The present randomized controlled clinical trial compared clinical/ radiological and patient centered outcomes following the application of Amniotic membrane/Collagen membrane, (porcine derived) in combination with Biphasic Calcium phosphate, (60 % Hydroxyapatitie: 40 % ß TCP) for the surgical reconstruction of periodontal intrabony defects.
The two surgical groups were compared in terms of mean differences in Probing Pocket Depth, Clinical Attachment Level and radiographic bone fill at 6 months follow-up. Patient centered outcomes in terms of Visual Analog Scale and Wound Healing Index were assessed at the 3 and 10 day follow-up of the surgical procedure. The types of bony defects included in the present study varied from three-walled to one-walled intrabony defects. It is noteworthy that most defects were also combined. It has been reported that three-walled defects show more predictable results after GTR procedures compared to combined or complex defects. However, the results of the present study revealed that morphologic variations of the bony defects did not influence the efficacy of GTR treatment in either group.
To date, there are no published data on the clinical use and assessment of patient centered outcome measures of AM alone or in conjunction with biphasic alloplastic graft material for the treatment of intrabony defects. The results of the present study showed a mean reduction in the PPD of 2.89 ± 0.69 mm in the CM + BiCP group and 2.95 ± 0.57 mm in the AM + BiCP group and CAL gain of 2.60 ± 1.43 mm in CM + BiCP group 3.18 ± 1.13 mm in the AM + BiCP group at 6 months follow-up. Although the reduction in the pocket depth and the gain in clinical attachment (clinical measures indicative of the healing) was better in the amniotic membrane with bone graft group as compared to collagen membrane with bone graft group, a statistical significance could not be established The above mentioned findings are in concurrence with the observations in systematic reviews by Laurell et al.,  and Parrish LC et al.,  wherein the authors reported the average CAL gain after treating periodontal intrabony defects with biodegradable membranes with and without graft material was 2.96 mm and 3.50 mm respectively.
Collagen membranes are the most commonly employed barrier membranes for GTR, as they facilitate migration of periodontal ligament fibroblasts and provide an early scaffold for neo-angiogenesis. However, these types of membranes act mainly as barriers, and they are considered biologically inactive, while owing to the unique characteristics of AM, it acts as a biological membrane, providing wound protection and bacteriostatic effect .
As described by Schultz et al. , dynamic reciprocity is an ongoing, bidirectional interaction among cells and their surrounding microenvironment. These interactions take several forms that may be categorized as direct or indirect. AM offers a scaffold for proliferation and differentiation owing to the presence of elastin, nidogen, collagen types I, III, IV, V, and VI, elastin, and hyaluronic acid. AM contains growth factors such as platelet-derived growth factors alpha and beta and transforming growth factor beta that hasten formation of granulation tissue by stimulating the growth of fibroblasts and stimulates neovascularization . Laminin-5 in amniotic membrane has a high affinity to gingival epithelial cells, producing an early physiologic seal with the wound surface and thereby facilitating cell migration, accelerated wound healing . AM also contains Fibronectin which acts as the recognition system that guides cell positioning . Fibronectin is a matrix protein which contributes to cell migration and attachment through the RGD sequence . One of the major advantages of AM, in comparison to other bio-degradable membranes, is its thickness (320 μm) and good adaptability which owes to the increased adaptability of the membrane to the defect morphology .
Randomised controlled trial have been performed by several authors [12, 25, 26] comparing AM/Biogide in combination with allograft and xenograft material for the management of grade II furcation defects and intrabony defects. After a post-operative period of 6 and 9 months, both the test and control groups showed significant reduction in PPD, CAL, and increased in percentage of bone fill, without any significant differences between the groups assessed.
The property of degradability of barrier membrane will influence the surgical outcome of GTR. Porcine- derived collagen membranes in GTR procedures have been shown to resorb within 4 to 6 months . The degradation period of amniotic membrane can be hypothesised from studies of wound dressing and bladder reconstruction , it can be put forth that placement of AM as barrier membrane beneath a periodontal flap, prevents the early exposure of the surgical site. The membrane shows reduction in structural stability by 14-21st day as a result of mucoid degeneration [29, 30]. The gains in CAL and reductions in PPD in the present study make it safe to speculate that the absorption of AM was slow enough to produce the desired effects.
In an attempt to further improve the clinical outcomes of GTR, the present study was designed to employ a combined periodontal regenerative technique. The study included one wall, two wall, three wall and combined osseous defects with an intrabony defect angle of ≤ 40°. Owing to the bioactive properties and chemical similarity to the mineral phase of bone, calcium phosphate based biomaterials (CaP) are widely used for bone regeneration .
By modifying the HA/β-TCP ratio and, thus, the solubility of ceramic, it is possible to influence the pattern of resorption. On comparison with different ratios of HA and β-TCP (HA100; 80:20; 60:40) in a dog model, the 60:40 group showed more new bone formation and less residual bone mineral remaining at the end of 24 weeks [32, 33]. In the present study we have used Biphasic calcium phosphate in the ratio of 60 % HA: 40 % β-TCP which would provide the same degree of osteoconductive property in both the intervention groups. The bone graft used had a porosity of 60–70 % and a pore size of 100–300 micrometers which allows for optimal revascularization and bone regeneration. In addition, the particle size used was 0.5 to 1mm which supplements the defect fill and the osteoconductive ability of the bone graft.
Surgical access to the intrabony defects was selected from three different surgical approaches: the simplified papilla preservation flap; the modified papilla preservation technique; and the crestal incision based on the available interdental space. The degree of wounding and flap reflection are minimised by these techniques and ensures wound stability, primary closure, and space maintenance .
Cortellini P et al., , in a multicenter randomized clinical trial, evaluated the outcomes of the simplified papilla preservation flap technique used with barrier membranes for intrabony defect management and reported favourable clinical outcomes and lesser post operative morbidity. Hence, in the present study we have performed the simplified papilla preservation technique to gain access to the periodontal intrabony defects.
Percentage of bone fill at the end six months of the surgical procedure was assessed in terms of Defect depth reduction. The present study utilised standardised radiography to diminish the variations of the projection geometry between pre- and post-surgical radiographs using the long cone parallel technique with a commercially available positioning guide and the radiographic analysis was done by means of computer aided program. (Image J software). Defect depth reduction in the present study at 6 months follow-up in CM + BiCP and AM + BiCP groups were 98.62 ± 6.51 % and 98.25 ± 7.21 % respectively with no statistical significance (p < 0.950).
At present there are no clinical studies evaluating the Patient Centered Outcome measures and wound healing following the Collagen membrane and Amniotic membrane in the management of periodontal intrabony defects. AAP Consensus report on Periodontal regeneration of intrabony defects, 2015  has concluded that, in future research designs, objective measures of postoperative pain evaluated in real time should be incorporated, inclusion of modulating factors such as treatment modality, operator experience, surgical technique, and patient-related factors. The patient centered outcomes were evaluated using Visual Analog Scale for pain at Day 3 and 10. The mean VAS score at Day 3 were 2.04 ± 0.88 and 2.60 ± 0.57 in the AM + BiCP and CM + BiCP groups respectively with no statistical difference between them (P value < 0.190).
Post-operative healing was evaluated using the Wound Healing Index . Wound healing after the surgical procedure was assessed (3 days, 10 days) and was quantified and graded. In the present study, the mean WHI scores at day 10 following the surgery were 3.80 ± 0.75 and 4.12 ± 0.43 for CM + BiCP and AM + BiCP groups respectively with no statistical difference (P value < 0.014). However, in one of the cases in the CM + BiCP group, on day 10 follow-up, there was membrane exposure (with no sign of infection) observed at the surgical site which subsequently healed (following removal of exposed portion of the membrane) allowing for no further complications in regeneration. No post-operative complications were observed during the early healing phase in the AM + BiCP group, owing to the easy adaptability of the amniotic membrane over the defect site providing an early physiologic seal.
A limitation of this study was the use of 2D imaging analysis with the help of Radiovisuography (RVG) instead of 3D analysis with CBCT. Re-entry was not considered, as none of the teeth included in this study were candidates for extraction, a histologic study was not performed.
Guided tissue regeneration in the present era can no longer be considered as a single treatment approach. There is paramount of evidence available to consider GTR as a multifactorial treatment approach comprising careful selection of patients and defects, different surgical techniques, various types of membranes and adjunctive materials and many suturing approaches. All the cited components could be combined to build up different treatment strategies in order to increase the predictability of the treatment outcome.