Clinical, Anti-Inflammatory And Anti-Infective Properties Of Amniotic Membranes Used For Guided Tissue Regeneration

Study design including Randomization and Blinding The study was designed as a split-mouth, double-blind, randomized controlled clinical trial. Randomization and blinding included computerized generation of the allocation sequence in random permuted blocks (block randomization) and blinded disbursement of medication. Allocation was performed by assigning the block of sites to study groups according to the specified sequence. Based on the sequence, the first operator selected two sites for each of the following experimental sites; the test site, in which amniotic membrane with the graft was placed and the control site which was treated by graft placement only. All the surgeries were performed by a designated operator for the sake of uniformity whereas the relevant readings were recorded by the first operator who was blinded to the nature of the site. The blind was not broken until this clinical trial was completely finished.

After the prospective interdental areas were probed buccally and lingually/palatally, the site was considered for the study if the average probing pocket depth (PPD) was ≥5 mm. All baseline (on the day of surgery) values were recorded before the surgical procedure. Gingival crevicular fluid (GCF) samples for IL-1β were taken at baseline and 1 week and for hBD-2 were taken at baseline, 1 week and 4 weeks. Non-invasive site-specific measures of the plaque index (PI) and modified gingival index (MGI) at the baseline, 1 week, 12 weeks and 24 weeks. The PPD, CAL and bone fill were recorded at the baseline (on the day of surgery) and at the end of 12 and 24 weeks. PPD and CAL were recorded at the baseline and at the end of 12 and 24 weeks using a UNC-15 color-coded periodontal probe. A custom acrylic stent limited to the occlusal 2/3rds of the clinical crown was prepared from a cast obtained from an alginate impression. A groove was prepared in the stent to standardize the probing angulation throughout the procedure and also during the follow up visits.

Radiograph Standardization Standard digital radiographs were taken at baseline, 12 and 24 weeks by the paralleling/long-cone technique at pre-set parameters using a commercially available RVG system. After the imaging plate was placed in the film holder for paralleling technique, addition silicon impression material was added around the biting surface and allowed to set. This arrangement ensured standardized alignment of the aiming device and the holder ensuring correct positioning of the collimator in subsequent radiographs.

Collection of GCF Samples:

After isolation, 6 mm diameter filter paper circles were used for collecting the samples by the intra-crevicular method. The fluid seeping out of the sulcus was collected and any paper contaminated with blood or saliva was discarded, and the collection was repeated after 30 minutes. GCF samples were eluted from the strips by placing them in Eppendorf tubes that contained 500 µL of buffer and stored at -80ºC. GCF Samples were collected at baseline, at the end of 1 and 4 weeks.

Enzyme Linked Immunosorbent Assay (ELISA) for IL-1ß and hBD-2 Commercially available ELISA kits were used to quantify GCF levels of IL-1ß and hBD-2. All assay procedures were conducted according to the manufacturer's instructions and in both the tests, 100μl of the samples or standards were added into the respective wells in duplicate. Color development was stopped using 1% H2SO4 and absorbance of the entire plate was measured within 30 minutes by an ELISA reader at 450nm. The amounts of IL-1ß and hBD-2 were calculated based on the dilutions, and the results were expressed as pico-gram/30 second and pico- gram/site in the 30-second GCF sample.

Amniotic membrane Lyophilized and irradiated AM (3×3 sq.cm) was obtained from a commercial tissue bank. The tissue was dispatched for clinical use from the tissue bank. The tissue conforms to the International Atomic Energy Agency (IAEA) recommendations and the Asia Pacific Association of Surgical Tissue Banks (APASTB) standards. AM is stable and can be stored at room temperature.

Surgical phase:

All surgical procedures were performed by one clinician. After recording relevant parameters, and following routine local anesthesia, sulcular incisions were given and full thickness mucoperiosteal flap was elevated for defect access. Thorough degranulation of the bone defect was done and root planing was done by using appropriate site-specific curettes.

The test site was treated as follows; After placement and proper condensation of natural Hydroxyapatite graft in the defect, AM was cut based on defect anatomy of surgical site and then adapted over the bone graft and alveolar bone extending from base of the flap reflection to the tooth surface. Flap was approximated and stabilized by placing direct loop sutures. Periodontal dressing was placed subsequently. For the control site, the procedure was identical except that the membrane was not placed. Patients received post-operative instructions and were prescribed medication. The sutures were removed at 1 week post-surgery and subsequent measures were recorded.

Radiographic assessment The evaluation of bone fill was performed by using digital subtraction technique and morphometric area analysis by using specific tools in two image processing softwares.