Comparative Evaluation of Clinical and Radiological Parameters Following the Use of Biphasic Alloplastic Material With Amniotic Membrane/ Collagen Membrane for the Management of Periodontal Intra-bony Defects – a Prospective Double Blinded Randomised Controlled Clinical Trial


 Background: The concept of periodontal regeneration has been revolutionised since the introduction of growth factors and bioactive bone substitutes which ensures optimal regeneration of the diseased periodontium. The aim of the present study was to evaluate the efficacy of Amniotic membrane + Biphasic Calcium phosphate as compared to Collagen membrane + Biphasic Calcium phosphate for the management of periodontal intrabony defectsMethods: 50 systemically healthy patients with localised moderate to severe periodontitis, sites which had a Probing Pocket Depth (PPD) ≥6 mm and an intrabony component of ≥3 mm as detected on Intra oral periapical radiographs (IOPAR) and bone sounding were recruited based on specific inclusion and exclusion criteria. They were randomly allocated by computer generated tables to CM+BiCP and AM+BiCP groups. The amount of bone fill and changes in Probing Pocket Depth, Clinical Attachment Level were measured at baseline and six months.Results: The results of the present study showed a mean reduction in the PPD of 2.89±.69 mm in the CM+BiCP group and 2.95±.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 with no statistical significance between the groups. In terms of Defect resolution, 98.62 ± 6.51 % was achieved in CM +BiCP group and 98.25 ± 7.21 % in AM +BiCP group. Conclusion: Within the limitations of the present study, it can be concluded that AM can be used as a barrier membrane, in conjunction with Biphasic calcium phosphate, and provides comparable results to Collagen membrane with Biphasic calcium phosphate when used in the management of periodontal intrabony defects. Trail registration: The study protocol was approved by the Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research (IEC/18/DEC/145/51) and was registered in the Clinical Trial Registry of India [Ref No: CTRI/2020/03/0240075].


Background
There has been a signi cant progress in various aspects of periodontal therapy in the last two decades, from resective periodontal surgical procedures to techniques aimed at regeneration and reconstruction of the lost periodontium. The concept of periodontal regeneration has changed from being passive to an active protocol with the introduction of tissue engineering and biomimetic concepts. It utilises mechanical, cellular and biologic mediators to facilitate reconstruction/regeneration of a particular tissue 1  Guided tissue regeneration with barrier membranes has been demonstrated to be effective in preventing epithelial and gingival connective tissue cells from migrating onto the instrumented root surface 3 . The primary outcomes in the treatment of intrabony defects achieved by guided tissue regeneration are (i) increase in functional tooth support (clinical attachment and bone levels); (ii) reduction in pocket depth; and (iii) minimal gingival recession 4 .
Various nonresorbable barrier membranes have been employed in animal and clinical studies however, membrane exposure and subsequent contamination and the need for second surgical procedure to remove the membrane are the most prevalent problems associated with the use of nonresorbable membranes. Among the various natural and synthetic biodegradable membranes, collagen based membranes are frequently applied 5 .
Type I collagen (from varied sources) serves as a potential barrier for use in GTR procedures. It acts as a, a) major extracellular macromolecule of the periodontal connective tissue, b) aids in broblast chemotaxis, c) cell occlusion d) weak immunogen 6 . Combining CM with bone substitutes may prevent soft tissue collapse, especially in non-contained infrabony defects, and thus ensure space maintenance (Bunyaratavej and Wang, 2001) 7 . However, collagen membrane act mainly as barriers, and they are considered biologically inactive. Fetal membranes as biodegradable materials have been employed in medicine for decades. Amniotic membrane (AM), the innermost layer of fetal membranes, was rst used for transplantation of skin in 1910. AM serves as a brillar scaffold and acts as a reservoir of stem cells, modulates angiogenesis and promote wound healing 8 .
In an attempt to further improve the clinical outcomes of GTR techniques, particularly in unfavourable and large defects, it has been suggested by clinicians the use of bone grafts with the barrier membrane to obtain an additive effect; this association is called Combined Periodontal Regenerative Therapy (CPRT) 9 .
This approach may lead to the assemblage of different regenerative principles, such as osteoconductivity and osteoinductivity provided by the bone grafts and space provision, wound stability provided by the barrier membranes.
Hence a combination of AM/CM with bone substitute may positively in uence the treatment of periodontal intrabony defects by enhancing wound healing and promoting robust periodontal regeneration. The aim of the present study was to evaluate the e cacy of Amniotic membrane + Biphasic Calcium phosphate as compared to Collagen membrane + Biphasic Calcium phosphate in the management of periodontal intrabony defects.

Materials And Methods
Trial design: The study was designed as a prospective double-blinded randomized controlled clinical trial with two arm parallel group design. The sample size 10 was calculated with an assumed power of 80% (β = 0.89) to detect a minimum clinically signi cant difference in probing depth of 1 mm and a standard deviation of 2.1 and alpha error (5%), a total of 40 (20 per group) patients were required and recruited in the following study. Allowing for dropouts, a total of 50 patients were recruited (25 in each group).

Randomization:
A random number table was used to select 50 subjects for intervention from the target population of 75 and selected subjects were allocated into blocks containing 4 subjects per block. Figure1 shows the CONSORT chart for the present study. In each block, randomisation of the intervention procedures was done using the following code of 1221; 1122; 2112; 1212; 2121, (wherein 1-represents Collagen membrane +Biphasic alloplastic bone substitute (Control) 2-represents Amniotic membrane + Biphasic alloplastic bone substitute (Test)).

Allocation concealment & Blinding:
Allocation concealment was done by placing the interventions in a sealed envelope which was opened by the surgeon prior to start of the surgery. Patients and statistician were blinded to the intervention. Pre-surgical preparation: Surgical guides were fabricated with cold cure resin on cast models of patients, obtained by alginate impressions. The guides covered the occlusal surface of the tooth being treated including atleast one tooth mesial and distal to it, extending both bucally and lingually/ palataly. A groove was made in the guide with a ssure bur extending in an apico-coronal direction at the point where probing pocket depth (PPD) and infrabony defect were identi ed. The groove serves to provide reproducible alignment of the probe (Hu-Friedy PCP-12) during pre-op and post-op measurements. All clinical parameters were analysed by a single trained examiner.
Clinical baseline and 6 month follow-up measurements, Probing Pocket Depth (PPD), Clinical Attachment Loss (CAL), were recorded by taking CEJ as the reference landmark and aligning the probe through the groove in the surgical guide, which ensures proper reproducible angulation.
Each patient involved in the study received a full diagnostic workup, which included Intra -oral periapical radiograph of the affected site, study casts and intraoral photographs. A thorough sub-gingival scaling was performed along with possible elimination of the etiological factors and oral hygiene instructions were given. Routine Blood parameters were checked pre-operatively (FBS/PPBS, BT, CT, CBC, INR). After oral prophylaxis, root planing was done under local anesthesia using Gracey's Area speci c curettes.
Occlusal adjustments were addressed when indicated. Approximately 3-4 weeks following the nonsurgical periodontal therapy, patients with persistent periodontal pockets (>5mm) were taken up for further periodontal ap surgery. 0.12% of 10ml of chlorhexidine mouth rinse was given to the patient as a preoperative preparation, where the patient was asked to swish for 30 seconds and then expectorated / suctioned to decrease the bacterial load prior to the start of the surgery.

Surgical Intervention:
All surgical procedures were performed by the same expert operator (VL) with a longstanding clinical experience (>10 years) in periodontal surgery.
Under local anesthesia, simpli ed papilla preservation ap (Coretellini1999) 11 was performed. No.63 microsurgical blade was used, the rst incision was given across the defect associated papilla in an oblique direction beneath the contact point and was carried out keeping the blade parallel to the long axis of the teeth to avoid excessive thinning of the remaining interdental tissues following which a full thickness mucoperiosteal ap was elevated exposing the intrabony defect. After which, defect debridement and root planing was performed using Gracey's area speci c curettes. Root conditioning was done using Tetracycline Hydrochloride 5% solution and decortication of the defect site was done with the low speed micromotor. The defect was lled with biphasic alloplastic bone substitute material (60% hydroxyapatite: 40% Beta TCP). Following grafting of the defect site, it was followed by placement of Amniotic membrane (Figure2: a-j) or Collagen membrane (Figure3: a-j). The aps were then trimmed and approximated by external vertical mattress using 4-0 vicryl resorbable sutures and Cyanoacrylate dressing was placed over the surgical site.
Post-surgical care: Each patient was prescribed analgesics & antibiotics after surgery. Antibiotic Cap. Amoxcillin 500mg thrice daily and analgesic T. Aciclofenac+Paracetamol combination is prescribed twice daily for three days following the surgery. A 60-second rinse with 0.12% CHX is prescribed 3 times/day for rst 2 weeks.
Statistical Analysis: Statistical analysis was performed with SPSS 20 software. Descriptive statistics were recorded as mean values, standard deviations, frequencies and percentages. Analysis was done at patient and technique levels. The primary outcome variable was change in Probing Pocket Depth (PPD) Clinical Attachment Level (CAL) and Radiographic bone ll. Secondary variables included assessment of VAS and WHI scores. The signi cance of the differences between the groups was evaluated using T-Test. P value <0.005 was considered signi cant.

Results
Fifty patients (22 males and 28 females) were included in the study. The patients were equally distributed within the AM+BiCP and CM+BiCP groups in terms of demographic characteristics and clinical parameters assessed at baseline (Probing depth/ Clinical Attachment Level and Defect angle) and distribution of the defect walls within AM+BiCP and CM+BiCP (Table1).
The results of the present study showed a mean reduction in the PPD of 2.89±.69 mm in the CM+BiCP group and 2.95±.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 with no statistical signi cance between the groups. In terms of Defect resolution, 98.62 ± 6.51 % was achieved in CM +BiCP group and 98.25 ± 7.21 % in AM +BiCP group (Table 2).
Secondary outcome variables measured were Visual Analog Scale (VAS) and Wound Healing Index (WHI) at Day 3 and Day 10 follow-up. Higher WHI scores and reduced VAS scores were observed in the AM+BiCP group than the CM+BiCP at day 3 and day 10 follow-up, no statistical signi cance were observed between the two groups ( Table 2).

Discussion
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 ll 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 in uence the e cacy of GTR treatment in either group. Collagen membranes are the commonly employed barrier membranes for GTR, as they facilitate migration of periodontal ligament brobalsts and provide an early scaffold for neoangiogenesis. 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 14 .
As described by Schultz et al 15 , 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 broblasts and stimulates neovascularization 16 . Laminin-5 in amniotic membrane has a high a nity to gingival epithelial cells, producing an early physiologic seal with the wound surface and thereby facilitating cell migration, accelerated wound healing 17 . AM also contains Fibronectin which acts as the recognition system that guides cell positioning (Hood et al 1977) 18 , Fibronectin-cell matrix protein contributes to cell migration and attachment through the RGD sequence (Ruoslahti et al) 19 . 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 20  The property of degradability of barrier membrane will in uence the surgical outcome of GTR. Porcinederived collagen membranes in GTR procedures have been shown to resorb within 4 to 6 months 24 . The degradation period of amniotic membrane can be hypothesised from studies of wound dressing and bladder reconstruction 25 , it can be putforth that placement of AM as barrier membrane beneath a periodontal ap, prevents the early exposure of the surgical site. The membrane shows reduction in structural stability by 14-21 st day as a result of mucoid degeneration 26,27 . 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 28 . Cortellini 2001 32 , 2008 33 in multicenter randomized clinical trials, evaluated the outcomes of SPPT to test the generalizability of the added bene ts of using barrier membranes in deep intrabony defects along with a variety of regenerative materials. Thus in the present study we have employed the simpli ed papilla preservation technique to gain access to the periodontal intrabony defects.
Percentage of bone ll at the end six months of the surgical procedure was assessed in terms of Defect resolution. 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 resolution in the present study at 6 months followup in CM+BiCP and AM+BiCP groups were 98.62 ± 6.51 % and 98.25 ± 7.21 % respectively with no statistical signi cance (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 34 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 ± .88 and 2.60 ± .57 in the AM+BiCP and CM+BiCP groups respectively with no statistical difference between them (pvalue < .190) Post-operative healing was evaluated using the Laundry's Wound Healing Index. Wound healing after the surgical procedure was assessed (3 days, 10 days) and was quanti ed and graded. In the present study, the mean WHI scores at day 10 following the surgery were 3.80 ± .75 and 4.12 ± .43 for CM +BiCP and AM +BiCP groups respectively with no statistical difference (pvalue < 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 drawback 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 variously combined to build up different treatment strategies in order to increase the predictability of the treatment outcome.

Conclusion
Within the limitations of the current study, it can be concluded that AM in conjunction with Biphasic calcium phosphate, appears to provide better patient related outcomes and comparable clinical and radiological outcomes as porcine derived Collagen membrane with Biphasic calcium phosphate in the management of periodontal intrabony defects. However future research studies, directed at overcoming the limitations of amniotic membrane which include the handling characteristics, structural stability and limited availability are needed to be performed with long term follow-up.