Functionally graded membrane: A novel approach in the treatment of gingival recession defects.

BACKGROUND: Guided tissue regeneration has recently been advocated in re-constructing soft-tissue dimensions in recession defects. Advancement in nanotechnology has led to increased zest for approaches such as electrospinning of biologically active; nanofibrous functionally graded regenerative membranes for periodontal tissue engineering. A functionally graded membrane (FGM) had been tailored by incorporating chitosan and nano-hydroxyapatite over Amnion membrane and used in gingival recession defects. STUDY
DESIGN: It was single-blind, randomized controlled study. Split-mouth study was conducted in nine patients and 22 sites with recession defects were selected. Sites were divided into Group A (Amnion membrane with coronal advanced flap) and Group B (FGM with coronal advanced flap).
MATERIALS AND METHODS: Sites were assessed clinically by recording plaque index (PI), gingival index (GI), vertical recession defect depth (VRDD), relative clinical attachment level (CAL), and width of keratinized tissue at baseline, 3-6 months; and radiographically by recording linear bone growth by dentascan at baseline and 6 months. RESULT: Both groups showed statistically significant reduction in PI, GI and VRDD, and CAL and nonsignificant reduction in width of keratinized tissue at 3 and 6 months postoperatively. Group A showed statistically significant linear bone growth at 6 months. Group B also showed gain in linear bone growth at 6 months; however, result was statistically nonsignificant.
CONCLUSION: FGM had shown favorable results by enhancing bone growth while preventing the gingival tissue downgrowth. Copyright:

INTRODUCTION

Gingival recession represents a condition in which the management is needed to address both biological and esthetic demands. The main goal should not be only to attain complete root coverage but also to regenerate the lost periodontal structures to minimize the chances of relapse of gingival recession. Thus, guided tissue regeneration (GTR) technique has been implicated to create the space for regenerating periodontal ligament cells to regenerate bone and attachment apparatus and prevent the down growth of migrating epithelial cells.[1]

Amnion is a thin and innermost lining of amniotic sac. The preserved human amniotic membrane is an innovative biomaterial that has abundant growth factors and stem cells that aids in hastening healing of wound with periodontal regeneration.[2] It is rich in collagen (Type I, IV, V, and VI), proteoglycans and glycoproteins like laminin and fibronectin.[3] It has exquisite properties like anti-microbial and anti-inflammatory that further enhances its utility.

Degradable membranes such as amnion membrane have the advantage of elimination of second surgical site as required in nonresorbable membranes, but the mechanical strength of such membranes is typically low and is difficult to maintain space underneath for regeneration of bone. Grafts are used underneath to maintain space and to prevent falling of membrane on the bone defect. It has been observed that cells adhere better on the fibers whose dimension is below their own. Thus, a GTR membrane will be preferred that contains bone graft and growth factors, whose dimensions comprise of nanometer-diameter protein fibers and have better mechanically properties.

Therefore, functionally graded three-layered membrane was tailored by electrospinning (e-spinning) Chitosan/Nano-hydroxyapatite over amnion membrane. It comprises of nanoparticles and resembles natural extracellular matrix (ECM).[45] Such a composite scaffold can stimulate healing, permits diffusion of materials across the membrane, stimulates formation of bone, and has good mechanical strength.[67]

Thus, an attempt had been made to evaluate the regenerative potential of functionally graded membrane (FGM) and to compare it with Amnion membrane in recession defects.

MATERIALS AND METHODS

Patient selection

Twenty-two sites in nine patients (eight males and one female) with gingival recession were selected. The selected sample size was based on the assessment of studies conducted earlier and the expected mean differences. The power of the study was fixed at 80% and error was fixed at 5%.

Patients were selected between the age group of 30–55 years, with buccal/labial vertical gingival recession defects more than or equal to 2 mm. Only co-operative and motivated patients committed to oral hygiene were selected. Patients with previous history of any periodontal treatment in last 6 months, any systemic ailment, smokers, chronic alcoholics, and pregnant and lactating females were excluded. This study was carried out according to revised Helsinki declaration (2013) and approved by ethical committee constituted by the institution. Patients signed the consent form enclosing detailed description of risks and benefits of the treatment.

Study design

Sites with gingival recession were divided randomly into Group A and Group B in a split-mouth design, such that each patient received treatment by both the membranes.

Group A

In 11 sites, amnion membrane was placed with coronally repositioned flap.

Group B

In 11 sites, FGM was placed with coronally repositioned flap.

Amnion membrane used in the study was procured from tissue bank in Tata Memorial Hospital, Mumbai, India [Figure 1]. FGM had been fabricated by e-spinning chitosan and nano-hydroxyapatite over Amnion membrane at IIT, Roorkee by multilayering spinning[8] [Figure 1]. It consisted of a core layer (Amnion membrane) and two functional surface layers (SLs) interfacing bone (nano-hydroxyapatite) and epithelium (chitosan).

Figure 1

Amnion membrane and functionally graded membrane

Assessment of clinical parameters was carried out using custom-made occlusal acrylic stents at baseline before the periodontal therapy and at 3 and 6 months postoperatively. The clinical parameters included plaque index (PI),[9] gingival index (GI),[10] relative clinical attachment level (from lower/apical/lower limit of occlusal stent to bottom of the pocket using University of North Carolina-15 [UNC-15] periodontal probe) (Relative attachment level [RAL]),[11] position of gingival margin/vertical recession defect depth (VRDD) (from the apical/lower most part of occlusal stent to free gingival margin using a UNC-15 periodontal probe) (VRDD),[12] and width of keratinized tissue[13] (from gingival margin at the deepest part of recession to the mucogingival [MG] junction).

Radiographic assessment at baseline and 6 months was done using computed tomography (CT) Dentascan imaging. Linear bone fill was measured as distance from the apical most end of cementoenamel junction (CEJ) to crest of buccal radicular bone.

Clinical procedure

After the completion of Phase I therapy, patients were subjected to surgical procedure. Adequate local anesthesia at the selected site was obtained. Two horizontal incisions were made from the line angles of involved tooth at a distance of 1 mm more than vertical depth of the recession from the tip of anatomical papilla. These incisions should not bisect the gingival margins of adjacent teeth. An intrasulcular (crevicular) incision was given joining the two horizontal incisions. Oblique releasing incisions were given on either side to release the pedicle flap. The resulting full-thickness trapezoidal flap was raised till the MG junction. It was followed by sharp dissection to obtain a split-thickness flap beyond MG junction to attain a freely movable flap. Coronal advancement was made possible by removing all muscle insertions. Fresh connective tissue beds were created by careful de-epithelization of the inter-dental papillae. The root planning of the involved surfaces was done with curettes.

Both membranes were cut to the desired dimensions to cover the defect area extending 2–3 mm onto the bone. Group A was treated with amnion membrane and Group B with FGM [Figures 2-3]. Flap was then coronally repositioned to wholly cover the membrane and sutured 1 mm coronal to CEJ. The flap was secured with direct interrupted sutures on the vertical incision line and sling suture for the papilla by 4-0 nonresorbable silk sutures. Surgical site was protected by the placement of the periodontal pack (coe-pak). Patients were advised proper postoperative instructions. Amoxicillin 500 mg and Diclofenac potassium were prescribed thrice daily each for 5 days. 0.12% chlorohexidine was also advised for 2 weeks to be used twice daily.

Figure 2

Group-A placement of amnion membrane on gingival recession on canine

Figure 3

Group B placement of functionally graded membrane on gingival recession on canine

One week postsurgically, sutures and pack were removed followed by irrigation of the surgical site. Patients were then periodically monitored at 3 and 6 months after surgery [Figures 4-7].

Figure 4

Group-A vertical recession defect depth at baseline

Figure 7

Group-B vertical recession defect depth at 6 months

Group-A vertical recession defect depth at 6 months postoperatively

Group-B vertical recession defect depth at baseline

On recall visits, oral hygiene was evaluated and related instructions were reinforced.

Statistical analysis

The arithmetic mean and standard deviation for the requisite assessment intervals were calculated. For intragroup variations, “Independent t-test” and for inter-group variations “Paired t-test” was done.

RESULTS

Patients showed satisfactory oral hygiene, with no signs of complications at the operated sites. For both the groups, the mean difference of PI, GI, VRDD, and RAL at 3 and 6 months from baseline was statistically highly significant (P < 0.01), whereas the mean difference from 3 months to 6 months was statistically nonsignificant (P > 0.05). The mean difference in the width of keratinized tissue at all the three time intervals was statistically nonsignificant (P > 0.05) in both the groups.

On comparison between the two groups, the mean difference of PI, GI, VRDD, RAL, and width of keratinized tissue at all the three time intervals were statistically nonsignificant (P > 0.05) [Tables 1 and 2].

Table 1

Intergroup comparison of mean differences of plaque index and gingival index at various intervals

Time interval	Group A versus Group B
	PI			GI
	Mean difference	T	P	Mean difference	T	P
Baseline to 3 months	0.023	0.199	0.734	0.045	0.188	0.473
Baseline to 6 months	0.023	0.108	0.648	0.068	0.279	0.359
3-6 months	0.000	0.000	1.00	0.023	1.00	0.038*

t - T value measured using Paired t test. P – Probability value. PI - Plaque index; GI - Gingival index

Table 2

Intergroup comparison of mean differences of vertical defect depth, relative clinical attachment level and width of keratinized tissue at various intervals

Time interval	Group A versus Group B
	VRDD			Relative CAL			Width of keratinized tissue
	Mean difference	T	P	Mean difference	T	P	Mean difference	T	P
Baseline to 3 months	−0.364	−1.054	0.317	−0.545	−1.215	0.418	0.091	0.176	0.869
Baseline to 6 months	−0.364	−1.118	0.630	−0.454	−1.102	0.894	0.000	0.000	1.000
3-6 months	0.000	0.000	1.000	0.091	0.447	0.824	−0.091	−0.373	0.713

t - T value measured using Paired t test. P – Probability value. VRDD - Vertical recession defect depth; CAL - Clinical attachment level

For Group A, the mean difference of linear bone fill from baseline to 6 months was 0.16 ± 0.18 that was statistically significant (P < 0.05); for Group B it was 0.11 ± 0.27, that was statistically nonsignificant (P > 0.05).

On comparison of linear bone fill between two groups, the mean difference from baseline to 6 months was 0.051 that was statistically nonsignificant (P > 0.05) [Table 3 and Figures 8-11].

Table 3

Intergroup comparison of mean differences of linear bone growth at various intervals

Time interval	Group A versus Group B Linear bone growth
	Mean difference	T	P
Baseline to 6 months	0.051	0.525	0.677

t - T value measured using Paired t test. P – Probability value

Figure 8

Group A - Linear bone at baseline

Figure 11

Group B - Linear bone at 6 months

Group A - Linear bone at 6 months

Group B - Linear bone at baseline

DISCUSSION

Coronal flap repositioning had shown to be a predictable technique that produce satisfactory results for coverage of denuded root surface as shown by Wennström and Zucchelli.[14] Moreover, donor site morbidity is prevented in comparison to free gingival graft and connective tissue grafts. The technique used in the present study as advocated by Pini Prato et al.[15] was preferred over the conventional coronal flap repositioning technique, as the former did not affect the marginal tissues of adjacent teeth, thus minimizing the chances of postoperative recession.

Currently, numerous biomaterials (membranes and grafts) are being used along with coronally repositioned periodontal flap to augment the results of root coverage procedure.

In the present study, two different types of membranes had been compared due to their unique properties. Amnion membrane comprises of various matrix proteins such as laminin, fibronectin, and collagen that helps in cellular migration and proliferation. It is also composed of various signaling molecules such as epidermal growth factor, transforming growth factor-β1, and fibroblastic growth factor. Favorable results had been shown with Amnion membranes by Gurinsky[16] in root coverage procedures, Wallace[17] in guided bone regeneration (GBR).

FGM has a nanofibrous structure that has advantage of high number of molecules on the surface that speed up positive interaction between cells and ECM, angiogenesis, differentiation of stem cells, support three-dimensional matrix adhesion and stimulate cell-signaling pathways required to achieve tissue regeneration.

Chitosan, a cationic natural biopolymer of N-acetyl glucosamine, is a structural component found in the glycosaminoglycans. Its cationic nature is responsible for attracting various negative charged proteoglycans. This suggests that chitosan may interact with growth factors, thus enhancing the healing property by the stimulation of fibroblasts and osteogenic differentiation.[18] Chitosan layer had been placed interfacing epithelial tissues as it had been shown to act as seal to prevent junctional epithelium from falling into the defect site and hence maintain space for bone regeneration.[19] It also exhibits biocompatibility, biodegradability, low toxicity, and antibacterial properties.[67] Being positively charged, chitosan combines with negatively charged red blood cells, thus quickly forming blood clot and shortening the homeostasis time.

Nanohydroxyapatite SL had been placed interfacing bone as it possesses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues, probably due to its structural similarity with the bone and being osteoconductive, is capable of forming a direct chemical bond with surrounding hard tissues. It improves mechanical strength of the membrane while maintaining its high porosity and microstructure. There is a possible interaction between the NH2 group and primary and secondary −OH group of chitosan with Ca2+ (metal coordination interaction) of nanohydroxyapatite responsible for the higher mechanical strength of the composite scaffold.

Nowadays, ternary systems with superior regenerative properties are getting immense attention as compared to binary systems like chitosan and HA scaffolds, by introducing components like growth factors.[20] Thus, in the present study, membrane has been fabricated comprising of chitosan, nano-hydroxyapatite and amnion membrane.

There is a paucity of literature regarding the use of these membranes in the field of periodontics. Noh et al.[21] suggested better wound healing with nanofibrous chitin as compared to microfibrous chitin. Baek et al.[22] supported the use of chitosan/hydroxyapatite membrane on new bone formation for GBR.

In this study, sites were selected where VRDD was greater than or equal to 2 mm. This was in accordance with Balaji et al.[23] where such recession defects were treated with successful results.

Dentascan is a CT software program that allows 3-planar view of the hard tissue structures of the oral cavity. It has an added advantage of interacting with images to make measurements, manipulate images to simulate treatment and to assess bone volume and density. Gingival recession is a consequence of loss of radicular bone. Thus, linear buccal bone loss was evaluated with the help of Dentascan.

In the present study, there was statistically highly significant reduction in mean PI, GI score of both Groups at 3 and 6 months postoperatively from baseline [Table 2]. These observations were in accordance with Agarwal et al.[24] who used coronally advanced flap procedure with or without Amnion membrane in gingival recession defects.

In both the Groups, no significant difference was observed between 3 and 6 months for both PI and GI [Table 2]. This might be because all patients must be complying with the oral hygiene instructions and maintaining health of the oral cavity.

The present study showed statistically highly significant reduction in mean VRDD, RAL of both Groups at 3 and 6 months postoperatively [Table 3]. Ghahroudi et al.[25] observed similar results with amnion allografts along with coronally repositioned flap procedure in recession defects. These changes were due to the coronal shift of the gingival apparatus owing to the recession coverage achieved.

Statistically nonsignificant reduction in RAL was observed between 3 months and 6 months in both groups Table 3. It must be due to relapse of gingival margin postsurgically due to thin tissues.[26]

There was statistically nonsignificant reduction in mean width of keratinized tissue of Group A and Group B at 3 and 6 months postoperatively from baseline. Gürgan et al.[27] studied the level of MG junction after coronally advanced flap procedure over the period of 5 years. They observed that keratinized tissue showed a significant decrease in width from baseline (3.68 ± 1.17) to 12 months (2.96 ± 1.19). However, a tendency of MG junction was seen to revert at 5 years period thereby nullifying the decrease.

The present study showed statistically significant gain in linear bone growth of Group A at 6 months postoperatively. Group B had also shown gain; however, results were nonsignificant (P > 0.05) at 6 months postoperatively [Table 4]. The results were similar to Saimbi et al.[28] and Kamboj[29] who evaluated bone defect fill in human intrabony defects using Dentascan.

Table 4

Intergroup comparison of mean differences of linear bone growth at various time interval

Group A vs Group B	Linear bone growth
Time Interval	Mean difference	T	P
Baseline to 6 months	0.051	0.525	0.677

t - T value measured using Paired t test. P – Probability value

CONCLUSION

The results showed the same regenerative potential of FGM and Amnion membrane. Since, this is the first reported use of FGM in periodontal defects, thus more clinical and histological studies with larger sample size and with different types of tissue engineered membranes are needed to explore the true regenerative potential of these third-generation GTR/GBR membranes, as they hold promise at solving the drawbacks of currently available membranes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.