Comparative Evaluation of Two Hyaluronic Acid Gel Products for the Treatment of Interdental Papillary Defects.

OBJECTIVES: The aim was to investigate the efficacy of single injections of two different hyaluronic acid products, Flex Barrier and Revident, in reducing the size of black triangles to treat Nordland-Tarnow Class I and II recessions.
MATERIALS AND METHODS: Forty adult patients were recruited with at least two upper and two lower interdental papilla defects in the front region between canine teeth. According to the Nordland-Tarnow classification of papillary defects, both Class I and Class II recessions were included in the investigation. Patients were randomly assigned to experimental groups to receive single injections of two different hyaluronic acid products, either Flex Barrier or Revident. The untreated sites served as controls. Photographs were taken before and immediately after the treatment, and again after one week and one month. To determine the size of the black triangles, Image J software was used. For statistical analysis, a mixed-design ANOVA was applied.
RESULTS: Both Flex Barrier and Revident significantly decreased the size of the treated defects immediately after the treatment and also one week later (p<0.001). The beneficial effect of Revident lasted longer than Flex Barrier as it remained significant even after one month in Revident-treated patients, however, not in the Flex Barrier-treated group. Furthermore, Nordland-Tarnow Class I lesions generally showed a greater improvement than Class II lesions.
CONCLUSION: In this proof-of-concept, randomized clinical trial we have demonstrated the clinical applicability of both Flex Barrier and Revident, although Revident gave longer-lasting improvements than Flex Barrier. Further trials are needed to optimize multiple-application protocols for treating gingival black triangles.

Introduction

Anterior esthetics has become one of the most important topics in contemporary dentistry (). While several sophisticated techniques exist to create “white esthetics”, the successful restoration of “pink esthetics” remains a challenge for clinicians, especially in the interdental area ().

The formation of a “black triangle” between teeth due to the absence of sufficient papilla may result from several factors, including age, periodontal disease, crown form and the course of the cemento-enamel junction. It appears because of the increased distance between the contact point and the alveolar bone crest (-). The prevalence of open embrasure areas appears to be relatively high, affecting 38% of adult orthodontic patients and 15% of the general adolescent population ().

Various treatment approaches have been developed in recent years to treat interdental papilla loss, including surgical techniques (), tissue engineering methods, such as the use of an injectable regenerative acellular dermal matrix (), and autologous fibroblast injections (). A non-surgical method introduced recently is the injection of hyaluronic acid fillers into the deficient papillae. The technique was first described by Becker et al. () in a pilot study treating 14 sites in 11 patients. Several other reports followed that work (-). Those studies reported variable outcomes with differing success rates, which are difficult to compare due to the lack of appropriate control sites and the different number of treatment sessions per individual.

In the present proof-of-concept study the two different hyaluronic acid preparations were used, Flex Barrier and Revident, as detailed below in Methods. Both gels were specifically developed for human clinical periodontal applications, including papilla reconstruction to correct “black triangles” in the interdental area. According to the manufacturers’ documentation, both Flex Barrier and Revident are bacteriostatic, antiseptic and promote wound healing, but neither of them was previously tested for their effectiveness in randomized clinical trials. Our study aimed to investigate the efficacy of the two different hyaluronic acid products in a randomized controlled clinical trial examining the effect of a single administration of injectable hyaluronic acid gel on the size of “black triangles” for the treatment of the Nordland-Tarnow class I and II recessions ().

Material and methods

We investigated the effects of the two hyaluronic acid gel products; both were compared to the untreated control papillae at four consecutive measurement time points. Based on a sample-size calculation using G*Power 3.1.9.7 software (Dusseldorf, Germany), the total number of papillae to be investigated for each hyaluronic acid group was 76 for an alpha of 0.05, a power of 0.90, an effect size of 0.3 (comparing the treated papillae to the untreated controls) and a correlation among the repeated measures of 0.5. For each patient, we aimed to treat at least one upper and one lower papilla and investigate at least one upper and one lower control papilla (at least 4 papillae/patient). Therefore, a minimum of 19 patients was needed for each hyaluronic acid group. In order to compensate for the loss to follow-up, we enrolled more patients and treated more papillae per patient. A total of 40 adult patients were involved (30 females and 10 males) with an average age of 44.4 (±12.8; inclusion criterion: age between 18-70), who had at least two upper and two lower interdental papillary defects in the front region between canine teeth. According to the Nordland-Tarnow classification of papillary defects, Class I or Class II recessions were included in the investigation.

Exclusion criteria were:

active periodontitis (Community Periodontal Index (CPI) grade 3 or 4),

acute oral and/or upper respiratory tract infection,

previous surgical treatment of the papillae to be investigated (due to the presence of scar tissue),

pregnancy or lactation,

smoking,

bleeding disorders or any medication that would affect blood coagulation (e.g.: coumarin-type drugs, platelet aggregation inhibitors, etc.),

systemic diseases that may affect periodontal health (e.g.: diabetes mellitus) or any kind of ongoing immunosuppression,

known or suspected allergy to local anesthetics and/or hyaluronic acid.

All the patients underwent non-surgical periodontal therapy three-six months prior to the present investigation. As a baseline examination, a CPI chart of the front teeth was recorded and digital photographs were taken in frontal and lateral views, centered to central incisors and lateral incisors, respectively. Camera settings were standardized (1:3 aspect ratio, Canon EOS 550D camera, Canon EF 100mm f/2.8 Macro USM lens, manual mode, 1/200 sec, f/22, ISO 100, Sigma EM-140 DG ring flash, manual mode, ½ power; the optical axis is parallel to the occlusal plane). Papillary defects were visually classified according to the criteria of the Nordland-Tarnow classification, following examiner calibration.

Two different hyaluronic acid preparations were used. As a reference material, the commercially available Flex Barrier gel (Naturelize GmbH and Bio Science GmbH, Ransbach-Baumbach, Germany) was purchased. Flex Barrier was specifically developed for clinical periodontal applications, including papilla reconstruction to correct “black triangles” in the interdental area. According to the manufacturer’s description, it is a safe, synthetic, and user-friendly alternative to resorbable membranes, and is bacteriostatic and antiseptic, and it promotes wound healing. The gel contains two-thirds cross-linked and one-third non-cross-linked hyaluronic acid. Since the two types of particle vary in size, they compress and form a flexible membrane on the area to be shielded. The barrier function remains active for three weeks ().

The other treatment group received Revident, a gift from its manufacturer, SLS LLC (Moscow, Russia). Revident is a 1% hyaluronic acid formulation for clinical use. The hyaluronic acid substrate in this gel was the same as in Flex Barrier. During its preparation, hyaluronic acid is modified by adding a water-soluble extract of wheat-germ root, principally a neutral hydrophilic complex of polysaccharides. Both of these ingredients are readily soluble in water (, ). The components of wheat-germ extract have been shown to exhibit anti-inflammatory effects (, ), to stimulate osteogenic differentiation (), to increase cell adhesion capability (, ) and also to enhance cell proliferation (, ). Such mixtures have already been used in vivo for targeted delivery to liver cells ().

After the first examination, the patients were randomly allocated to one of the test groups by coin tossing. In the Flex Barrier group (Flex Barrier; n=20) at least one upper and one lower papilla was treated with Flex Barrier gel following the “Three Step Technique” (TST), as recommended by the manufacturer, while at least one upper and one lower papillary defect was left untreated and served as a negative control. The TST method consisted of the following steps:

(1) injection of the gel with a 30G needle along the mucogingival junction at the base of the papilla at 4-5 sites, creating depots of 0.1ml per site, () injection of the gel into the attached gingiva at the base of the papilla at 2-3 sites, creating depots of 0.1ml per site, () injection of the gel into the papilla 2-3 mm from its tip at one site, creating a depot of 0.1 ml. In the Revident group (Revident; n=20) the same treatment was performed using the Revident gel. Again, at least one upper and one lower untreated papilla served as negative controls. All the treatments were performed by the same clinician in the same clinical setting.

Photographic documentation of treated papillae was performed immediately after the injection of hyaluronic acid gel, applying exactly the same settings as before treatment. The follow-up assessments of the volumetric changes in the treated and control papillae were carried out one week and one month after the treatment and consisted of photo documentation and a visual reassessment according to the Nordland-Tarnow classification. A retreatment of previously treated papillae and the treatment of untreated negative control papillae was performed upon patient’s request after the one month follow-up.

The analysis of digital photographs was performed using ImageJ software (). All measurements were performed by the same investigator. First, a standard distance (the mesiodistal width of a tooth neighboring the papillary defect) was measured on each photograph to set the scale. Using this scale setting, outlines of the interdental “black triangle” indicating the papillary defect were drawn with the help of the Image J polygon tool. The area of the polygon was automatically calculated by the software. Changes in the “black triangle” area were expressed as a percentage of the initial lesion area. Decreases in lesion size indicated increases in papilla size.

A mixed-design ANOVA analysis (IBM SPSS Statistics 19) was used with the Bonferroni post-hoc tests to evaluate the differences between the hyaluronic acid-treated and the control papillae (the between-subjects factor) and between the time points (within-subjects factor).

Results

Nine of the 40 patients included in the study were excluded from the analysis. Eight of them did not attend the control appointments, while one patient had herpetic lesions of the attached gingiva around the treated area developed by the time of the one-week recall appointment. Therefore, the exact measurements of the papillae dimensions could not have been performed for that individual. Data from 31 patients and a total of 160 papillae were analyzed (Revident test: n=48, Revident control: n=32, Flex Barrier test: n=50, Flex Barrier control: n= 30). Altogether 98 papillae were treated with one of the two hyaluronic acid gels, while 62 were used as control sites. The average age of the 31 patients who completed the study was 44.0 (±13.03; Revident group: 46.1 ±12.3, Flex Barrier group: 41.8 ±13.8). 23 of them were females (Revident group: 11, Flex Barrier group: 12) and eight of them were males (Revident group: 5, Flex Barrier group: 3) (Table 1).

Table 1

Data from the patients who completed the study. The pixel count of the interdental papillary defect was expressed as a percentage of the baseline value. Both Revident- and Flex Barrier-treated defects show a decreased defect size compared to the untreated control defects in both the upper and the lower jaws (CPI: community periodontal index).

Treatment group (patient)		Revident (n=16)				Flex Barrier (n=15)
Gender distribution		f=11; m=5				f=12; m=3
Average age		46.1 (±12.3)				41.8 (±13.8)
Average CPI score		1.81 (±0.54)				1.80 (±0.41)
Papilla		test (n=48)		untreated control (n=32)		test (n=50)		untreated control (n=30)
Jaw		upper	lower	upper	lower	upper	lower	upper	lower
Interdental papillary defect size in the percentage (%) of baseline pixel count	Before treatment	100 (±0)	100 (±0)	100 (±0)	100 (±0)	100 (±0)	100 (±0)	100 (±0)	100 (±0)
		100 (±0)		100 (±0)		100 (±0)		100 (±0)
	Immediately after treatment	79.4 (±16.5)	82.7 (±16.4)	100 (±0)	100 (±0)	81.8 (±19.8)	86,0 (±13.7)	100 (±0)	100 (±0)
		81.0 (±16.38)		100 (±0)		83,8 (±17,1)		100 (±0)
	1 week	88.0 (±15.3)	81.35 (±22.0)	98.5 (±4.83)	100.0 (±1.7)	91.6 (±12.8)	90.7 (±14.0)	99.9 (±1.4)	100.1 (±0.6)
		84.8 (±18.9)		99.3 (±3.6)		91.2 (±13.24)		100.0 (±1.1)
	1 month	90.3 (±17.0)	81.5 (±27.2)	101.0 (±6.5)	99.9 (±0.5)	97.5 (±7.0)	94.4 (±10.4)	100.1 (±0.9)	100.0 (±0.4)
		86.1 (±22.6)		100.4 (±4.6)		96.0 (±8.8)		100.1 (±0.9)

In the Flex Barrier group, the results of the mixed-design ANOVA showed that interdental papillary defect size was significantly affected by the administration of the gel (between-subjects factor): F(1, 78)=23.27, p<0.001 with a large effect size (η2=0.230). There was also a significant interaction between time and Flex Barrier administration F(1.96, 152.63)=15.45, p<0.001. Since the Mauchly’s test of sphericity was violated, a Greenhouse-Geisser test was used. The partial η2 effect size (η2=0.165) indicated that the effect of the interaction was moderate. A Bonferroni post-hoc test revealed that the defect size decreased significantly right after the administration of the gel (p<0.001). The treated defect areas then increased significantly (p=0.43) over the first week of follow-up but remained significantly smaller (p<0.01) than the control defects. There was also a significant increase (p<0.01) in the defective area between one week and one month at the treated sites, and there was no significant difference (p=0.127) between the test and the control sites at the end of the investigation (Figure 1).

Figure 1

Changes in the mean papillary defect size in the Flex Barrier group: the treated defects (the green line) showed a significant decrease compared to the control baseline. The initially beneficial effect had diminished after one week and at the one-month follow-up. However, the mean defect size did not return to the initial size measured at baseline. The mean size of the untreated (control) papillary defects in the same patients (the blue dashed line) did not reveal any change throughout the follow-up period. The error bars represent the standard error of the mean for both the treated and the untreated defects. *p<0.05; **p<0.01 versus control.

In the Revident group, the results of the mixed-design ANOVA showed that interdental papillary defect size was significantly affected by the administration of the gel (between-subjects factor): F(1, 78)=26.26, p<.001 with a large effect size (η2=0.252). There was also a significant interaction between time and Revident administration F(1.91, 148.79)=13.84, p<.001. Since the Mauchly’s test of sphericity was violated, a Greenhouse-Geisser test was used. The partial η2 effect size (η2=0.151) indicated that the effect of the interaction was moderate. A Bonferroni post-hoc test revealed that defect size decreased significantly right after the administration of the gel (p<0.001). The improvement showed a slight, but statistically insignificant, decrease by the one week (p=1.000) and the one month (p=0.697) follow-ups. At the end of the investigation, the treated defect areas remained significantly smaller (p<0.01) than the untreated control defects (Figure 2).

Figure 2

Changes in the mean papillary defect size in the Revident group: the treated defects (the green line) showed a significant decrease compared to the control baseline. The slight relapses in the beneficial effects could be seen after one week and at the one-month follow-up, however the mean defect size did not return to the initial size measured at the baseline. The mean size of the untreated (control) papillary defects in the same patients (the blue dashed line) did not show any change throughout the follow-up period. The error bars represent the standard error of the mean for both the treated and the untreated defects. *p<0.05; **p<0.01 versus control.

The comparison of the two treatment groups demonstrated that there was no significant difference between the control defects (F(1,60)=0.075, p=0.786), while the type of gel used had a significant effect on the decrease in the defect size (F(1, 96)=0,032, p=0.032), where Revident proved more effective. However, the partial η2 effect size (η2=0.047) indicated that the effect of the gel type on defect size was small.

We performed these evaluations in both the Nordland-Tarnow Class I type and II type patients (Figures 3-4). Most of the papillae included in the study were the Nordland-Tarnow Class II type. Our results presented that in this type of papillary lesion neither of the investigated hyaluronic acid gels caused improvements that were noticeable to the patients, although we could measure improvements by image analysis (Figure 4). Class I lesions exposed a greater improvement that was noticeable for the patients by subjective judgement (Figure 3). As a general rule, we can state that the smaller the initial lesion, the greater the improvement in the reduction of the “black triangle” lesion. However, the long-term results are largely dependent on the patient’s individual interdental oral hygiene rather than on the type of hyaluronic acid gel. In the cases where plaque accumulation was observed during the recall visits, no improvement has been detected compared to the baseline lesion, irrespective of the type of the gel used.

Figure 3

Changes in the appearance of a small (Nordland-Tarnow Class I) papillary defect after the Revident treatment (patient 5). (a) “Black triangles” at the baseline; (b) immediately after the injection: the paleness of the papillae and the attached gingiva is due to the injected gel; (c) at the one-week check-up: both defects were noticeably decreased; (d) at the one-month follow-up, suggesting further improvement. Complete resolution of small papillary defects is achievable with a single administration of Revident gel in this case.

Figure 4

Changes in the appearance of a large (Nordland-Tarnow Class II) papillary defect after the Revident treatment (patient 13). (a) “Black triangles” at the baseline; (b) immediately after the injection; (c) at the one-week follow-up; (d) at the one-month follow-up. Large defects do not show clinically noticeable changes after a single administration of hyaluronic acid gel.

We also investigated many other papillae, both in the upper and lower jaws, to exclude possible biasing effects of the choice of the site for treatment. Our results show that both the upper and lower sites responded similarly.

In the preliminary screening for possible long-term effects of treatment, four patients in the Revident group had been regularly checked for up to 18 months after the initial application of the hyaluronic acid gel. No harmful long-term effects were observed.

Discussion

As Bertl et al. stated in their systematic review, having investigated the adjunctive effects of hyaluronan on periodontal therapy, recommendations for clinical application should be based on repeated positive outcomes in multiple controlled clinical trials (). Therefore, there has been a strong need for robust scientific data to evaluate the use of hyaluronan injections for interdental papillary augmentation. By using two different, injectable hyaluronic acid-containing gel preparations, Flex Barrier and Revident, we have shown that even a single administration of these gels effectively decreases the size of “black triangles” in patients with Nordland-Tarnow Class I and II type recessions.

Advantageous physicochemical properties of hyaluronic acid include biodegradability, non- toxicity, biocompatibility, and non-immunogenicity. These properties make them suitable for various biomedical applications (), such as intra-articular (osteoarthritis) (), cosmetic (dermal implantation and wrinkle correction) (), and topical uses (wound dressing and treatment of burns) (, ). Hyaluronan production increases in proliferating cells, and the polymer may play a role in mitosis (). Recent investigations have found that cross-linked hyaluronic acid hydrogel is also an excellent biodegradable scaffold for tissue engineering and regenerative medicine (, ).

A new systematic review clearly identifies the main application areas of hyaluronic acid in dentistry (), such as adjuvant treatment of gingivitis and periodontitis (-), and in various surgical procedures including dental implantation, sinus lift and impacted third molar surgeries (), as well as the treatment of aphthous ulcers (). Although the data show some beneficial results in the treatment of temporomandibular joint disorders (), the FDA approved this therapy only for the use in knee osteoarthritis ().

Our study is the first randomized clinical trial demonstrating that clinically applicable hyaluronic acid preparations are effective, with single administration, in the treatment of gingival “black triangles” in humans. Furthermore, our data indicate that although both preparations are effective, Revident gave longer-lasting effects than Flex Barrier.

Since the technique for papillary augmentation by hyaluronan fillers was first introduced by Becker and co-workers (), several single- or multiple-case, noncontrolled follow-up studies have been conducted to investigate the effectiveness of hyaluronic acid injections. Becker et al. treated 14 sites in 11 patients. Sites adjacent to teeth () and implants () were both included. The number of treatment sessions ranged between one and three, depending on the results seen at control visits, but, unfortunately, no control sites were investigated. The follow-up period ranged from six to 25 months and success ranged between 57 and 100% ().

Mansouri et al. investigated 21 interdental papillae in 11 patients treated with hyaluronic acid gel injections, again without the assessment of the control sites. Injections were repeated up to three times on control visits, based on an individual assessment, and all patients were followed-up for six months. A mean success rate of 47.3±20.2% (range 22-100%) in papilla reconstruction was reported.

Lee and co-investigators treated 43 maxillary anterior papillary defects in 10 patients, also without the investigation of control sites. Administration of the gel was repeated up to five times (mean 3.4), based on individual assessment, at three-week intervals, and patients were followed up for six months. Complete reconstruction rates were recorded at 29 sites (100%) and partial reconstruction rates (39-96%) at 14 sites, with a mean of 92.5%. They also reported another study involving 57 upper anterior sites in 13 patients, with no control group, where administration of the gel was repeated up to five times (mean 3.33) at three-week intervals. That study reported an 88.8% mean papillary reconstruction rate after six months.

Awartani and co-workers treated 17 sites (13 maxillary, four mandibular) in nine patients with no control group. The hyaluronic-acid-containing gel was administered three times, with repeats at 21 and 42 days after the initial session, and patients were followed up for six months. Papillary defects showed a 0-100% reduction at six months with a mean of 41±37% ().

Finally, a recent study by Bertl et al. investigated 21 patients with papillary deficiencies between implants and natural teeth. 11 papillae were treated with hyaluronic acid gel while 10 more were treated with physiological saline solution as a control. The injections were repeated four weeks after the initial treatment, and the patients were followed up for a period of six months. This study did not discover any significant reduction in the papillary defects, and no significant differences were observed between the test and the control values ().

There is a large degree of variability in these studies: in the number of sites and patients involved, and in the number of hyaluronic acid gel administrations. Except for Bertl et al. (), none of the research settings included control sites, but the Bertl et al. study only investigated papillae adjacent to implants. It is therefore hard to draw any firm conclusions from these reports, but it is clear that hyaluronic acid gel injection could have some beneficial effects since the mean success rate ranged from 0% to 93%.

The two major reasons for the huge variation in the effectiveness of the treatment may be inconsistencies in gel administration and different intervals between retreatment visits. Therefore, in the present work one of our major aims was to investigate the effect of a single administration. Our data present that a single administration of the hyaluronic acid gel is effective when measuring “black triangles” in a standardized and reproducible way. But we should also admit that the outcome of the present work, in terms of the magnitude of changes, is inferior compared to some other studies in which multiple administration was performed (-). In our study, the effect also seemed to last for a very short time and shrinking of the augmented papillae was observed even in the short follow-up period.

The main inclusion criterion for the present study was the subjective esthetic complaint of the patients that they had “black triangles” between their teeth. We therefore included both the Nordland-Tarnow class I and class II patients in our work. Considering that the larger the lesion, the more disturbing it is for the patient, most of the cases were Nordland-Tarnow Class II types. Our present proof-of-concept study clearly demonstrates the effectiveness of both Revident and Flex Barrier gels on the periodontal defect. Additionally, it also shows that the single injection application of these compounds results in only a moderately positive clinical outcome. The results of the digital image analysis showed significant superiority of the Revident gel over the Flex Barrier gel. But we must also note that, using the single injection protocol, the difference between the two materials was not detectable by physical examination or a subjective visual evaluation by the patients. The reasons for this are discussed above. Large, disturbing Nordland-Tarnow class II “black triangle” defects decreased in size but did not disappear. Therefore, in clinical practice it seems to be reasonable to inform patients in advance about the possible necessity of multiple treatment sessions for better results. An exact recommendation on the number of re-treatments has to be determined by well-designed randomized clinical studies in the future. Although the average age of the patients in the two treatment groups was comparable, it has to be noted that the relatively wide age range may have had an effect on the results as well, because the healing capacity and collagen density of gingival tissues change greatly with age. In the present study, the youngest patient treated was 20, while the oldest was 66 years old. Future studies need to focus on a narrower age range to exclude this potentially confounding factor.

Conclusion

In this proof-of-concept randomized clinical trial, the clinical applicability of two hyaluronic acid preparations, Flex Barrier and Revident has been demonstrated. In a single-injection protocol Revident showed longer lasting effects than Flex Barrier. Apart from showing effectiveness, this study clearly points out the need for further, well-designed randomized clinical trials in order to determine the optimal arrangements for treating gingival “black triangles” with multiple injections of hyaluronic acid.