The Adjunctive Use of Hyaluronic Acid in Periodontal Therapy: A Systematic Review

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Annex No.2

Xinni Lu

5th Year, Group 12

The Adjunctive Use of Hyaluronic Acid in Periodontal Therapy:

A Systematic Review

Master’s Thesis

Supervisor assist. Egle Ramanauskaite

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FINAL MASTER‘S THESIS IS CONDUCTED

AT THE DEPARTMENT OF DENTAL AND ORAL PATHOLOGY

STATEMENT OF THESIS ORIGINALITY

I confirm that the submitted Final Master‘s Thesis: The adjunctive use of hyaluronic acid in periodontal therapy: a systematic review.

1. Is done by myself.

2. Has not been used at another university in Lithuania or abroad.

3. I did not used any additional sources that are not listed in the Thesis, and I provide a complete list of references.

I confirm by e-mail, and the work will be signed after the end of the quarantine and emergency situation due to the COVID-19 pandemic in the republic of Lithuania.

2020.04.30 Xinni Lu

(date) (autthor‘s full name) (signature)

CONCLUSION OF FINAL MASTER‘S THESIS ACADEMIC SUPERVISOR

ON THE DEFENSE OF THE THESIS

(date) (author‘s full name) (signature)

FINAL MASTER‘S THESIS IS APPROVED AT THE DEPARTMENT

(date of approval) (name of the Department and full name of the Head of the Department) (signature)

Final Master‘s Thesis reviewer

(full name) (signature)

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Evaluation of Final Master‘s Thesis Defense Board:

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Annex No. 8

EVALUATION TABLE OF THE MASTER’S THESIS

OF THE TYPE OF SYSTEMIC REVIEW OF SCIENTIFIC LITERATURE Evaluation: ...

Reviewer: ...

(scientific degree. name and surname)

Reviewing date: ...

No. MT parts MT evaluation aspects

Compliance with MT requirements and evaluation Yes Partially No 1 Summary (0.5 point)

Is summary informative and in compliance with the

thesis content and requirements? 0.3 0.1 0

2 Are keywords in compliance with the thesis

essence? 0.2 0.1 0

3 Introduc-

tion, aim and tasks (1 point)

Are the novelty, relevance and significance of the

work justified in the introduction of the thesis? 0.4 0.2 0

4 Are the problem, hypothesis, aim and tasks formed

clearly and properly? 0.4 0.2 0

5 Are the aim and tasks interrelated? 0.2 0.1 0

6 Selection criteria of the studies, search methods and strategy (3.4 points)

Is the protocol of systemic review present? 0.6 0.3 0

7

Were the eligibility criteria of articles for the selected protocol determined (e.g., year, language, publication condition, etc.)

0.4 0.2 0

8

Are all the information sources (databases with dates of coverage, contact with study authors to identify additional studies) described and is the last

search day indicated?

0.2 0.1 0

9

Is the electronic search strategy described in such a way that it could be repeated (year of search, the last search day; keywords and their combinations; number of found and selected articles according to

the combinations of keywords)?

0.4 0.1 0

10

Is the selection process of studies (screening, eligibility, included in systemic review or, if applicable, included in the meta-analysis)

described?

0.4 0.2 0

11

Is the data extraction method from the articles (types of investigations, participants, interventions, analysed factors, indexes) described?

0.4 0.2 0

12

Are all the variables (for which data were sought and any assumptions and simplifications made) listed and defined?

0.4 0.2 0

13

Are the methods, which were used to evaluate the risk of bias of individual studies and how this

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information is to be used in data synthesis, described?

14 Were the principal summary measures (risk ratio,

difference in means) stated? 0.4 0.2 0

15 Systemiza- tion and analysis of data (2.2 points)

Is the number of studies screened: included upon assessment for eligibility and excluded upon giving the reasons in each stage of exclusion presented?

0.6 0.3 0

16

Are the characteristics of studies presented in the included articles, according to which the data were extracted (e.g., study size, follow-up period, type of respondents) presented?

0.6 0.3 0

17

Are the evaluations of beneficial or harmful outcomes for each study presented? (a) simple summary data for each intervention group; b) effect

estimates and confidence intervals)

0.4 0.2 0

18

Are the extracted and systemized data from studies presented in the tables according to individual tasks?

0.6 0.3 0

19

Discussion (1.4 points)

Are the main findings summarized and is their

relevance indicated? 0.4 0.2 0

20 Are the limitations of the performed systemic

review discussed? 0.4 0.2 0

21 Does author present the interpretation of the

results? 0.4 0.2 0

22

Conclusions (0.5 points)

Do the conclusions reflect the topic, aim and tasks

of the Master’s thesis? 0.2 0.1 0

23 Are the conclusions based on the analysed material? 0.2 0.1 0

24 Are the conclusions clear and laconic? 0.1 0.1 0

25

References (1 point)

Is the references list formed according to the

requirements? 0.4 0.2 0

26

Are the links of the references to the text correct? Are the literature sources cited correctly and precisely?

0.2 0.1 0

27 Is the scientific level of references suitable for _{Master’s thesis?} 0.2 0.1 0

28

Do the cited sources not older than 10 years old form at least 70% of sources, and the not older than 5 years – at least 40%?

0.2 0.1 0

Additional sections, which may increase the collected number of points

29 Annexes Do the presented annexes help to understand the

analysed topic? +0.2 +0.1 0

30

Practical recommen-

dations

Are the practical recommendations suggested and

are they related to the received results? +0.4 +0.2 0

31

Were additional methods of data analysis and their results used and described (sensitivity analyses, meta-regression)?

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32

Was meta-analysis applied? Are the selected statistical methods indicated? Are the results of each meta-analysis presented?

+2 +1 0

General requirements, non-compliance with which reduce the number of points

33

General require- ments

Is the thesis volume sufficient (excluding annexes)?

15-20 pages (-2 points)

<15 pages (-5 points)

34 Is the thesis volume increased _{artificially?} -2 points -1 point

35 Does the thesis structure satisfy the _{requirements of Master’s thesis?} -1 point -2 points

36 Is the thesis written in correct language, _{scientifically, logically and laconically?} -0.5 point -1 points

37 Are there any grammatical, style or _{computer literacy-related mistakes?} -2 points -1 points

38 Is text consistent, integral, and are the _{volumes of its structural parts balanced?} -0.2 point -0.5 points

39 Amount of plagiarism in the thesis. _{(not evaluated)}>20%

40

Is the content (names of sections and sub- sections and enumeration of pages) in compliance with the thesis structure and aims?

-0.2 point -0.5 points

41

Are the names of the thesis parts in compliance with the text? Are the titles of sections and sub-sections distinguished logically and correctly?

42 Are there explanations of the key terms _{and abbreviations (if needed)?} -0.2 point -0.5 points

43

Is the quality of the thesis typography (quality of printing, visual aids, binding) good?

*In total (maximum 10 points):

*Remark: the amount of collected points may exceed 10 points. Reviewer’s comments:

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1

Abstract

Objective:

To evaluate the effect of adjunctive application of hyaluronic acid (HA) on clinical parameters in non-surgical, surgical, and plastic periodontal surgery.

Materials and methods:

A systematic search was performed in both MEDLINE (Ovid) and Cochrane Central Register of Controlled Trials (CENTRAL) databases. The literature search was performed according to PRISMA guidelines. Weighted mean differences (WMDs) and 95% confidence intervals (CIs) between the test and control groups were estimated by using the random-effect model for probing depth (PD) reduction, clinical attachment level (CAL) gain, reduction of bleeding on probing (BOP), and gingival recession (GR) reduction.

Results:

Ten randomized controlled trials (RCTs) were included in the meta-analysis: 6 on non-surgical periodontal treatment, 2 on surgical periodontal treatment, and 2 on periodontal plastic surgery. In non-surgical periodontal therapy significant changes were observed for the changes in PD, favoring the adjunctive use of HA (WMD -0.67mm, p<0.05). No significant change was found in the changes of CAL(p>0.05) and BOP (p>0.05). In surgical periodontal treatment significant changes were found only for PD values (WMD -0.61mm, p<0.05). In periodontal plastic surgery, compared to control groups, the adjunctive application on HA resulted in greater recession coverage (WMD -0.56mm, p<0.05).

Conclusion:

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3

K

EYWORDS

:

Hyaluronic acid, Periodontitis, Non-surgical periodontal therapy, Surgical periodontal therapy, Periodontal plastic surgery, Gingival recessions

A

BBREVIATIONS AND ACRONYMS

:

BOP: bleeding on probing; C: control group;

CAF: coronally advanced flap surgery; CAL: clinical attachment level;

CENTRAL: Cochrane Central Register of Controlled Trials;

CI: confidence interval; GR: gingival recession; HA: hyaluronic acid; IQR: interquartile range; OFD: open flap debridement; PD: probing depth;

PICO: Population, Intervention, Comparison, Outcome;

PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; RCT: randomized controlled trial;

rhFGF-2: recombinant human Fibroblast growth type 2; SD: standard deviation;

SRP: scaling and root planing; T: test group;

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4

1 Introduction

Periodontitis is a chronic multifactorial inflammatory disease, which is characterized by progressive destruction of the tooth-supporting apparatus [1]. It leads to destruction of periodontal tissues (gingiva, periodontal ligament, root cementum, and alveolar bone proper) and eventually results in tooth loss [2]. In addition, periodontitis negatively affects chewing function and aesthetics, becoming a source of social inequality, and diminishing the quality of life [3].

The goals of periodontal therapy include the reduction or elimination of tissue inflammation, correction of defects or anatomical problems, and regeneration of lost periodontal tissues [4]. In general, reduction of bleeding on probing (BOP), reduction of pocket probing depths (PD), gain of clinical attachment level (CAL) of periodontal tissues and elimination of open furcation in multirooted teeth, are the clinical parameters to evaluate the quality of periodontal therapy [5]. Scaling and root planing (SRP) is a cornerstone in periodontal therapy [6]. Its primary goal is to remove soft and hardened microbial deposits from the pathologically exposed root surfaces [7]. Ideally and theoretically, periodontal therapy can reduce and even eliminate the pathogenic species, which cause sustain periodontal diseases [7]. However, this therapy is technically demanding and has certain limitations. Bacterial plaque cannot be sufficiently eliminated from deep pockets, infrabony defects, or furcation areas. Therefore, it is important to use adjunctive antimicrobial chemotherapeutic agents to eliminate or inactivate pathogenic microflora in sites where mechanical instrumentation is invidious.

It has been shown that a successful periodontal treatment requires not only a meticulous mechanical removal of subgingival deposits but also an application of adjunctive chemotherapeutic agents [8]. Recently, there has been receiving increasing attention from applications of hyaluronic acid (HA) in the field of dentistry [9]. It has been shown that, HA has a great potential of being applied as the adjunctive material in periodontal treatment [10,11].

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5 connective tissue, the umbilical cord synovial fluid, embryonic mesenchyme, vitreous body of the eye, heart valves, skin, and most organs and tissues including periodontium [12,13]. Many studies demonstrate various properties of HA. It presents viscoelastic properties reducing the penetration of viruses and bacteria into the tissue [14]. High concentration of the medium molecular weight HA has the greatest bacteriostatic effect [15]. Strikingly, HA plays a key role in inflammatory moderation [16]. Moreover, HA shares bone induction characteristics with osteogenic substances. Besides it has biocompatibility and non-antigenicity properties [10]. These properties allow HA to become an ideal material of wound healing and tissue regeneration.

HA plays a key role in inflammation moderation, granulation tissue formation, epithelization, and tissue remodeling. It has been shown that HA has a positive effect on the acceleration of periodontal wound healing (i.e., the use in gingival graft surgery) [17–19]. Because of the anti-inflammatory and bacteriostatic properties of HA, it was suggested to support tissue regeneration such as bone regeneration in periodontal bone defect and interpapillary reconstruction of either teeth or implant-support crowns [20–22]. Furthermore, HA improves periodontal parameters during non-surgical periodontal treatment such as used in combination with ultrasonic scaling and root planing [23]. This study aims to estimate the current evidence evaluating the potential clinical benefits of hyaluronic acid in periodontal therapy (including non-surgical, surgical treatment of periodontitis, and periodontal plastic surgery).

2 Materials and Methods

The report of this systematic analysis adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [24].

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6 2.1 Objectives

The aims of the current systematic review are as follows:

1. To evaluate the effect of adjunctive application of HA on clinical parameters in non-surgical periodontal therapy;

2. To evaluate the effect of adjunctive application of HA on clinical parameters in surgical periodontal therapy;

3. To evaluate the effect of adjunctive application of HA on clinical parameters in periodontal plastic surgery.

2.2 The PICO (Population, Intervention, Comparison, Outcome) study design

Research question addressing the research objectives are stated as follows:

Population: systemically healthy individuals, diagnosed with periodontitis, and/or with mucogingival defects.

Intervention (test group): surgical or non-surgical periodontal therapy or periodontal plastic surgery with the application of HA.

Comparison (control group): surgical or non-surgical periodontal therapy or periodontal plastic surgery.

Outcome: the primary outcome variable was the probing depths (PD) reduction (mm); secondary outcome variables included clinical attachment level (CAL) gain (mm) and/or bleeding on probing (BOP) reduction (%) and/or gingival recession (GR) reduction (mm).

2.3 Information sources

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7 following journals: International Journal of Periodontics and Restorative Dentistry, Journal of

Clinical Periodontology, Journal of Periodontology, and Journal of Periodontal Research.

2.4 Search

The keywords used for the relevant articles search in the selected databases were: (“periodontitis” OR “periodontitis”[MeSH Terms] OR “periodontal disease” OR “periodontal disease” [MeSH Terms] OR “periodontal treatment” OR “periodontal treatment”[MeSH Terms] OR “periodontal therapy” OR “periodontal therapy”[MeSH Terms] OR “periodontal surgical treatment” OR “periodontal surgical treatment”[MeSH Terms] OR “periodontal surgical therapy” OR “periodontal surgical therapy”[MeSH Terms] OR “periodontal plastic surgery” OR “periodontal plastic surgery” [MeSH Terms] OR “gingival recession” OR “gingival recession”[MeSH Terms] OR “ infrabony defects”* OR “periodontal tissue regeneration” OR “periodontal tissue regeneration”[MeSH Terms]) AND (“hyaluron”* OR “hyaluronic” OR “hyaluronic”[MeSH Terms] OR “hyaluronan” OR “hyaluronan”[MeSH Terms] OR “hyaluronic acid” OR “hyaluronic acid”[MeSH Terms] OR “sodium hyaluronate” OR “sodium hyaluronate”[MeSH Terms]).

2.5 Inclusion and exclusion criteria

Inclusion criteria:

1. Randomized controlled trials (RCTs) (parallel or split mouth); 2. Studies including systemically healthy patients;

3. Studies reporting on the application of HA as adjunctive to surgical, non-surgical periodontal therapy and periodontal plastic surgery;

4. Studies reporting either on PD (mm), CAL (mm), BOP (%) or GR (mm) as outcomes; 5. Follow-up period ≥3 months after initial therapy;

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8 Exclusion criteria:

1. Animal studies, laboratory studies;

2. Studies including individuals with systemic diseases that could influence the outcomes of periodontal therapy;

3. Studies not reporting clinical outcomes. 2.6 Sequential search strategy

The titles and abstracts of studies of possible relevance were obtained and screened. Studies without abstracts however with titles suggesting relevance to the subject of the review were selected for full-text screening. At the final stage, the full full-texts were screened based on the inclusion criteria to confirm the eligibility of each study. Studies that fulfilled all the selection criteria were processed for data extraction.

2.7 Data collection and data items

The data was extracted to previously defined templates according to the aims of the current review. For non-surgical and surgical periodontal therapy, the date was extracted as follows:

“Study”—revealed author and year of publication; “Study design”—indicated the type of study;

“Periodontitis”—specified clinical parameters according to which periodontitis diagnosis was confirmed and clinical examination criteria of included patients;

“Patients/sites (control/test)”—revealed the number of patients and sites treated in test and control groups;

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9 “Follow-up”—revealed the time in months in which the test and control group patients were followed;

“Treatment outcomes”—revealed clinical parameters and their values at baseline and after the follow-up period.

For periodontal plastic surgery, the data was extracted as follows: “Study”—revealed author and year of publication;

“Study design”—indicated the type of study;

“Defect”—the characteristic of clinical examination criteria of included patients;

“Patients/sites (control/test)”—revealed the number of patients and sites treated in test and control groups;

“Intervention”—treatment protocols in the test and control groups;

“Follow-up”—revealed the time in months in which the test and control group patients were followed;

“Treatment outcomes”— revealed clinical parameters and their values, at baseline and after the follow-up period.

2.8 Risk of bias assessment

The quality of all included studies was assessed during the data extraction process and involved evaluation of the methodological elements that could influence the outcome of each study. The Cochrane risk of bias tool [25] was used to assess the methodology of the included trials. The following criteria were included:

1. Random sequence generation (selection bias);

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10 3. Blinding of participants and the investigator (performance bias);

4. Blinding of outcome assessments (detection bias);

5. Incomplete outcome data (attrition bias);

6. Selective reporting (reporting bias);

7. Other bias (examiner calibration, standardized probing force, and standardized radiographic assessment).

The potential risk of bias was categorized as “low,” “unclear,” or “high” depending on the quality and detailed explanation of provided information about all above mentioned parameters. Each relevant domain per trail was judged either as low risk (if all criteria were met), moderate risk (only one criterion was missing), or high risk (two or more criteria were missing).

2.9 Data analysis

To summarize and compare studies, meta-analysis was used. Treatment outcomes of non-surgical therapy used in meta-analysis were PD reduction, CAL gain, and BOP reduction from baseline to 3 months, and from baseline to 6 months after treatment. Treatment outcomes of surgical therapy used in meta-analysis were PD reduction and CAL gain from baseline to up to 12 months, and from baseline to up to 24 months after treatment. Treatment outcomes of plastic surgery used in meta-analysis were PD reduction and GR reduction from baseline to at least 12 months after treatment. The alternative research hypothesis of this study, i.e.: there are differences in the treatment outcomes

between the test groups (with HA) and the control groups (without HA).

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11

A forest plot was created to present results with weighted means and 95% CIs. Heterogeneity across studies was evaluated using I2 statistic (I2 ≥ 50% denoting substantial heterogeneity). STATA 16 (StataCorp LP, Lakeway Drive, College Station, Texas, USA) software was used to perform all

analyses. Statistical significance was defined as a p-value <0.05.

3 Results

3.1 Search results

A total of 543 studies were identified in selected databases. After the elimination of duplicates, 368 studies could be assessed. 355 studies had to be excluded in the process of titles and abstracts reading. Those studies were case reports, systematic reviews, animal studies, in vitro studies, in vivo studies, and written in a language other than English. 13 full-text publications were further assessed for eligibility. After full eligibility assessment, 3 studies were excluded [26–28] (Table 1).

Table 1. Excluded studies and reasons for exclusion

Study Reason for exclusion

Bender (2005) [26] HA combined with bone matrix material

De Santana (2015) [27] HA combined with recombinant human

Fibroblast Growth Factor type-2

Prato (2003) [28] case series

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12 Fig. 1. Flow chart of the screened relevant publications

Non-surgical n=6 Surgical n=2 Inc lud e d

Records identified in selected database

n =543

Titles and abstracts are screened n =368

Irrelevant titles and abstracts; reviews and case reports are removed

n =355

Full-text articles assessed for eligibility

n =13

3 studies were excluded n =3

Studies included for meta-analysis

n = 10

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13 3.2 Results of quality assessment

Ratings of 2 studies [35,37] were categorized as “low risk”, another 2 studies [33,36] were categorized as “moderate risk”, and 6 studies [29–32,34,38] demonstrate a high risk of bias. (Table 2)

Table 2. Results of quality assessment

Study Random sequence generation Allocation concealment Blinding of participants and the investigator Blinding of outcome assessments Incomplete outcome data Selective outcome reporting Other bias General risk assessment Al-Shammari (2018) [30] + + - - + + ? High risk Briguglio (2013) [35] + + + + + + + Low risk

Eick (2013) [31] + + - ? - + ? High risk

Engström (2001) [29]

+ + - ? - + ? High risk

Fawzy (2012) [36] + + + + + + ? Moderate

risk

Kumar (2014) [38] + + - ? + + ? High risk

Lobato (2019) [33] + + ? + + + + Moderate

risk

Pilloni (2019) [37] + + + + + + + Low risk

Rajan (2014) [32] + + ? ? + + ? High risk

Xu (2004) [34] + + - - + + ? High risk

“+”: low risk; “?”: unclear risk; “-”: high risk

3.3 Characteristics of included studies 3.3.1 Non-surgical therapy

Six RCTs have been evaluated for the effectiveness of HA, in non-surgical treatment on patients with periodontitis which are outlined in Table 3 [29–34]. One study was of a parallel design [31], while the remaining studies were of a split-mouth design [29,30,32–34].

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14 follow oral hygiene instructions for at least a week after treatment [30-34]. In one study, patients were instructed to rinse with 0.12% chlorhexidine, twice daily for a week [31].

The mode of application of adjunctive HA and time intervals differed among the explored research. In one study, patients received HA gel one time [33], twice in two studies [30,32] and three times in one study [29] during and post treatment procedure in a week. One of the studies concluded, HA gel had been applied seven times in 6 weeks [34], another study of HA was applied twice a day for 2 weeks [31]. Two types of HA-containing products were used in the included studies: 0.2% HA in two studies [32,34], 0.8% HA in another two [30,33], whereas both 0.2% and 0.8% HA were applied in one study [31] and one study did not report the content of HA that had been used [29].

With regards to the follow-up period of the investigations, four studies had a follow-up period of 3 months [30,32–34], one study had a up period of 6 months [31], and one study had a follow-up period of 12 months [29].

Table 3. Characteristics of individuals on HA as an adjunct to non-surgical periodontal therapy

Study Study design Periodontitis Patients/sites (test/control)

Intervention Follow up Treatment outcomes

Al-Shammari (2018) [30]

Split mouth At least 4 sites with PD >5 mm, having at least 20 teeth 22/44（22/22） C: SRP T: SRP+HA(x2) 1.5, 3 months At baseline PD0 (mm): C:4.85967a, T:4.92046a CAL0 (mm): C:3.34a, T:3.78a After 3 months PD1 (mm): C:3.5a, T:3.05255a CAL1 (mm): C:2.69a, T:2.365a Eick. (2013) [31]

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15 C: 1.34 ± 0.57b, T: 1.24 ± 0.58b ΔBOP0-2 (%) C: -5.18 ±19.33b, T: -7.46 ± 24.73b Engström (2001) [29] Spilt mouth PD≥5mm 9/18(9/9) C: SRP T: SRP + HA(x3) 1,3,6,12 months At baseline PD0 (mm): C:6.8±1.5b, T:6.4±1.3b After 6 months PD1 (mm): C:4.2±1.4b, T:3.9±1.2b After 12 months PD2 (mm): C:3.7±1.5b, T:3.9±1.4b Rajan (2014) [32]

Split mouth At least 5 sites with PD ≥5mm, having at least 20 teeth 33/66(33/33) C: SRP T: SRP+HA（x2） 1,3 months At baseline PD0(mm): C: 6.09±1.26b, T: 6.33±0.99b CAL0 (mm): C: 9.12±1.67b, T: 10.18±2.08b After 3 months PD1 (mm) C: 4.36±1.29b, T: 2.49±0.51b CAL1 (mm): C: 7.48±1.51b, T: 6.91±1.16b Lobato (2019) [33]

Split mouth The criteria for diagnosis of periodontitis followed by Eke et al [39] 16/32(16/16) C: SRP T: SRP+HA 1.5, 3 months At baseline: PD0 (mm)： C:3.9±0.6b, T: 3.8±0.6b CAL0(mm) ： C: 4.8±1.2b, T: 4.9±1.1b BOP(%)： C: 33.2±19.5b, T: 32.1±17.3b After 3 months: PD1 (mm)： C: 3.3±0.6b, T: 3.2±0.5b CAL1 (mm)： C: 4.3±1.0b, T: 4.2±1.0b BOP1 (%)： C: 14.9±8.9b, T:9.4 ±4.0b Xu (2004) [34]

Split mouth At least 2 sites in each quadrant with ≥5 mm, having at least 20 teeth 20/40(20/20) C: SRP T: SRP+HA(x7) 1.5, 3 months At baseline PD0 (mm)： C:5.2 ± 1.62b, T: 5.3 ± 1.61b CAL0 (mm)： C: 5.4 ± 1.97b, T: 5.5 ±.1.79b BOP0 (%)： C: 72b, T: 78b After 3 months PD1 (mm)： C: 4.2 ± 1.57b, T: 4.3 ± 1.46b CAL1 (mm)： C: 4.5 ± 1.90b, T: 4.5 ± 1.68b BOP1 (%)： C: 23b, T:19b

SRP: scaling and root planing; HA: hyaluronic acid gel; PD: probing depth; CAL: clinical attachment level; BOP: bleeding on probing; C: control group; T: test group; ΔPD: changes of probing depth; ΔCAL: changes of clinical attachment level; ΔBOP: changes of bleeding

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16 3.3.2 Surgical therapy

Two RCTs have been conducted to assess the effectiveness of HA in surgical treatment on patients with periodontitis with an infrabony defect, which are outlined in Table 4 [35,36]. One study performed a parallel study design [35], while another one performed a split-mouth design [36]. The included studies involved 54 patients, in which all of the patients completed the trial. In one study, the subgingival debridement was accomplished by ultrasonic and Gracey curettes [35]. In another study, subgingival debridement was solely performed by Gracey curettes and post-operative rinsing with 0.12% chlorhexidine was instructed to patients [36]. Patients in both studies were instructed to follow oral hygiene protocol.

With regards of the technique of application of HA and time interval, HA was applied a single time with surgical procedures for both studies. 0.8% HA was used in one study [36], the other study did not report the HA content [35].

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17 3.3.3 Periodontal plastic surgery

Two studies have evaluated the effectiveness of HA in periodontal plastic surgery on patients with Miller class I gingival recession which are outlined in Table 5 [37,38]. One investigation performed a parallel study design [37], and the other investigation performed a split-mouth design [38].

Table 4. Characteristics of individuals with HA to surgical therapy

Study Study design

Periodontitis Patients/sites (control/test)

Briguglio (2013) [35] Parallel group PD≥7mm, CAL≥7mm, infrabony defect ≥5mm 40/40(20/20) C: OFD T: OFD+HA 12,24 months Baseline: PD0 (mm)： C:8.0±0.7a, T:8.6±1.5a CAL0 (mm)： C:8.3±1.2a, T:7.2±1.5a After 12 months PD1 (mm)： C:7.1±1.3a, T:7.4±0.6a CAL1 (mm): C:6.9±1.8a, T:6.5±0.9a After 24 months PD2 (mm): C:7.2±0.5a, T:7.0±1.2a CAL2 (mm): C:7.2±0.7a, T:5.3±1.8a Fawzy El- Sayed (2012) [36] Split mouth PD >5 mm infrabony defect ≥3mm 14/28(14/14) C: OFD T: OFD + HA 3,6months Baseline: PD0 (mm): C:5.00(5.00/6.00)b, T:5.00(5.00/6.00)b CAL0 (mm): C:5.50(5.00/8.00)b T:5.50(2.00/7.00)b After 3 months PD1 (mm): C:3.00(1.75/3.00)b T:2.50(1.73/3.00)b CAL1 (mm): C:3.50(3.00/5.00)b T:3.00(1.75/3.50)b After 6 months PD2 (mm): C:3.00(2.00/4.00)b T:2.00(1.00/3.00)b CAL2 (mm): C:3.00(2.00/5.00)b T:2.00(1.00/3.50)b

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18 The included studies involved 40 patients, in which all of the patients completed the trial. In both of the two studies, subgingival debridement was accomplished by Gracey curettes. Post-operative rinsing with 0.12% chlorhexidine was instructed to patients of both studies.

With regards of the technique of application of HA and time interval contrasted among the studies, HA was applied a single time with the treatment procedure for both studies. 0.2% HA was used in one study [38], while a cross-linking high concentrated HA gel was used in the other study [37]. Regarding the follow-up period one study had a follow up period of 6 months [38], the other presented 18 months follow-up period [37]

Table 5. Characteristics of individuals with HA to periodontal plastic surgery

Study Study design Defect Patients/sites (control/test)

Pilloni (2019) [37]

Parallel group Gingival recession(depth≥2 mm), Miller class I, PD <4mm 30/30(15/15) C: CAF T: CAF +HA 18 months At baseline PD0 (mm): C:1.0[0.0]a, T:1.0[0.0]a CAL0: C:4.0[1.0]a, T:4.0[1.0]a GR0(mm): C:3.0[1.0]a, T:3.0[1.0]a At 18 months PD1: C:2.0[1.0]a, T:1.0[1.0]a CAL1: C:2.0[0.0]a, T:1.0[0.0]a GR1(mm): C:1.0[1.0]a, T: 0.0[0.0]a Kumar (2014) [38]

Split mouth Gingival recession, Miller class I 10/20(10/10) C: CAF T: CAF +HA 3, 6 months At baseline PD0 (mm): C: 2.00±0.47b, T:1.80±0.42b GR0(mm): C:2.90±0.73b, T:3.20±0.78b At 3 months PD1(mm): C: 1.80±0.91b, T:1.60±0.69b GR1(mm): C:0.90±0.73b, T: 0.90±0.87b At 6 months PD2(mm): C: 2.00±0.81b, T:1.70±0.82b GR1(mm): C:1.00±0.66b, T: 1.10±0.99b

PD: probing depth; CAL: clinical attachment level; HA: hyaluronic acid gel; CAF: coronally advanced flap; GR: gingival recession; C:

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19 3.4 Results of meta-analyses for non-surgical therapy

3.4.1 PD reduction

Six studies [29–34] reported data on PD reduction for sites treated with scaling and root planing either with or without the adjuvant use of HA. WMD of the included studies was -0.67 mm (95% CI −1.20 to -0.14 mm; p<0.05). Therefore, the PD reduction was significantly enhanced by applying HA. However, considerable heterogeneity was identified among studies (chi-squared test p<0.05). (Fig. 2)

Fig. 2. Forest plots for PD reduction following non-surgical therapy

3.4.2 CAL gain

Four studies [31–34] reported data on CAL gain for sites treated with HA as test groups versus without HA as control groups. WMD was 0.39 mm (95% CI -0.89 to 0.10 mm; p>0.05). Considerable heterogeneity was identified among studies (chi-squared test p<0.05). (Fig. 3)

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20 Fig. 3. Forest plots for CAL gain following non-surgical therapy

3.4.3 BOP reduction

Three studies [31,33,34] reported data on BOP reduction. WMD was -22% (95% CI -65 to 22%; p>0.05). No significant difference was observed between the control and test groups. A low heterogeneity among studies was observed (chi-squared test p=0.97). (Fig. 4)

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21 Fig. 4. Forest plots for BOP reduction following non-surgical therapy

3.5 Results of meta-analyses for surgical therapy 3.5.1 PD reduction

Two studies [35,36] reported data on PD reduction for sites treated with HA as test groups versus without HA as control groups. WMD was -0.61 mm (95% CI −0.96 to -0.27 mm; p<0.05). Therefore, PD reduction was significantly enhanced in the application of HA. A low heterogeneity among studies was observed (chi-squared test p=0.62). (Fig. 5)

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22 Fig. 5. Forest plots for PD reduction following surgical therapy

3.5.2 CAL gain

Two studies reported data on CAL gain for sites treated with HA as test group versus without HA as control groups [35,36]. WMD was -0.16 mm (95% CI −0.67 to 0.34 mm; p>0.05). There is no significant difference between test and control groups. Furthermore, the comparison presented low heterogeneity among the two studies (chi-squared test p=0.08). (Fig. 6)

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23 Fig. 6. Forest plots for CAL gain following surgical therapy

3.6 Results of meta-analyses for periodontal plastic surgery 3.6.1 PD reduction

Two studies [37,38] reported data on PD reduction for sites treated with HA as test groups versus without HA as control groups for up to 18 months. WMD was -0.94 mm (95% CI −2.57 to 0.70 mm; p>0.05). There was no significant difference between control and test groups. Despite that, there is considerable heterogeneity among the two studies (chi-squared test p<0.05). (Fig.7)

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24 Fig. 7. Forest plots for PD reduction following plastic surgery

3.6.2 CAL gain

There was only one study [37] reported data of CAL gain in the included studies. Therefore, the meta-analysis was not considered for CAL gain in plastic surgery.

3.6.3 GR reduction

Two studies [37,38] reported data on GR reduction for sites treated with HA as test groups versus without HA as control groups up to 18 months. WMD was -0.56mm (95% CI −1.11 to -0.01mm; p<0.05). The GR reduction is significantly higher in treatment with HA compare to without. Low heterogeneity was observed among studies (chi-squared test p=0.07). (Fig. 8)

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25 Fig. 8. Forest plots for GR reduction following plastic surgery

4 Discussion:

Most recently, the hyaluronic acid (HA) application has been recognized as an adjuvant in several invasive or non-invasive procedures in dental and oral health treatment. The present systematic review is composed of a meta-analysis, which was aimed to evaluate the potential effect of local application of HA to periodontal treatment, including non-surgical, surgical, and plastic surgery. Four clinical parameters (PD reduction, CAL gain, BOP reduction, and GR reduction) were used to evaluate the effect of HA as an adjuvant in each treatment.

The meta-analysis revealed that non-surgical treatment with adjunctive HA resulted in additional PD reduction (WMD -0.79mm; p<0.05) and CAL gain (WMD 0.53mm; p<0.05) compared with conventional scaling and root planing after 3 months. However, after 6 months the data of CAL gain does not indicate any significant difference between the test and control groups, which can be due to the limitation of data availability post-treatment during the 6-month period. The recently published studies have observed that HA has additional benefits as an adjuvant in CAL gain and PD reduction undergoing periodontal treatment in the short term, which is consistent with the findings of the present study [40,41].

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26 The results from the meta-analysis on surgical treatment have shown that after 3-24 months, the PD reduction (WMD -0.61mm; p<0.05) was significantly enhanced in the treatment with the application of HA. However, the effect of HA in CAL gain (WMD 0.16mm; p>0.05) showed a slight effect, but no statistical difference compared to groups without HA application. It can be due to the limited number of included RCTs and the various follow-up periods. Also, the heterogeneity among the studies may be attributed to differences in the treatment protocol.

It is important to note that when HA combined with other biomaterials, it also manifests additional effects on patients with infrabony defect. Such as HA combines with rhFGF-2(recombinant human Fibroblast growth type 2) or with a membrane showing significantly more in PD reduction, CAL gain, and increase of bone height [27,29].

In periodontal plastic surgery, the meta-analysis revealed that the adjunctive application of HA yielded the significant improvement of recession coverage (WMD -0.56mm; p<0.05). Interestingly, the follow-up period after initial treatment among the two studies of plastic surgery varied from 3 to 18 months. It explains a high heterogeneity between the studies. The study of Pilloni et al. [37] demonstrated PD and GR reduction were significantly higher in using of HA at 18 months. While the results of the study of Kumar et al. [38] with 3 months follow up showed the effect of HA was not significant. It is well established that, a post-operation tissue regeneration requires a longer timeframe to proceed, in other words, 3 months is insufficient for recovery. Moreover, the risk of bias assessment indicated a lower risk for the study of Pilloni et al. [37] compared to the study Kumar et al. [38]. Therefore, the final results of plastic surgery are more biased toward the findings of Pilloni et.al [37] that the application of HA has additional benefits in both reduction of PD and GR.

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27 (WMD -0.89mm; p<0.05) after 6-24 months in surgical treatment [42]. The results are similar to our findings that CAL gain (WMD 0.53mm; p<0.05) and PD reduction(WMD -0.79mm; p<0.05) enhanced remarkedly after 3 months of non-surgical treatment by using of HA, and PD reduction (WMD -0.61mm; p<0.05) also enhanced notably after up to 24 months in surgical treatment by using of HA.

In surgical treatment, the present review and Eliezer et al. [42] share common aims objective issues: 2 RCTs [35,36] are the same. However, the result of CAL gain of Eliezer et al. (WMD 0.85mm; p<0.05) is different compared to the present study (WMD 0.22mm; p>0.05) [42]. There exists a difference in results, which could conceivably be due to different data extraction methods. Eliezer et al. [42] extraction of data was done at the last months of visitation from both RCTs (at 6 months in one study [36], and 24 months in the other study [35]), whereas the present study extracted the data from each follow-up visits (at 3 and 6 months in one study [36], and 12 and 24 months in the other study [35]) from both RCTs. However, due to limited available data, a conclusion on the reliability of the analysis results cannot be drawn.

Another systematic review Bertl et al. also evaluated the benefits of locally delivered HA on non-surgical and non-surgical periodontal treatment [43]. According to Bertl et al., there are 4 RCTs [31,34-36] coincided with the present study. Bertl et al. indicates that the treatment favored HA as an adjunctive, in terms of PD and BOP reduction in both surgical and non-surgical treatment [43]. However due to the large heterogeneity, the systematic review Bertl et al. was not able to perform a meta-analysis [43].

As stated by the present study and the systematic reviews mentioned above, PD reduction was remarkedly enhanced by local application of HA, despite being in non-surgical or surgical treatment. Notably, the results of HA effect in non-surgical treatment in this study, may be explained by an

in-vitro and clinical studies, which indicated that 0.8% HA has effects of antiplaque, such as reducing P. gingivalis, and anti-inflammatory in patients exhibiting biofilm-induced periodontal diseases

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28 dental implantation, and in peri-implantitis treatments [46–49].

In molecular biology, HA is a key element in both mineralized and non-mineralized periodontal tissues (alveolar bone, cementum, periodontal ligament). HA was demonstrated in maintaining the stemness of mesenchymal stromal and pre-osteoblastic cells by regulating the BMP/Smad pathway [50].

This systematic review and meta-analysis meet rigorous inclusion/exclusion criteria. However, there still exist limitations in the present systematic review. The main limitation was the small number of included studies in surgical treatment and plastic surgery. Besides, short follow-up periods were reported in most of the studies of non-surgical treatment. Furthermore, the percentage content of HA of HA-containing products, study design (split-mouth/parallel), application frequency of HA (from 1 to 29 times), and follow-up period (from 3 to 24 months) varied among the included studies. Only 2 out of 10 studies had a low risk of bias, which included a relatively small number of patients. Other studies were evaluated as having a moderate (n=2) or high (n=6) risk of bias. These aspects are important for detecting the methodological weaknesses of the included studies that might alter therapy outcomes. According to the results of a bias risk assessment, allocation concealment and the blinding of participants and personnel appeared to be the most critical domains.

5 Conclusion:

Within the limitations of this systematic review, the present data suggests that the adjunctive use of HA may lead to additional clinical benefits in non-surgical, surgical periodontal treatment, and periodontal plastic surgery. Furthermore, future studies should be based on adequate methodological procedures to improve the overall quality of the reporting and to reduce the risk of bias.

6 Clinical recommendations:

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29

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The Adjunctive Use of Hyaluronic Acid in Periodontal Therapy: A Systematic Review

Xinni Lu

The Adjunctive Use of Hyaluronic Acid in Periodontal Therapy:

A Systematic Review

FINAL MASTER‘S THESIS IS CONDUCTED

AT THE DEPARTMENT OF DENTAL AND ORAL PATHOLOGY

STATEMENT OF THESIS ORIGINALITY

CONCLUSION OF FINAL MASTER‘S THESIS ACADEMIC SUPERVISOR

ON THE DEFENSE OF THE THESIS

FINAL MASTER‘S THESIS IS APPROVED AT THE DEPARTMENT

Final Master‘s Thesis reviewer

Evaluation of Final Master‘s Thesis Defense Board:

TABLE OF CONTENTS

Abstract

K

:

A

:

1 Introduction

2 Materials and Methods

3 Results

4 Discussion:

5 Conclusion:

6 Clinical recommendations:

R

: