Amnion Membrane as a Novel Barrier in the Treatment of Intrabony Defects: A Controlled Clinical Trial Farin Kiany, DMD, MSc1/Fatemeh Moloudi, DMD2 Purpose: The purpose of this 6-month randomized, controlled, blinded, clinical trial was to evaluate and compare the efficacy of amnion membrane (AM) with deproteinized bovine bone mineral (BBM) and a collagen membrane (CM) with BBM in guided tissue regeneration (GTR) for the treatment of intrabony periodontal defects. Materials and Methods: Ten chronic periodontitis patients with bilateral intrabony defects with radiographic evidence of intrabony component ≥ 4 mm and probing pocket depths (PPDs) ≥ 6 mm were randomly divided into two groups. The test group was treated with AM+BBM, and the control group was managed with CM+BBM. Periodontal clinical parameters were recorded at baseline and at 6 months after treatment. Results: PPD, clinical attachment level (CAL), and probing bone (PB) showed significant improvements after 6 months in both the test and control groups. Gingival recession showed a significant increase in the control group but not in the test group. The changes in mean PPD, PB, and CAL preoperatively and postoperatively between the groups were not significant. There was no significant relationship between the depth of the baseline bony defect and CAL gain. Conclusion: Both AM and CM in conjunction with BBM provided improvement of clinical periodontal parameters. AM did not induce significant gingival recession and is suggested as a new barrier membrane in GTR treatment. Int J Oral Maxillofac Implants 2015;30:639–647. doi: 10.11607/jomi.3590 Key words: amnion membrane, bovine bone mineral, collagen membrane, guided tissue regeneration, intrabony defect

S

everal treatment modalities have been applied over the years to reconstruct intrabony periodontal defects surgically. The ultimate goal is regeneration of the attachment apparatus.1 One of the most effective and predictable surgical procedures for the treatment of periodontal intrabony defects is guided tissue regeneration (GTR).2 In this surgical approach, the placement of a barrier membrane creates a space around the diseased root surface, and selective repopulation of the periodontal wound by progenitor cells occurs.3 Various nonabsorbable barrier membranes have been used

1 Assistant

Professor, Department of Periodontics, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran. 2Postgraduate Student, Department of Periodontics, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran. Correspondence to: Dr Farin Kiany, Department of Periodontics, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran. Fax: +71-16270325. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.

successfully in animal3 and clinical studies.4 However, several problems are associated with the use of nonabsorbable barrier membranes. Membrane exposure and subsequent contamination and/or infection and the need for a second surgical procedure to remove the membrane are the most prevalent problems.5 Various natural and synthetic biodegradable membranes are now available, among which collagen-based membranes are frequently applied. Most existing collagen membranes are made of collagen of xenogeneic origin.6 GTR can be improved through the development of better GTR devices and refined surgical procedures. To this end, novel membranes with special qualities are often sought out. Fetal membranes as biodegradable materials have been utilized in medicine for decades. Amniotic membrane (AM), the innermost layer of fetal membranes, was first used for the transplantation of skin in 1910.7 It is now used successfully in the treatment of burns; creation of biologic surgical dressing; reconstruction of the oral cavity, bladder, and vagina; tympanoplasty; arthroplasty; abdominal surgery; and corneal transplantation.8 Furthermore, it has been introduced as a suitable membrane for vestibuloplasty surgery in dentistry.9,10 In a comparative study, AM was used in The International Journal of Oral & Maxillofacial Implants 639

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conjunction with bovine bone mineral and demineralized freeze-dried bone allografts in the treatment of class II furcation defects.11 A case report and a case series have also been published investigating AM in combination with a coronally positioned flap for root coverage.12,13 In another case series, BioXclude, which consists of dehydrated amnion plus chorion, was successfully used for socket preservation.14 Two case reports discussed the application of BioXclude in ridge augmentation with simultaneous implant placement and in the treatment of an intrabony periodontal defect.15,16 AM is composed of avascular stroma, a thick layer of collagen, and an overlying thick basement membrane with a single layer of epithelium.17 The unique biologic properties of AM include the ability to reduce scarring, inflammation, and pain; enhance wound healing and angiogenesis; and act as a scaffold for cell proliferation and differentiation.17 In addition, it is antiadhesive and antibacterial and causes no immunologic reactions. AM is also readily obtainable in large amounts, and its preparation and storage are relatively low in cost.18 The GTR technique is often combined with the placement of bone grafts and/or bone graft substitutes underneath the membrane. These grafts help support the barrier material and prevent its collapse into the defect or onto the root surface.19 One of the most popular xenograft materials is Bio-Oss (Geistlich), a deproteinized bovine bone mineral. Its morphology, porosity, and crystalline structure are identical to those of natural bone mineral, and its chemical composition is devoid of protein.20 It has been reported to be biocompatible and very well tolerated, with no allergic reactions reported. It is osteoconductive; that is, it promotes the healing of bony defects.21 Bio-Gide (Geistlich) is a highly purified natural product that contains collagen of porcine origin. It is used in GTR treatments. Its benefits include high biocompatibility and good attachment to the soft tissue.22 In this study, AM was considered as a suitable membrane in GTR because of its excellent biologic properties. These properties include the presence of various growth factors and its ability to stimulate bone induction and acceleration of wound healing. To the authors’ best knowledge, a comparison of the effect of AM with a collagen membrane (Bio-Gide), in combination with Bio-Oss as a grafting material, on the clinical outcomes of regenerative treatment of intrabony periodontal defects has not yet been published. Therefore, the purpose of this study was to evaluate and compare the clinical effects of these two treatment modalities in intrabony defects in terms of changes in probing pocket depth (PPD), clinical attachment level (CAL), probing bone level (PB), and gingival recession (REC).

MATERIALS AND METHODS Study Design

A double-masked parallel-group (split-mouth) design was used to assess the efficacy of AM with Bio-Oss in the treatment of intrabony defects and also to compare it with the efficacy of Bio-Gide membrane with Bio-Oss. The Ethics Committee of Shiraz University of Medical Sciences approved the protocol of the study. All surgical procedures were done in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000.

Study Population

Patients who were referred to the Postgraduate Department of Periodontology of the Dental School of Shiraz University for the treatment of advanced chronic periodontitis were screened for possible participation in this study. All participants received instructions in proper oral hygiene performance. Supragingival and subgingival scaling and root planing were performed, and potential participants were reevaluated after 1 month. Finally, 10 patients were selected according to predefined inclusion and exclusion criteria (see following). The patients received detailed information about all the aspects of the study and signed informed consent forms. The final study population consisted of six men and four women with an age range of 35 to 55 years and an average age of 45 years. Inclusion Criteria. Patients had at least two intrabony defects in interproximal areas with radiographic evidence of an intrabony component of ≥ 4 mm, PPD ≥ 6 mm, and bleeding on probing to the bottom of the defect at 1 month after nonsurgical periodontal treatment. In each patient, the selected teeth were positioned in different quadrants and had an identical number of roots. In multi-rooted teeth, the intrabony defect did not include the furcation area. The depth of the intrabony component of the defect and absence of furcation involvement were preliminarily evaluated during the screening phase but had to be confirmed during surgery. All the selected teeth had at least 2 to 3 mm of keratinized gingiva to allow appropriate surgical handling, flap adaptation, and suturing. Exclusion Criteria. Patients exhibiting any of the following criteria were excluded: (1) history of any systemic disease that affects the health of the periodontium or would interfere with periodontal surgery or therapeutic outcomes; (2) smoking; (3) current pregnancy or lactation; (4) poor compliance or failure to maintain good oral hygiene, as ascertained by the presence of full-mouth plaque score ≥ 20% on at least two consecutive appointments; (5) restorations or caries on root surfaces or untreated endodontic infections,

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occlusal disharmony, severe attrition and facets, or excessive mobility; (6) periodontal treatment within the previous 6 months; (7) use of systemic or local antibiotics during the past 3 months.

Fig 1   The occlusal surgical guide.

Preparation of AM

The AM was obtained from placentas of prescreened, full-term pregnant mothers just after elective cesarean delivery. Placentas from normal vaginal delivery were not procured because of possible contamination by normal vaginal flora. All tissue procurement and processing were in compliance with federal regulations and tissue standards of the American Association of Tissue Banks. Potential donors were identified after a complete history of high-risk sexual behavior, intravenous drug abuse, blood transfusions, or malignant diseases was obtained. All donors were informed about the manner of obtaining the tissue and its usage and provided written informed consent. All donor serum samples were tested to ensure the absence of HIV types 1 and 2 antibodies, hepatitis B surface antigen, hepatitis B core antigen, anti-hepatitis C virus antibody; also, a rapid plasma regain test for syphilis was done. All serologic tests were repeated 6 months later to identify infections if the donor had been in the window period of the serologic tests. Until the seronegative status of the donor was confirmed, the AM was preserved at –80°C. The AM (Iranian Tissue Bank, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences) was prepared by the lyophilized (freeze-dried) method.23 Under a lamellar flow hood in a clean room with a neatness class of 100 and under sterile conditions, the placenta was washed off blood clots with isotonic solution. Then, the inner AM was separated from the rest of the chorion by blunt dissection and washed again by isotonic solution that contained a cocktail of antibiotics (50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin, and 2.5 μg/mL amphotericin B). The separated membranes were cut into different sizes and kept in an M199 environment containing 1,000,000 IU specific antibiotic composition at 4°C for 24 hours. Before treatment with antibiotic solution and after a 24-hour period, the samples were cultured to check for microbial contamination. After confirmation of the negative microbial culture results, AM was packaged and frozen at –80°C. It was then dried under high vacuum using a freeze-drying device. Tissue water was extracted through sublimation to reach a final water content of 5% to 10%. Finally, packing and sterilization by gamma irradiation were performed.

Clinical Measurements Before Surgery

Occlusal surgical guides were fabricated with coldcured acrylic resin on cast models obtained from

alginate impressions. The guides covered the occlusal surface of the tooth being treated, as well as at least one tooth mesial and distal to it. The guides covered the coronal third of the teeth both buccally and lingually. A groove was made in the guide with a fissure bur in an apico-occlusal direction at the point where the graft material and the membrane had to be placed. The grooves served to provide reproducible alignment of a probe (Fig 1). A single calibrated examiner performed clinical baseline and 6-month follow-up measurements. O’Leary plaque index was assessed. PPD, CAL, PB, and REC were recorded to the nearest millimeter with a Williams probe. The investigator was blinded with respect to the treatment modalities. The measurements were performed on the buccal and lingual surfaces of each interproximal defect, but only the deeper measurement (buccal or oral) was reported for each defect. Measurements of PPD, CAL, and PB were done by inserting the probe through the groove in the surgical guide, which ensured proper angulation. With the aid of the guide and the prepared groove, the probe was able to reach the deepest portion of the interproximal pocket at a reproducible angulation. The reference landmark for CAL was the cementoenamel junction (CEJ), and the lower margin of the guide acted as the reference landmark for measuring PB. PB was measured after the area was anesthetized, and sounding to the bone level was performed. All sites had recession, and there were no root fillings in the area of the measurements, so the use of the CEJ as the reference point was applicable. Intraexaminer agreement was checked by examining standard deviations of repeated measurements; this was better than 0.4 mm, and measurements were similar at > 90% of sites.

Randomization

Before surgery, the defects were randomly (through toss of a coin) assigned to one of two treatment groups. In the test group, defects were filled with Bio-Oss and covered with AM, and in the control group, defects were filled with Bio-Oss and covered with Bio-Gide. The International Journal of Oral & Maxillofacial Implants 641

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Kiany et al

Fig 2   AM and Bio-Oss.

Surgical Procedure

Fig 3   Two layers of trimmed AM.

A single investigator, not involved with the clinical measurements, performed all the surgeries. The intrabony defects were treated according to the principles of GTR with the application of biodegradable membranes. All the patients were treated under local anesthesia (2% lignocaine with 1:80,000 adrenaline). After intracrevicular incisions were made, full-thickness mucoperiosteal flaps were raised buccally and lingually; the surgeon tried to preserve the maximum extent of the marginal and interdental gingival tissue to obtain primary closure and membrane coverage. There was no need for releasing incisions. The alveolar bone was exposed at least 3 mm beyond the edges of the defect, and periosteal releasing incisions were used to ensure complete membrane coverage at the time of suturing. All granulation tissue was removed, the defects were debrided, and the roots were thoroughly scaled and root planed by hand instruments and ultrasonic devices. No root conditioning was performed. When debridement was complete, the defects were filled with Bio-Oss (0.25- to 1-mm-diameter granules). The grafted material, impregnated with saline, was placed with only light condensation into the defect. The defect was filled only to the most coronal level of the existing alveolar bone, and overfilling was avoided. AM was available in three rectangular sizes and was not pretrimmed. Following placement of the grafting material in the test group, the AM of the most appropriate size was trimmed according to the size and form of the defect. AM has self-adhering properties after it becomes moist. Upon contact with tissue fluid, the dry AM was easily placed without shriveling (Fig 2). It could be tucked underneath the flap margins, so that there was complete adaptation and coverage of the defect and interproximal area. The hydrated AM sealed itself over the defect. Double layers of AM were used in the test group to delay its degradation (Fig 3). In some cases, the excess AM could be folded onto itself without any consequences, which might further improve the

Fig 4   AM adapted over the bony defect.

healing (Fig 3). The AM was always placed coronal to the interproximal bone crest so that it completely covered the defect and extended 2 to 3 mm beyond the residual bone. No sutures, pins, or tacks were used for membrane fixation or stabilization. The orientation of the membrane did not matter. In the control group, the Bio-Gide membrane was also trimmed and adapted over the defects to cover 2 to 3 mm of the surrounding bone. It was stabilized without sutures or pins (Fig 4). When necessary, flap elevation was completed in a split thickness to allow coronal displacement of the flap and obtain coverage of the membranes. Vertical or horizontal mattress sutures with 4-0 silk were placed in the interproximal spaces to obtain primary closure of the interdental tissues over the membranes. A periodontal dressing (Coe-Pak, GC America) was used for wound stabilization and patient comfort.

Intrasurgical Clinical Measurements

Intrasurgical clinical measurements were performed following debridement of the defects as previously described24: (1) distance from the CEJ to the bottom of the defect (CEJ-BD) and (2) distance from the CEJ to the most coronal extent of the interproximal bone crest (CEJ-BC). These measurements were performed at the deepest interdental point of the defect (ie, the deepest point of the site demarcated by the interdental line angles of the affected tooth). The intraosseous component of the defects (INTRA) was defined as INTRA = (CEJ-BD) – (CEJ-BC).

Postoperative Care and Maintenance

All the patients received antibiotics (500 mg amoxicillin three times/day) for 1 week. Analgesics (400 mg ibuprofen three times/day) were also prescribed for 2 days. Patients were advised to rinse twice daily with 0.12% chlorhexidine for 4 weeks after surgery. Dressings and sutures were removed after 2 weeks. Patients were instructed to abstain from brushing the teeth in the surgical area for 2 weeks after suture removal. After that, they were advised to start brushing with

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an extra-soft toothbrush. Subjects were recalled at 4-week intervals for a period of 6 months for plaque scoring, oral hygiene instruction, and professional prophylaxis (when needed). No subgingival probing or instrumentation was performed at the experimental sites until the 6-month follow-up appointment.

Statistical Methods

Means and standard deviations for all parameters were calculated. The paired-sample t test was used to assess the statistical significance of differences between the initial and the 6-month visit within each group. The paired-sample t test was used to determine the statistical significance of differences between the test and control groups. The Pearson correlation coefficient was used to assess the relationship between INTRA and CAL gain. Differences were considered statistically significant at the P < .05. All data analysis was performed using statistical software (SPSS, version 14.0).

Table 1  Clinical Parameters at Baseline and 6 Months Postsurgery in the Test and Control Groups Parameter/ group

Baseline

6 mo postsurgery

P

PPD  Control  Test

7.3 ± 1.494 7.3 ± 1.947

REC  Control  Test

1.9 ± 1.524 1.6 ± 1.776

3.4 ± 1.838 1.9 ± 1.287

.009 .279

CAL  Control  Test

9.2 ± 1.687 8.9 ± 2.424

6.9 ± 1.197 5.9 ± 1.969

< .001 < .001

PB  Control  Test

13.5 ± 3.136 13.8 ± 2.044

10 ± 2.160 9.9 ± 1.792

< .001 < .001

3 ± 1.033 4 ± 11.563

< .001 < .001

RESULTS

DISCUSSION

None of the patients participating in this study showed a significant inflammatory response after surgery or during the follow-up period. Negligible inflammatory reactions subsided after proper oral hygiene maintenance. The mean values for PPD, CAL, PB, and REC at baseline and at 6 months in both groups are shown in Table 1. After 6 months, the mean PPD in the test group showed a significant reduction from 7.3 ± 1.947 mm to 4 ± 1.563 mm (P < .001). The reduction in mean PB was also significant and was from 13.8 ± 2.044 mm to 9.9 ± 1.792 mm (P < .001). In addition, mean CAL change in the test group was significant (8.9 ± 2.424 mm to 5.9 ± 1.969 mm; P < .001). However, mean REC did not increase significantly in the test group (1.6 ± 1.776 mm at baseline and 1.9 ± 1.287 at 6 months; P = .279). In the control group, the changes in PPD, PB, CAL gain, and REC were significant. Mean PPD showed a reduction, from 7.3 ± 1.494 mm at baseline to 3.8 ± 1.033 at 6 months (P < .001). Mean PB values decreased from 13.5 ± 3.136 mm to 10 ± 2.160 mm (P < .001). Mean CAL reduction was from 9.2 ± 1.687 to 6.9 ± 1.197 (P < .001). Mean REC increased from 1.9 ± 1.524 mm to 3.4 ± 1.838 mm (P = .009). A comparison of differences in PPD, CAL, PB, and REC after 6 months in the two groups is shown in Table 2. The changes in mean PPD, BP, and CAL gain were not significantly different between groups. However, the increase in REC in the control group after 6 months was significant (P = .009). Evaluation of the relationship between INTRA and CAL gain showed no significance (P = .440).

There has been significant progress in various aspects of periodontal therapy in the last two decades. Perhaps the most obvious shift has been from resective periodontal surgical procedures to techniques and methods aimed at regeneration and reconstruction of the lost periodontium. This randomized clinical trial compared the application of AM with Bio-Gide (in combination with Bio-Oss) for the surgical reconstruction of intrabony periodontal defects. There are difficulties in the standardization of the clinical evaluation of CAL, PPD, PB, and REC. The utmost effort was made to overcome this problem in this study. One masked calibrated examiner performed all the clinical measurements, and acrylic stents were used to guide the probe during all examinations. The split-mouth design mitigated the influence of patientspecific characteristics. Thus, each patient served as his or her own control. Both membranes were compared under the same regeneration and recall conditions. After this investigation was completed, it was found that both barrier membranes produced comparable clinical results. Both treatment modalities resulted in statistically significant improvements in CAL and reductions in PPD and PB after 6 months. There were no significant differences between the test and control groups regarding CAL gain or PPD and PB reductions, but treatment with AM did not result in significant REC. The most practical clinical parameter for assessing the results of regenerative periodontal treatment methods is CAL.25 To determine the effect of the baseline depth of osseous defects on changes in CAL after treatment, the relationship of the depth of the osseous The International Journal of Oral & Maxillofacial Implants 643

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Table 2  Comparison of Test and Control Groups Change in PPD

Control group

Test group

3.5 ± 0.972

–3.3 ± 1.418

P .735

Change in REC

1.5 ± 1.434

3 ± 0.823

Change in CAL

–2.3 ± 1.337

–3.00 ± 1.764

.009* .343

Change in PB

–3.5 ± 1.354

–3.9 ± 2.025

.599

defect (INTRA) to CAL gain was also evaluated. In fact, INTRA approximates the available space under the membrane, which in turn indicates the maximum possible attachment gain.26 It has been claimed that greater baseline depth of the osseous defects results in greater CAL gain.25 However, there was no statistically significant relationship between the baseline depth of defects and gain in CAL 6 months after GTR treatment in this study. This suggests that the differences in the depth of bony defects that were present at baseline did not influence the clinical outcomes. The types of bony defects in this study varied from three-walled to one-walled. It is noteworthy that most defects were combined. It has been reported that strictly three-walled defects improve more predictably after GTR procedures compared to combined or complex defects.20 However, the results of this study revealed that morphologic variations of the bony defects did not influence the efficacy of GTR treatment in either group. During the immediate postoperative period, no membrane exposures were observed. In both groups, uneventful healing and subsequent integrity of soft tissue were noted. The unremarkable clinical response of the periodontal tissue proved the biocompatibility of AM. Despite the lack of histologic clues and with respect to clinical healing, it can be claimed that, in both groups, cellular adhesion to the membrane surface, blood clot stabilization, and integration of the membrane with the proliferating connective tissue of the gingiva occurred. To date, there are no published data on the use of AM alone or in conjunction with graft material for the treatment of intrabony defects. Also, there are several variations that make it difficult to compare the results of this clinical study with those of other studies that examined other biodegradable membranes. Among these variables are study design, patient population, measurement techniques, pattern of bony defects, and differences in healing patterns and microbial pathogens. In a meta-analysis performed by Laurell et al,27 average CAL gain after treating interproximal intrabony defects with biodegradable membranes with and without graft material was reported to be 2.96 mm. In

another meta-analysis,28 GTR of intrabony defects with collagen membranes with grafts resulted in average CAL gain of 3.50 mm. In the present study, the mean CAL gain of the test group was 3 ± 1.764 mm, which is in the range of CAL gain obtained in the aforementioned meta-analyses.27,28 Shaila et al11 used AM and Bio-Oss in grade II furcation defects and compared it with a combination of AM and demineralized freeze-dried bone allografts. After 9 months, there was significant reduction in PD and gain in CAL and percentage of bone fill in both groups, without any significant differences. Collagen membranes appear to be suitable for GTR, as they are chemotactic for periodontal ligament fibroblasts and can serve as fibrillar scaffolds for early vascular ingrowth.29 However, these types of membranes act mainly as physiologic barriers, and they are considered biologically inactive.30 In contrast, AM has unique characteristics, including bacteriostatic effects, antiadhesive properties, wound protection, and epithelialization effects, that make it different from routinely used barrier membranes.17 Since its introduction there have been immense alterations in the processing of AM, which have resulted in improved clinical outcomes. Inefficient tissue processing would lead to a loss of the biologic properties of AM. The lyophilized (freeze-dried) method of preparation of AM induces minimal changes in its biologic properties. This method results in loss of epithelial cells and lack of immunogenicity, and the product can be stored at room temperature.23 It has been shown that AM, through adhesion to the wound surface, can act as an antibacterial barrier and reduce bacterial infiltration.31 Thus, it has been speculated that AM, because of its antibacterial properties, could decrease the risk of infection and, based on the presence of growth factors, promote healing. AM contains growth factors that hasten formation of granulation tissue by stimulating the growth of fibroblasts.32 In the meantime, AM vascularizes healthy granulation tissue and stimulates neovascularization in the neighboring tissues.33 Also, AM provides a protein-enriched bioactive matrix that facilitates cell migration.34 Hence, it can be speculated that the use of AM as a membrane for GTR could stimulate vascularization of the granulation tissue in the defects and promote cell migration and wound healing. The presence of laminin-5 in high concentrations throughout AM, with its high affinity for gingival epithelial cells, could accelerate healing and integration of the membrane with gingival tissue.33,35 In other words, it has been claimed that AM has the ability to form an early physiologic “seal” with the host tissue. This precludes bacterial contamination.14 This quality of good integration of AM with the overlying gingiva

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may account for the smaller amount of REC observed in the test group. For confirmation of this hypothesis, further studies with histologic examination at different points during the healing period are necessary. It can also be claimed that the presence of various growth factors in AM, such as platelet-derived growth factors alpha and beta and transforming growth factor beta,36 is likely to induce faster sealing of the defects and limited loss of the grafting material in the test group. Thickness of normal AM is 0.02 to 0.5 mm, which is equivalent to six to eight layers of cells.37 One of the major advantages of AM, in comparison to other biodegradable membranes, is its thinness (320 μm) and good adaptability. The significant lack of REC observed in the test group might be a result of the thinness of the AM, which resulted in better adaptation of the membrane over the bony defect and consequently better coverage of gingiva over the membrane. Although in this study a double layer of AM was used, no postoperative membrane exposure or uneventful healing was observed. AM is suggested for use in areas with limited thickness and height of gingiva, as full coverage of membrane is more easily accomplished. Considering the advantages of AM, it can be introduced as an inexpensive and readily available membrane. Its unique physical nature permits the clinician to ignore many of the recognized guiding principles for the application of customary barrier membranes. From a clinical and practical viewpoint, the ease of handling, trimming, and adapting of this form of AM renders it an easy-to-use and operator-friendly membrane. The AM used in this study, after being hydrated, needs less defined trimming and adapts tightly to the underlying grafting material, bony margin, and tooth surface. It is naturally self-adhesive to the underlying surfaces. However, it must be noted that this physical property of AM does not afford any space maintenance capabilities. Essentially, both of the membranes used in this study are characterized by a lack of stiffness. Thus, after becoming dampened by the tissue fluid, they adapt to the surgical field. This event was more prominent when AM was used; therefore, some kind of space-saving grafting material was needed under both membranes. In an attempt to further improve the clinical outcomes of GTR, this study was designed to employ a combined periodontal regenerative technique.38 It means that the study did not include a nongrafted group. There was no defect selection according to the number of walls or configuration of the defect in the protocol of the study, so it was expected that unfavorable and large defects might be encountered during surgery. According to the rationale for combined periodontal regenerative therapy, the bone material that is used in conjunction with membranes enhances the

stability of the coagulum, sustains the membranes in the presence of noncontained defects, and prevents collapse of the membranes onto the root surface or into the defects during wound healing, thereby preserving the space necessary for regeneration. Reduced or limited space might result in compromised healing as a consequence of inadequate space for tissue ingrowth.16 Moreover, a lack of osteoinductive activity in the anorganic bovine bone was desirable,39 because it was expected that this graft material would act mainly as filler and scaffold. It was assumed that the grafting material would enhance and facilitate the proliferation of osteogenic cells through its osteoconductive quality in both groups to the same degree. The presence of physical support by the graft material under such membranes allows the flaps to be sutured over the defects without exerting pressure on the membranes or displacing them. It was the clinical impression of the authors that, despite the pliability and delicacy of AM, the presence of Bio-Oss as a biocompatible filler in the defect provided good support for AM and prevented its collapse into the defect. One of the important factors in the outcome of GTR is the speed at which the biodegradable membranes are absorbed. Researchers disagree on the precise bioabsorption time of barrier membranes. Porcinederived collagen membranes in GTR procedures have been shown to resorb within 4 to 6 months.40 Chen et al41 reported that a collagen barrier of type I bovine tendon is either integrated into the healing connective tissue or is degraded by macrophages in 6 to 8 weeks. There is not enough evidence to determine the time to degradation of AM when it is used as graft, wound dressing, or membrane. In fact, it is difficult to determine how long the barrier effect of the AM lasts. It has been claimed that the protective function of AM, acting as a skeletal substructure, diminishes by the 14th to 21st days as a result of mucoid degeneration.7,42 The two reports of the degradation time of AM were obtained from studies of bladder reconstruction and wound dressing. It can be hypothesized that placement of AM as a membrane under a periodontal flap and prevention of exposure to the oral cavity might lead to longer degradation time. The gains in CAL and reductions in PPD in this study make it safe to speculate that the absorption of AM was slow enough to produce the desired effects. Logically, histologic studies are required to characterize the process of bioabsorption of AM in different clinical situations. A drawback of this study was the lack of pressuresensitive probe in clinical measurements. Although acrylic resin guides were used to confirm the reproducibility of the measurements and the examiner was calibrated, the application of pressure-sensitive probes might have resulted in a smaller margin of error. The International Journal of Oral & Maxillofacial Implants 645

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Reentry was not considered in this study, so the changes in crestal bone levels and bony defect depths could not be measured directly. Certainly, the nature of the attachment between the newly regenerated tissue and the root surface cannot be determined without biopsy of the treated teeth. Because none of the teeth included in this study were candidates for extraction, a histologic study was not performed. Histologic studies are needed to claim true periodontal regeneration. Further studies with a larger sample size over a longer postoperative follow-up period are needed to conclusively proclaim the efficacy of this membrane. Moreover, the authors suggest that AM be combined with other grafting materials in GTR and guided bone regeneration procedures. The results of this investigation indicated that AM as a novel barrier membrane, in conjunction with Bio-Oss, is quite predictable and comparable to Bio-Gide when used in intrabony defects in bringing improvements in clinical parameters. In addition to its various biologic properties, AM is easy to use. It could be folded and compressed into narrow spaces between the roots. AM, with its diverse characteristics, can influence the clinician’s decision, but ultimately, the choice between the two membranes would be mainly surgeon preference.

CONCLUSIONS Amniotic membrane (AM), as a biologic membrane, may open new perspectives in guided tissue regeneration (GTR) procedures. In view of the results of this study, the authors would like to propose AM as a promising membrane in GTR. AM possesses multiple biologic and physical characteristics that might not be found in other conventional barrier membranes, making it preferable for GTR. Positive benefits were seen when AM was used; statistically significant clinical outcomes were achieved in GTR treatment of intrabony periodontal defects that were comparable to the results seen with a collagenbased resorbable membrane (Bio-Gide). Both barrier membranes, when used in the treatment of intrabony defects, resulted in a reduction of probing pocket depths and probing bone and gains in clinical attachment levels, and both techniques produced sites that healed without complications. AM resulted in less recession.

ACKNOWLEDGMENTS The authors thank the Vice-Chancellery of Shiraz University of Medical Sciences for supporting this research (grant #4273). This manuscript is based on the thesis of Dr Fatemeh Moloudi. The authors would like to thank Dr Shahram Hamedani, DDS, MSc, from the Dental Research Development Center for his editorial suggestions and English writing assistance and Dr Mehrdad Vossoughi for the statistical analysis. The authors also express their gratitude to Dr Hamid Reza Aghayan and Dr Amir Hossein Tavakoli (from Iranian Tissue Bank, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences) on their valuable comments. The authors reported no conflicts of interest related to this study.

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The International Journal of Oral & Maxillofacial Implants 647 © 2015 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

Amnion membrane as a novel barrier in the treatment of intrabony defects: a controlled clinical trial.

The purpose of this 6-month randomized, controlled, blinded, clinical trial was to evaluate and compare the efficacy of amnion membrane (AM) with depr...
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