of stitches (first control), and the presence of tissue adhesive was verified. Signs of inflammation such as edema, pain, erythema, suppuration and loss of the collagen membrane were evaluated, according to Early-Wound Healing Index (EHI) and Early Wound Healing Score (EHS) by a single calibrated operator. The calibration was performed by the Kappa index for the intra-operator correlation. The EHI of each patient was classified according to different scores considering closure of the flap:
- Complete closure of the flap without fibrin line
- Complete closure of the flap with fibrin line
- Complete closure of the flap with fibrin clots present
- Incomplete flap clot with partial tissue necrosis
- Incomplete closure of the flap with total tissue necrosis, more than 50% of the flap.
The EHS was classified according to clinical evidence of epithelium formation (SCR); clinical evidence of hemostasis (SCH); and clinical evidence of inflammation (SCI):
SCR:
- 0 pts: distance between the margin of the incision,
- 3 pts: margins of the incision in contact,
- 6 pts: connected margins of the incision.
SCH:
- 0 pts: bleeding of the incision margins,
- 1 pt: fibrin in the margins of the incision,
- 2 pts: absence of fibrin in the margin of the incision.
SCI:
- 0 pts: redness in >50% of the length of the incision and/or pronounced inflammation,
- 1 pts: redness of the implies < 50% of the length of the incision,
- 2 pts; absence of the redness along the incision length.