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: Purpose: There is no consensus on the clinical and radiological benefits of hyaluronic acid (HA) in patients with periodontitis having infrabony defects. Hence, this study examined the effects of HA in periodontitis patients with infrabony defects through a comprehensive systematic review process. Methodology: A systematic literature search was performed using PubMed/Medline, Scopus, Embase and Cochrane Library from inception to March 2022. Randomized or non-randomized clinical trials and single-arm clinical trials that assessed the clinical and radiological benefits of HA in periodontitis patients with infrabony defects with a minimum of 6 months follow-up were included in this study. Surgical regenerative therapy was considered as the comparator. The Cochrane risk of bias assessment tool and Downs and Black checklist was used for the quality assessment of randomized and non-randomized interventional studies, respectively. A subgroup and sensitivity analyses were performed to explore the heterogeneity and robustness of the findings, respectively. Results


INTRODUCTION
Hyaluronic acid (HA) is one of the promising agents in the management of periodontitis in view of its biocompatibility, biodegradability, antimicrobial and wound healing properties. HA is a bio-molecule observed in various parts of the human body. It can be found in the gingiva, periodontal ligaments, cementum, alveolar bones, and in unstimulated saliva. It is a major constituent of the extracellular matrix and have a substantial role in cell migration and proliferation [1]. Ulti-controlled clinical trials (RCTs) with adequate sample sizes are needed to confirm these promising results. Additionally, the available primary research provides conflicting conclusions regarding the effects of HA in periodontitis patients with infrabony defects [3, 5 -7].
The systematic review conducted by Eliezer et al. [8] with 13 studies indicated a beneficial effect of HA with respect to the CAL gain and PPD reduction following the non-surgical and surgical management of periodontitis. However, high heterogeneity and risk of bias were the limitations of the studies. Another meta-analysis by Onisor et al. [9] from a limited number of studies recorded a significant increase in CAL gain and non-significant PPD reduction with adjuvant HA administration compared to the open-flap debridement.
To date, there are no meta-analyses known to the authors that assessed the radiological effects of HA in patients with infrabony defects following their surgical management. In view of these conflicts and limitations in the available evidence, we aimed to assess the clinical and radiological effects of the HA administered alone or in combination for the management of infrabony defects following the surgical intervention through a comprehensive systematic review and meta-analysis process.

MATERIALS AND METHODS
We followed a PICOS framework (Population, Intervention, Comparator, Outcome, and Study Design) for the inclusion of relevant studies and adapted the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guidelines [10] to report this systematic review.

Study Designs
Randomized or non-randomized clinical trials and singlearm clinical trials with pre-post analysis having a minimum of 6 months follow-up were considered in this review. Only the studies with full text available in the English language were considered. The observational studies, reviews, descriptive studies, commentary, guidelines, and qualitative analyses were excluded.

Participants
Adult patients with generalized periodontitis with infrabony defect who needed regenerative therapy irrespective of their gender were considered in this review. Studies involving the paediatric population were excluded.

Intervention
Application of HA or its derivative in different forms (like gel or mixed with other agents) or in different doses irrespective of the method of administration (alone or as an adjuvant therapy to surgical regenerative therapy) was considered. Studies that used HA along with non-surgical methods such as subgingival instrumentation or scaling and root planning were excluded.

Comparator
Surgical regenerative periodontal therapy without HA was considered as the comparator in the case of controlled trials. The pre-post analyses were considered in the case of singlearm studies.

Outcomes
Both clinical and radiological parameters were considered as outcomes which include i) bleeding on probing; ii) PPD reduction; iii) gain in CAL; iv) plaque accumulation/plaque index; v) Gain in bone probing depth from the cementoenamel junction (CEJ); vi) bone defect reduction/fill from CEJ; vii) changes in alveolar crest level; vii) reduction in marginal gingival level; viii) bone fill; ix) gingival recession; x) recession depth; and xi) bone crest depth.

Search Strategy
The databases such as PubMed/Medline, Scopus Embase, and Cochrane Library were accessed in March 2022 through a comprehensive search strategy. The strategy was prepared using all the possible keywords and entry terms for "Hyaluronic Acid" AND "Periodontitis". We also did a snowball search in Google, Google Scholar, and ResearchGate to identify any relevant articles. Additionally, the Clinical Trial registry (https://clinicaltrials.gov/) and reference lists of relevant articles were also screened to identify additional relevant citations. The search strategy was prepared by the author with the support of the librarian. No date limit was used, and the search was limited to the English language. A detailed search strategy in various databases is provided in Supplementary file S1.

Study Selection
All articles identified following the database search were retrieved to an Excel sheet and screened against the pre-defined criteria. The studies were screened by reading its title and abstracts initially and followed by reviewing the full text of those included. Only those studies that passed both stages were considered for final inclusion in the review. Two independent reviewers were involved in the study selection, and any disagreements were resolved through discussion or consultation with another reviewer.

Data Extraction
The data were abstracted to a validated, comprehensive data extraction form. The author's first name and year of publication were used to identify the studies. The data regarding the publication, study settings, participants, intervention, and outcomes were captured from the studies or calculated from the available data. The above data extraction form was finalized by trial and error by piloting on the first 2 articles.

Risk of Bias and Quality Assessment
The Cochrane Risk of Bias Assessment Tool was used to assess the methodological quality of included RCTs [11], and The Downs and Black checklist was used to assess the methodological quality of non-randomized clinical trials and single-arm studies [12]. The RCTs were graded as high, moderate, and low risk of bias based on the assessment. The single-arm studies were graded as excellent (26-28), good (20-25), fair (15-19) and poor (≤14) based on the score achieved.

Evidence Synthesis and Meta-Analysis
All the evidence extracted through the systematic process was summarized narratively and presented in tabular form. RevMan 5.4 was used to conduct the meta-analysis [13] wherever possible. The data were used as the difference in mean with standard deviation (SD), and outcomes were presented as standardised mean difference (SMD) along with a 95% confidence interval (CI). In studies that did not report an SD, the corresponding SD was calculated as per Cochrane guidelines [14]. If the same study reports the endpoints in different follow-ups, then the longest follow-up was considered for meta-analysis. Only studies that provided comparative data between the intervention and control group were considered for the meta-analysis, and the remaining evidence was presented narratively. The I 2 statistics were used to estimate the heterogeneity in the analysis. We used the random effect model in case of substantial heterogeneity (I 2 >50%; P<0.10) and the fixed effect model in case of low heterogeneity (I 2 <50%; P>0.10). To explore the sources of heterogeneity, subgroup analysis was carried out based on the length of follow-up, wherever possible [15].

Publication Bias and sensitivity analysis
Assessment of evidence for publication bias through visual analysis of funnel plots or statistical methods was not performed, as less than 10 studies were used for the metaanalysis. Cochrane and previous literature recommend not performing funnel plot analysis in such cases [16 -18]. The sensitivity analysis was performed to check the robustness of the findings by removing the study with the lowest weight in each analysis, and results were provided [18].

Study Selection Process
A total of 838 studies were identified from the databases and 4 studies from the additional searches. Out of these, 725 studies were screened using their title and abstract after removing the duplicate. Overall, 630 studies were excluded for various reasons, while 95 studies were eligible for full-text screening. A total of 82 studies were excluded during this stage; 13 studies were considered for systematic review, and 9 studies were included in the meta-analysis. The reason for excluding 4 studies from the meta-analysis is presented in Table 1. A detailed study selection process is depicted in Fig. (1).

Characteristics of the Included Studies
The studies were published between the years 2001 to 2021, with a majority of studies from India (n=4) [5 -7, 19 -22], followed by Egypt (n=3) [5,21,22], Italy (n=3) [3,23,24], and one each from Croatia [25],Brazil [26], and Sweden [2]. Among the included studies, 11 were RCTs, and the remaining two [24,25] were single-arm studies. The duration of follow-up of the studies ranged from 6 months to 24 months. A detailed study and subject characteristics are presented in Table 2.

Characteristics of the Treatment and Outcomes
Strict inclusion and exclusion criteria were used by all the studies to include their participants. All studies included adult participants with periodontitis. HA was administered alone or in combination with other agents such as bioresorbable membrane [19], deproteinized porcine bone mineral xenograft [25], recombinant human Fibroblast Growth Factor type 2 [26], β-tricalcium phosphate [20], and bone allograft [22] following surgical procedure as an intervention. Various clinical and radiological outcomes were considered by the studies, as presented in Table 3.

Quality Assessment of Included Studies
Among the included RCTs, 45% (n=5) of the studies were observed to have a high risk of bias and low risk of bias (n=5, 45%), whereas only a single study [21] had a moderate risk of bias in terms of randomization. Allocation was concealed in 54% (n=6) of the studies, which is observed to have a low risk of bias in this domain, and the remaining studies did not report the allocation. Six (54%) of the studies were blinded (the study participants did not know the treatment they received), whereas the remaining five (46%) studies didn't report it. The risk of bias on outcome assessor blinding was high, moderate, and low in three (27%), five (46%) and three (27%) of the studies, respectively. The risk of bias with respect to the incomplete outcome data, selective reporting and other biases was observed to be low. The two single-arm interventional studies [24,25] were observed to have a fair quality, as both of them scored 17 out of 28. The quality assessment analysis is provided in Supplementary file S2.

Probing Pocket Depth
A meta-analysis of 9 studies with 296 participants indicated that treatment with HA significantly reduced PPD by 1.12 mm (95%CI: 0.60 to 1.65; p<0.0001; (Fig. 2A) compared to the control group in patients with infrabony defects. However, there was a significant level of heterogeneity (I 2 : 84%; 76%); hence random effect model was applied.

Hyaluronic Acid in Periodontal Infrabony Defects
The Open Dentistry Journal, 2023, Volume 17 11 Fig. (2). Effect of HA on reduction in probing pocket depth.
A single-arm study by Božić D et al. [25] recorded a significant reduction (p<0.001) of 4.54 ± 1.65 mm in PPD following 6-month treatment in 23 patients with 27 infrabony defects who were treated with a combination of HA and deproteinized porcine bone mineral following regenerative periodontal surgery. Another study by Vanden Bogaerde L et al.
[24] among 19 defects indicated a mean reduction of 5.8 mm in PPD following one-year treatment with HA in patients with infrabony defects. None of the studies recorded PPD reduction at 9 months. The study conducted by Pilloni A et al. [23] recorded a significantly (p= 0.001) better PPD reduction in the enamel matrix derivatives group (4.5±0.97 mm versus 3.31±0.70 mm) compared to the HA group. In contrast, a nonsignificant effect was reported by Mohamed et al. [21] with hyaluronan gel.

Gain in CLinical Attachment Level
A meta-analysis of 8 studies with 284 participants indicated that treatment with HA was significantly effective in a CAL gain of 1.40 mm (95%CI: 0.33 to 2.47; p<0.00001; Fig.  3A) compared to the control group in patients with infrabony defects. However, there was a significant level of heterogeneity (I 2 : 84%; 93%); hence random effect model was applied.

Plaque Accumulation Index
A meta-analysis of 2 studies with 66 participants recorded no significant difference between the HA-treated group and control group in terms of plaque accumulation index at 6 months (SMD: 0.00; 95%CI: -0.48 to 0.48; 2 studies; 66 participants; I 2 : 83%; Fig. 4) in patients with infrabony defects.

Bleeding on Probing
Pilloni et al. [23] assessed the effect of HA versus enamel matrix derivative following the single-flap in patients with infrabony defects. They recorded a non-significant change in 3A: Overall effect of HA on CAL gain 3B: Subgroup analysis on CAL gain BOP at 12, 18, and 24 months in the groups. Similarly, the study conducted by  recorded a nonsignificant benefit following the HA application in conjunction with periodontal surgery. The number of bleeding sites decreased over the treatment in a study reported by Engström et al. [2], whereas it increased as per the findings by Briguglio et al. [3].

Reduction in Bone Probing Depth
The meta-analysis of 2 studies with 92 participants revealed an additional reduction of 3.22mm (95%CI: -2.10 to 8.54) bone probing depth from CEJ at 12 months in the the HA group compared to the control group in patients with infrabony defects (Fig. 5).

Reduction in Bone Defect Depth
A meta-analysis of 3 studies with 102 participants revealed a significantly greater reduction of 1.04 mm (95%CI: 0.62 to 1.47) in bone defect depth from CEJ at 12 months in patients treated with HA compared to the control group in patients with infrabony defects (Fig. 6).

Changes in Alveolar Crest Level
A meta-analysis of 2 studies among 81 participants indicated a change of 0.42mm (95% CI: -0.03 to 0.87) in alveolar crest level in HA treated group compared to the control group following 12 months of treatment in patients with infrabony defects (Fig. 7).

Gingival Recession
A meta-analysis of 2 studies with 100 participants indicated no significant difference in gingival recession (SMD: -0.39 (95%CI: -1.19 to 0.42)) between the HA-treated group and control group following 12 months of treatment in patients with infrabony defects (Fig. 8).

Sensitivity Analysis
The sensitivity analyses were performed by removing the study with the lowest weight. The study by Engström et al. [2], with a weight of 8.6%, and de Santana RB et al. [26], with a weight of 11.2%, were removed in PPD reduction and CAL gain analysis, respectively. The results were robust, as there was no alteration in the overall effect. The result of sensitivity analyses is provided in Supplementary file S3 Fig. (4). Plaque accumulation index at 6 months.

DISCUSSION
HA is a natural polysaccharide of the extracellular matrix of connective tissue, synovial fluid, and other tissues. HA exhibits many biological functions, such as cellular and extracellular interactions, growth factors interaction, osmotic pressure homeostasis, and tissue lubrication. These properties helped HA to achieve wide therapeutic applications in various fields of cosmetics, medicine and pharmaceuticals [27].
HA has a wide variety of applications in many medical conditions, such as osteoarthritis as a dermal filler [28], ophthalmic, dermal, burns, wound repair, skin aging and other health conditions [29,30]. Recently, HA has become an attractive topic in the dental industry and has become a boon in periodontal therapy due to its anti-inflammatory and antibacterial effects [27].
This systematic review assessed the clinical and radiological effects of HA in periodontitis patients with infrabony defects. We included a total of 13 studies published from 2001 to 2021 from various parts of the world. Majorly, of the studies were from India, Egypt and Italy. The HA was used with various other treatment options following surgical management. The included RCTs were observed to have a low risk of bias, and single-arm interventional studies included in the review had fair quality.
Our meta-analysis indicated a significant PPD reduction of 1.12 mm in the HA-treated group compared to the control group. A similar significant reduction of PPD was observed in the subgroup analysis at 6 months (1.28 mm), 12 months (0.89 mm) and 24 months (0.85 mm) of therapy with HA compared to the control group in patients with infrabony defects. This was in line with the findings reported by Onsior et al. [9]. In which, they have reported a 1.11 mm PPD reduction in HA treated group compared to the open flap detriment. All the single-arm studies also recorded a significant PPD reduction following the HA treatment with surgical procedure, though not with reports by Mohammed et al. [21]. Similarly, another previous meta-analysis by Eliezer et al. [8] recorded a 0.89 mm PPD reduction after 6-24 months of treatment with HA compared to surgery.
There was a significantly greater CAL gain (1.40 mm) in the HA-treated group compared to the control group who received the surgical procedure in patients with infrabony defects. A similar CAL gain was observed in the subgroup analysis at 6 months (0.94mm), 12 months (1.52mm) and 24 months (0.57mm) of the management. Similar findings were recorded by the meta-analysis conducted by Onsior et al. [9] and Eliezer et al. [8], where they recorded a 1.38 mm and 0.85 mm CAL gain in the HA group compared to surgery, respectively.
The previous meta-analyses [8,9] did not quantify other efficacy parameters of HA, and here we tried to assess the clinical and radiological efficacy parameters of HA with the available data. Our meta-analysis indicated that the treatment with HA was significantly effective in reducing the bone defect depth in 12 months (Fig. 6) in patients with infrabony defects. Only 3 studies [6,7,22] recorded this outcome, and further clinical studies should analyze the effect of interventions on reducing bone defect depth as it is an important parameter to decide the efficacy of management. However, the treatment with HA was not effective in improving the plaque index or bone probing depth (Fig. 5), and alveolar crest level (Fig. 7) over surgical intervention. It should be noticed that all studies included patients with good oral hygiene and low plaque index in starting with, which could explain the lack of significant changes in plaque index after the intervention.
This study has several strengths. i) All the 9 included RCTs in the meta-analysis were observed to have a good methodological quality with a low risk of bias. Hence, the interpretation drawn from this meta-analysis can be adopted in clinical practice. ii) In this study, we tried to capture the maximum possible clinical and radiological benefits of HA in patients with infrabony defects. iii) Our sensitivity analysis by removing the studies with the lowest weight revealed the robustness of our findings by yielding a non-differing result from the original results. iv) A subgroup analysis with respect to the length of follow-up could help us to understand the effect of HA with respect to its treatment duration; however, future studies should report detailed subgroup data to strengthen these findings.
The restriction to the English language studies was a limitation in our meta-analysis. However, a comprehensive search in numerous databases might have helped us to collate the maximum available resources. There was a high level of heterogeneity in the analyses; hence caution should be taken while interpreting the results. Moreover, a subgroup analysis based on the length of follow-up showed variation in the heterogeneity; hence future studies should focus on other factors that can influence the outcomes. Additionally, only a few studies recorded the radiological benefits of the interventions, and future studies should also focus on these outcomes, as this is also an important parameter regarding the effectiveness of the treatment's success. Further studies are needed across different populations of the world to better characterize the treatment outcomes in all different settings with more focus on the radiological outcomes of HA.

CONCLUSION
The current evidence indicates that the administration of HA in the periodontal regenerative treatment of infrabony defects was significantly effective in increasing clinical attachment levels and reducing probing pocket and bone defect depth.

CONSENT FOR PUBLICATION
Not applicable.

STANDARDS OF REPORTING
PRISMA guidelines are followed.

FUNDING
No financial support was received for this manuscript.