Effectiveness of Hyaluronic Acid Application in Coronally Advanced Flap Combined With a Subepithelial Connective Tissue Graft Versus Coronally Advanced Flap Combined With a Subepithelial Connective Tissue Graft Alone: A Clinical Trial
Phase: NA
Conditions: Gingival Recession, Mucogingival Surgery
Sponsor: Universidade do Porto

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Official title: Effectiveness of hyaluronic acid application in Coronally Advanced Flap Combined with 
a subepithelial connective tissue graft Versus Coronally Advanced Flap Combined with a 
subepithelial connective tissue graft alone: A clinical trial 
Date: 02-03-2026 
Clinical trial registration: NCT to be assigned  
Document type: Study Protocol 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. INTRODUCTION AND RATIONALE  
 
1.1. Introduction 
Gingival recession (GR) is a frequent mucogingival deformity characterized by the apical 
displacement of the gingival margin with exposure of the root surface beyond the cemento-
enamel junction (CEJ) [1–3]. Its prevalence increases with age and may lead to dentin 
hypersensitivity, root caries, plaque retention, aesthetic impairment, and patient discomfort [4,5]. 
Among the available treatment modalities, the coronally advanced flap (CAF), particularly when 
combined with a subepithelial connective tissue graft (SCTG), is a well-documented and widely 
adopted approach for the management of single and multiple recession-type defects, providing 
predictable root coverage, gain of keratinized tissue, and long-term stability [6–8]. However, CAF 
+ SCTG procedures may still be associated with postoperative morbidity, donor-site discomfort, 
and incomplete root coverage in some cases [9,10]. Therefore, adjunctive strategies aimed at 
enhancing wound healing and increasing the probability of complete root coverage (CRC) remain 
of significant clinical interest. 
Hyaluronic acid (HA), a high-molecular-weight, non-sulfated glycosaminoglycan, is a key 
component of the periodontal extracellular matrix and plays a pivotal role in angiogenesis, cell 
migration, proliferation, and differentiation during tissue healing [11–13]. HA has been shown to 
accelerate granulation tissue formation, modulate inflammatory responses, and stimulate 
fibroblast and keratinocyte activity [14–16]. Preclinical studies demonstrated that HA enhances 
vascularization, supports osteoinduction, and upregulates osteogenic markers such as Runx2, 
alkaline phosphatase (ALP), and osteocalcin [17,18]. In vitro studies further confirm that HA 
promotes fibroblast proliferation, migration, and extracellular matrix synthesis [19,20], while 
cross-linked formulations prolong its bioactivity in surgical wounds [21]. Moreover, HA exhibits 
bacteriostatic 
properties 
against 
periodontopathogens 
such 
as Aggregatibacter 
actinomycetemcomitans and Porphyromonas 
gingivalis [22], 
potentially 
reducing 
early 
contamination of surgical sites. 
The adjunctive use of HA in periodontal regenerative and mucogingival surgery has been 
investigated in several clinical studies. Randomized controlled trials (RCTs) demonstrated 
significantly greater clinical attachment level (CAL) gain, mean root coverage (MRC), and CRC when 

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HA was used in combination with CAF compared to CAF alone [23,24]. Case series also reported 
high root coverage percentages and enhanced esthetic outcomes when HA was combined with 
SCTG in tunnel and minimally invasive techniques [25,26]. Beyond recession coverage, clinical 
studies on intrabony defects showed that HA, particularly when combined with xenografts, 
promoted the formation of new bone, cementum, and periodontal ligament [27]. Moreover, HA 
application has been associated with reduced postoperative pain and improved palatal wound 
healing in donor sites following free gingival grafting [28]. A recent systematic review and meta-
analysis concluded that HA may provide additional clinical benefit in both non-surgical and surgical 
periodontal therapy, although heterogeneity across studies highlights the need for further high-
quality trials [29]. 
Parallel to clinical findings, mechanistic studies provide biological plausibility for the observed 
effects. Fibroblasts play a central role in periodontal wound healing through extracellular matrix 
synthesis, wound contraction, and secretion of growth factors [30]. In vitro investigations 
demonstrated that HA enhances fibroblast proliferation, stimulates type I and III collagen 
synthesis, and modulates cytokine and matrix metalloproteinase activity [19,20]. Furthermore, 
periodontal ligament (PDL) cell and cementoblast studies confirmed that HA supports cell viability 
and induces the expression of mineralization-related genes [16,31]. Culturing fibroblasts derived 
from SCTGs obtained in clinical settings offers a unique translational approach, allowing donor-
specific responses to HA to be studied under standardized conditions and correlated with clinical 
outcomes [32,33]. This bench-to-bedside model strengthens the biological rationale for HA-
mediated wound healing and regeneration. 
Based on this rationale, the present randomized controlled clinical trial was designed to evaluate 
the potential benefits of adjunctive HA application in CAF combined with SCTG for the treatment 
of single RT1 and RT2 gingival recession defects. In parallel, an in vitro study using fibroblasts 
isolated from SCTGs of the same patients will investigate cell proliferation, migration, and 
expression of wound healing–related genes in the presence or absence of HA. Together, these 
complementary investigations aim to provide a comprehensive understanding of the clinical and 
biological effects of HA, potentially paving the way for optimized regenerative protocols in 
periodontal plastic surgery. 
1.2. Study Rationale  

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The rationale for this study is to evaluate whether the adjunctive application of cross-linked 
hyaluronic acid gel (hyaDENT BG®) enhances clinical outcomes of coronally advanced flap 
combined with subepithelial connective tissue graft (CAF + SCTG) in single RT1/RT2 gingival 
recessions. By promoting fibroblast migration, proliferation, and angiogenesis, HA may increase 
complete root coverage rates, improve patient-reported outcomes, and reduce donor-site 
morbidity. A parallel in vitro study using fibroblasts from the same patients will explore the cellular 
and molecular mechanisms underlying the clinical findings, strengthening the biological plausibility 
of the results. 
 
2. STUDY PRODUCT DESCRIPTION 
 
The investigational product is hyaDENT BG®, a sterile, transparent, highly viscous gel containing 
highly purified hyaluronic acid of non-animal origin with specifically adjusted molecular weight. 
 
2.1 Study Product Description 
 
The hyaDENT BG® gel (Fig. 1) is a sterile, resorbable, cross-linked hyaluronic acid preparation 
specifically designed for dental applications. It is primarily used to support soft tissue healing and 
regeneration, particularly in mucogingival procedures such as coronally advanced flap (CAF) 
surgery combined with connective tissue grafting. Its biocompatibility and prolonged residence 
time at the surgical site enable sustained stimulation of fibroblast migration, angiogenesis, and 
extracellular matrix synthesis, which are critical for optimal wound healing outcomes. 
In periodontal plastic surgery, hyaDENT BG® serves as an adjunctive biomaterial to enhance root 
coverage predictability and keratinized tissue gain, while reducing patient morbidity associated 
with donor-site harvesting. Evidence supports its clinical efficacy in improving complete root 
coverage, reducing postoperative pain and edema, and accelerating the healing process. In 
addition, its bacteriostatic properties may limit early microbial colonization of the surgical wound. 
 
The product is supplied as a ready-to-use 1.2 ml glass cartridge, containing a transparent, highly 
viscous gel with the following composition per 1 ml: 

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• 16 mg cross-linked hyaluronic acid 
• 2 mg natural hyaluronic acid 
• 6.9 mg sodium chloride 
• Water for injection up to 1.0 ml 
 
 
Fig 1: hyaDENT BG® 
 
Manufacturer: 
BioScience GmbH 
Walsmühler Straße 18, 19073 Dümmer, Germany 
www.bio-science.org 
BioScience GmbH is a leading manufacturer specializing in the development and production of 
medical devices based on hyaluronic acid. All manufacturing activities are carried out under a 
rigorous Quality Management System, certified according to ISO 13485, ISO 10993, and in 
compliance with European Medical Device Regulation (MDR). The product is CE-marked (CE 2797), 
reflecting its adherence to the highest quality and regulatory standards. 
 
Characteristics: 
hyaDENT BG® is a cross-linked hyaluronic acid gel produced through a proprietary stabilization 
process that ensures a prolonged residence time in the surgical site while maintaining the 
biological activity of native HA. This cross-linking technology provides controlled, slow degradation, 
allowing sustained bioactivity during the critical phases of wound healing. 
The product exhibits excellent biocompatibility and tissue integration, forming a viscoelastic 
scaffold that supports fibroblast migration, angiogenesis, and collagen deposition. Its rheological 

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properties ensure that the gel remains localized at the application site, offering mechanical stability 
and protection of the underlying graft or connective tissue during the early healing phase. 
Additionally, hyaDENT BG® exerts a bacteriostatic effect against common periodontal pathogens 
and modulates the inflammatory response, contributing to a favorable environment for 
regeneration. Its hydrophilic and hygroscopic nature facilitates moisture retention and enhances 
cell–matrix interactions, guiding orderly tissue regeneration and reducing the risk of scar tissue 
formation. 
 
The study product will be used for its indication. hyaDENT BG® is a sterile medical device intended 
for single use. When applied in the following dental fields, hyaDENT BG® supports wound healing: 
• 
In the treatment of gingival recession using the coronally advanced flap technique. 
• 
After tooth extraction, due to its positive effect on postoperative swelling, pain, and 
trismus. 
• 
In the context of aesthetic dentistry, hyaDENT BG®, when injected into a receded 
interdental papilla, provides a regenerative effect. 
 
Contraindications: 
hyaDENT BG® must not be used in patients with: 
• 
Autoimmune diseases or those undergoing immunotherapy 
• 
Known hypersensitivity to hyaluronic acid 
• 
Severe allergy predisposition 
• 
Acute inflammatory or infectious processes 
• 
Coagulation disorders or patients undergoing anticoagulant therapy 
• 
Hormonal disorders 
 
There are no clinical data regarding the use of this product in pregnant or breastfeeding women, 
or in adolescents under 18 years of age. 
 
Adverse Events: 
The use of hyaDENT BG® may, in very rare cases, lead to tissue reactions. As with any similar 
procedure, the use of hyaDENT BG® carries an inherent risk of infection.  

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During papilla reconstruction, temporary pressure pain may occur as a result of the injection. No 
interactions of hyaDENT BG® with other medications are known. 
 
2.1. Product Registration Status 
 
All articles used in this clinical trial are CE marked. 
 
2.2. Instruction for Use, Handling, Labelling Packaging Type, Contents and Storage 
 
2.2.1. Instruction for Use 
 
Remove the glass cartridge from the blister and insert it into a cartridge syringe. For application of 
the product to the treatment area, blunt needles (23G) are recommended. For papilla 
reconstruction, sharp needles (27G) are recommended. The product is completely degraded over 
time. 
 
2.2.2. Handling 
 
REGEDENT AG will provide the study site with the required number of hyaDENT BG® cartridges for 
use exclusively in patients enrolled in the study and strictly according to the approved protocol. 
Each unit is supplied as a single-use, sterile 1.2 ml glass cartridge packaged in a sealed blister. 
The product must not be used if the sterile packaging is opened or damaged prior to use. In such 
cases, the cartridge shall be discarded following institutional biohazard procedures or returned to 
the manufacturer for documentation. hyaDENT BG® is intended for single use only and must not 
be re-sterilized or reused, as reuse may pose a risk of patient infection. 
Before application, the glass cartridge is inserted into a compatible cartridge syringe. For 
application to the recipient site, blunt cannulas (23G) are recommended to deliver the gel directly 
into the surgical area. In the test group, immediately after harvesting, the subepithelial connective 
tissue graft is fully soaked in sterile hyaluronic acid gel prior to its placement at the recipient site, 
as described in detail in Section 7.4 (Surgical procedure). hyaDENT BG® is applied immediately after 
flap elevation and prior to suturing, ensuring direct contact with the connective tissue graft and 
the recipient bed. 

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The gel should be dispensed slowly to achieve an even distribution, creating a thin, homogeneous 
layer covering the graft. Once applied, the flap is coronally advanced and sutured to ensure 
complete coverage of the treated site, minimizing the risk of contamination and optimizing healing. 
 
2.2.3. Labelling 
 
The study product is not relabeled specifically for use in this clinical study, since it is a product 
already available in the market. 
 
2.2.4. Packaging Type and Contents 
 
hyaDENT BG® is supplied ready-to-use in a 1.2 ml glass cartridge, packaged in a blister for single 
use. The syringe content of hyaDENT BG® is heat sterilized. 
The instructions for use, as well as labels with the batch number and expiry date, are included 
inside the box together with the blister. One of the labels is handed over to the patient to ensure 
product traceability. 
hyaDENT BG must not be resterilized for reuse. If the packaging is open or damaged, or if the blister 
is compromised, the product must not be used, as sterility can no longer be guaranteed and the 
risk of infection is increased. One package contains two glass cartridges. 
 
2.2.5. Storage 
 
Do not freeze and do not expose to extreme heat. Store at room temperature (2 °C – 25 °C). 
 
2.2.6. Important Information 
 
The scale on the glass cartridge serves as a guide for the user and refers to the final volume. It is 
not intended for measurement purposes but merely indicates the amount used relative to the 
nominal content of 1.2 ml. 
 
2.3.  Device Accountability 
 

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The Investigator must maintain an accurate and up-to-date accountability record of all hyaDENT 
BG® cartridges received, used, discarded (opened but unused), and returned throughout the 
duration of the study. This information shall be documented in the Study Product Accountability 
Log, including product name, lot number, expiration date, date of use, and patient identification 
code. 
At each monitoring visit, the study monitor will verify the completeness and accuracy of the 
accountability records and cross-check them with source documentation. At study close-out, the 
monitor or designated delegate will ensure full reconciliation of all investigational product units, 
confirming that all cartridges have been either used, appropriately discarded, or returned to the 
sponsor according to Good Clinical Practice (GCP) requirements 
 
2.4.  Return of Study Product 
 
After treatment of the last enrolled patient, any remaining unopened hyaDENT BG® cartridges at 
the study site must be returned to REGEDENT AG. The sponsor will issue an acknowledgement of 
receipt, which must be filed in the Investigator Site File (ISF) as part of the essential study 
documentation. 
 
3. RISKS AND BENEFITS OF THE STUDY PRODUCT AND CLINICAL STUDY 
 
According to the hyaDENT BG® Clinical Evaluation Report (CER) and post-market clinical follow-up 
(PMCF) data, the product has demonstrated a favorable safety profile and proven performance for 
its intended use in periodontal and mucogingival procedures. Reported adverse events are rare 
and limited to mild, transient local reactions such as temporary pressure pain or localized swelling, 
with no serious device-related events documented. 
There are no new or unforeseen risks identified that require additional clinical assessment. The 
overall risk–benefit ratio remains highly favorable, and the residual risk acceptability, as 
documented in the Risk Management Report (RMR), is unchanged. 
The potential benefits of adjunctive hyaDENT BG® include increased probability of complete root 
coverage, enhanced soft tissue healing, reduced postoperative discomfort, and decreased donor-
site morbidity, which outweigh the minimal associated risks. 
 

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3.1. Risks Linked to the Product 
 
Risk management for hyaDENT BG® is conducted in accordance with ISO 14971. A comprehensive 
description of the risk analysis, identified hazards, and control measures is presented in the Risk 
Management Report (RMR) provided by the manufacturer. 
For both the investigational and control interventions, residual and overall risks are considered 
acceptable, and no additional risks were identified that would require further clinical investigation. 
Residual risks cannot be further mitigated by the manufacturer; additional risk reduction relies on 
proper handling and clinical use by trained dental professionals. Therefore, the product must be 
used exclusively by well-trained clinicians and strictly following the Instructions for Use (IFU). 
 
Relevant side effects identified in the risk analysis and IFU include: 
• 
Allergy or hypersensitivity to hyaluronic acid or excipients 
• 
Localized edema 
• 
Hematoma or transient swelling 
• 
Gingival irritation or inflammation 
• 
Postoperative pain or discomfort 
• 
Localized or systemic infection 
• 
Esthetic dissatisfaction due to partial root coverage 
• 
Delayed healing at donor site (if SCTG used) 
• 
Transient sensory alterations (paresthesia/dysesthesia) 
 
Except for allergic or hypersensitivity reactions to hyaluronic acid, these potential adverse events 
are considered inherent to mucogingival surgical procedures and may occur regardless of whether 
hyaDENT BG® is used. 
All identified residual risks have been minimized to the lowest achievable level and are considered 
acceptable when weighed against the expected clinical benefits, in accordance with Essential 
Requirement 6 of the Medical Device Directive (MDD) 93/42/EEC and MDR requirements. 
To further mitigate risks, all investigators will receive training in the correct handling and 
application of hyaDENT BG® prior to the initiation of the study. 
 
Alternatives to the use of hyaDENT BG® include: 

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• 
Standard CAF + SCTG procedure without adjunctive material 
• 
Other soft tissue substitutes or biomaterials 
• 
No surgical treatment (maintaining the recession defect) 
 
3.2. Risks Linked to the Study 
This clinical investigation is conducted as part of the ongoing Post-Market Clinical Follow-Up (PMCF) 
activities for hyaDENT BG®, designed to detect any previously unreported risks and to further 
confirm the clinical performance and safety profile documented in the Risk Management Report 
(RMR). 
The investigational product will be applied in conjunction with the coronally advanced flap and 
subepithelial connective tissue graft (CAF + SCTG) technique, which represents a standard and well-
documented surgical procedure in periodontal plastic surgery. Therefore, no additional clinical risks 
are anticipated beyond those normally associated with routine mucogingival surgery. 
Compared to current practice, the only specificities introduced by this study are the patient 
selection criteria (inclusion/exclusion), standardized follow-up schedule, and additional data 
collection procedures (photographs, questionnaires, and in vitro cell analyses). These activities pose 
minimal risk to participants and are not expected to have any negative impact on patient safety. 
3.3. Benefits 
 
Patients participating in this study may benefit from adjunctive application of hyaDENT BG® during 
CAF + SCTG surgery for the treatment of single RT1/RT2 gingival recessions. The use of cross-linked 
hyaluronic acid has the potential to enhance soft tissue healing, increase the probability of complete 
root coverage, and reduce donor-site morbidity by accelerating wound healing and minimizing 
postoperative pain and swelling. 
By improving the quality and stability of the soft tissue seal around teeth, this approach may 
contribute to better plaque control, reduced gingival inflammation, and improved long-term 
periodontal stability. Enhanced aesthetic outcomes and greater patient satisfaction are also 
expected. 

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Taken together, these data support a favorable benefit–risk ratio for the use of hyaDENT BG® as an 
adjunct in CAF + SCTG procedures for the proposed clinical indication. 
4. OBJECTIVES 
 
4.1. Primary Objectives 
 
• Complete Root Coverage (CRC) at 12 months (proportion of sites with RD = 0). 
4.2. Secondary Objectives 
• 
Mean Root Coverage (MRC) – percentage of root coverage achieved at 12 
months. 
• 
Recession Reduction (RecRed) – reduction in gingival recession depth (mm) from 
baseline to follow-up. 
• 
Clinical Attachment Level (CAL) gain – improvement in CAL (mm) from baseline to 
12 months. 
• 
Probing Depth (PD) reduction – decrease in PD (mm) from baseline to 12 months. 
• 
Keratinized Tissue Width (KT) gain - increase in the width of keratinized gingiva 
(mm) from baseline to follow-up. 
• 
Operative and postoperative factors – total procedure time (minutes) and 
postoperative morbidity, evaluated via Visual Analog Scale (VAS) scores for pain 
and discomfort and by tracking analgesic use in the first week. 
• 
Early wound healing – Early Wound Healing Score (EHS) at early time points (3 
days, 14 days, 6 weeks) to assess initial healing quality. 
• 
Esthetic outcome – Root Coverage Esthetic Score (RES) at 12 months to appraise 
the esthetic success of root coverage. 
Periodontal health indices (FMPS, FMBS, BOP) will be monitored throughout the study to 
ensure consistent oral hygiene and periodontal stability. These variables are not subject 
to formal hypothesis testing. 
4.3. Safety Objectives 
 

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This study aims to confirm the safety profile of hyaDENT BG® when used as an adjunct to coronally 
advanced flap combined with subepithelial connective tissue graft (CAF + SCTG). 
Through continuous adverse event (AE) and serious adverse event (SAE) monitoring, the study seeks 
to: 
• 
Identify any previously unknown or unexpected side effects and continuously monitor those 
already documented in the Instructions for Use (IFU). 
• 
Analyse emerging risks using factual clinical data to enable timely intervention and risk mitigation. 
• 
Detect any potential systematic misuse or off-label application of hyaDENT BG®, allowing for 
corrective measures or additional training if required. 
By systematically documenting and reviewing adverse events throughout the trial, this study ensures 
the ongoing verification of product safety and contributes to the overall post-market clinical follow-
up (PMCF) activities of the manufacturer. 
By closely monitoring adverse events and analysing emerging risks, we are committed to ensuring 
the ongoing safety and efficacy of the device. 
 
4.4. Hypotheses in both the clinical and the in vitro sections 
 
4.4.1. Clinical trial 
 
Primary Hypothesis/Alternative Hypothesis (H1) 
H1: Adjunctive application of hyaDENT BG® during CAF + SCTG surgery results in a higher proportion 
of sites achieving complete root coverage (CRC) at 12 months compared to CAF + SCTG alone. 
 
Secondary Hypotheses (H2-H9) 
H2: Adjunctive hyaDENT BG® leads to greater mean root coverage (MRC) and greater recession 
reduction (RecRed) at 12 months versus control. 
H3: Adjunctive hyaDENT BG® yields greater CAL gain and PD reduction compared to control sites at 
12 months. 
H4: Adjunctive hyaDENT BG® results in greater height of keratinized tissue (HKT) gain compared to 
CAF + SCTG alone. 
H5: Patients receiving hyaDENT BG® report lower postoperative pain, swelling, and discomfort (VAS) 

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H6: Adjunctive hyaDENT BG® improves oral health–related quality of life, as measured by the OHIP-
14 questionnaire at early postoperative time points (3, 5, 14 days, 6 weeks). 
H7: Adjunctive hyaDENT BG® improves early wound healing score (EHS) at 3, 5, 14 days, and 6 weeks 
compared to control. 
H8: Adjunctive hyaDENT BG® provides better aesthetic outcomes (Root Coverage Esthetic Score, RES) 
compared to CAF + SCTG alone. 
H9: Adjunctive hyaDENT BG® is associated with a lower incidence of surgical complications (e.g., flap 
dehiscence, infection, graft necrosis). 
 
Null Hypothesis (H₀): No statistically significant differences are observed between test and control 
groups in any of the evaluated outcomes 
 
Multiplicity Control: To manage the risk of Type I error across multiple endpoints, a hierarchical 
testing 
strategy 
will 
be 
applied 
to 
the 
following 
prioritized 
hypotheses: 
H2 
(MRC), 
H3 
(CAL 
and 
PPD), 
H4 
(HKT), 
H5 
(VAS), 
and 
H8 
(RES). 
Each hypothesis in this sequence will be tested at the α = 0.05 level, and testing will proceed only if 
the 
previous 
hypothesis 
in 
the 
hierarchy 
is 
statistically 
significant. 
Remaining secondary outcomes (H6, H7, H9) will be analyzed descriptively. 
 
4.4.2. In vitro study 
 
Primary Hypothesis/Alternative Hypothesis (H1) 
H1-in vitro: Exposure of primary human gingival fibroblasts to hyaDENT BG® (4 mg/mL) enhances cell 
proliferation, migration, and extracellular matrix production compared to untreated control cells, 
supporting its role in soft tissue healing and regeneration. 
 
Secondary Hypotheses (H2-H5) 
H2-in vitro: hyaDENT BG® treatment results in higher cell viability (CellTiter-Blue assay) compared to 
control. 
H3-in vitro: hyaDENT BG® induces upregulation of wound-healing-related genes, including COL1A1, 
COL3A1, TGFB1, TGFB3, PDGFB, FGF2, EGF, relative to untreated control fibroblasts. 
H4-in vitro: hyaDENT BG® modulates inflammatory mediator expression (IL1A, IL1B, TNF, IL-8) and 

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MMP activity (MMP1, MMP2, MMP3, MMP8) toward a profile compatible with controlled matrix 
remodeling. 
H5-in vitro: hyaDENT BG® activates key intracellular signaling pathways involved in cell proliferation 
and migration (p-AKT, p-ERK1/2, p-p38) as shown by Western blot analysis. 
 
Null Hypothesis (H₀): Exposure to hyaDENT BG® does not significantly affect proliferation, migration, 
ECM synthesis, inflammatory profile, or signaling activity of fibroblasts compared to untreated 
controls. 
 
5. STUDY DESIGN 
 
5.1. Type and Design of the Study 
 
This is a prospective, randomized, controlled, post-market clinical follow-up (PMCF) clinical trial 
designed to compare the outcomes of CAF + SCTG with adjunctive hyaDENT BG® application versus 
CAF + SCTG alone for the treatment of single RT1/RT2 gingival recessions. 
The study is planned to last approximately 2 years, including a patient enrolment phase and a follow-
up period of 12 months for each participant, starting from the date of surgery. Periodical follow-up 
visits will be scheduled as outlined in the study flow chart (Figure 2), ensuring standardized clinical 
measurements, photographic documentation, and patient-reported outcomes collection throughout 
the study timeline. 

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During each appointment, among the clinical procedures, several data will be recorded to accomplish 
the study goals. 
 
Figure 2: Study Flow Chart 
 
5.1.1. Endpoints 
 
5.1.1.1. Primary Endpoint 
Complete Root Coverage (CRC) at 12 months, defined as the proportion of treated recession sites 
presenting Recession Depth (RD) = 0 mm relative to the cemento-enamel junction (CEJ). 
Measurements will be performed with a CP-15 calibrated periodontal probe by a blinded examiner, 
using the CEJ as a fixed reference point.  
Photographic documentation will be standardized using the same digital camera model, lens, and 
magnification settings. All images will be taken at a fixed distance with the aid of a custom acrylic 
stent and include a millimeter scale for calibration. Consistent lighting and angulation will be 
maintained across all time points to ensure reliable visual assessment of root coverage. 
5.1.1.2 Secondary Endpoints 
 

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• Mean Root Coverage (MRC) and Recession Reduction (RecRed) at 12 months (T7), expressed in 
millimeters and as a percentage of baseline RD. 
• Clinical Attachment Level (CAL) gain and Probing Depth (PD) reduction from baseline (T0) to 12 
months (T7). 
• Height of Keratinized Tissue (HKT) gain, measured from the gingival margin to the mucogingival 
junction (MGJ) at T4 (12 weeks), T5 (6 months), T6 (9 months), and T7 (12 months). 
• Periodontal parameters: Full-Mouth Plaque Score (FMPS), Full-Mouth Bleeding Score (FMBS), 
and Bleeding on Probing (BOP) at T4, T5, T6, T7 to evaluate maintenance of periodontal health. 
• Patient-Reported Outcomes (PROMs): 
o Visual Analog Scale (VAS) for pain, swelling, and discomfort at T1 (3 days), T2 (14 days), and 
T3 (6 weeks). 
o Oral Health Impact Profile-14 (OHIP-14) questionnaire at the same time points to assess oral 
health-related quality of life. 
o Number of analgesics and days of medication recorded at T1. 
• Early Wound Healing Score (EHS): assessed at T1 (3 days), T2 (14 days), and T3 (6 weeks) to 
monitor soft tissue healing dynamics. 
• Surgical and postoperative complications: any intraoperative or postoperative complications 
(e.g., flap dehiscence, graft necrosis, infection) will be recorded in the adverse event log, 
including clinical management and outcome. 
• Aesthetic outcomes: standardized intra-oral photographs will be analyzed to calculate the Root 
Coverage Esthetic Score (RES) at T7 (12 months) using fixed camera-to-subject distance, identical 
lighting conditions, and reference positioning. At T7 (12 months), the esthetic outcome will be 
assessed using the Root Coverage Esthetic Score (RES), as described by Cairo et al. 2009 [34]. 
o The RES system evaluates five parameters: 
§ Gingival margin (GM): 0 points = failure of root coverage (gingival margin apical or 
equal to baseline recession), 3 points = partial root coverage, and 6 points = 
complete root coverage (CRC). 
§ Marginal tissue contour (MTC): 0 = irregular/flat margin, 1 = scalloped, physiologic 
contour. 
§ Soft tissue texture (STT): 0 = scar formation or keloid-like surface, 1 = normal texture 
without scarring. 

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§ Mucogingival junction (MGJ) alignment: 0 = misaligned MGJ, 1 = MGJ aligned with 
adjacent teeth. 
§ Gingival color (GC): 0 = color mismatch with adjacent gingiva, 1 = normal, 
harmonious color. 
o The total RES ranges from 0 to 10, with higher scores indicating better esthetic integration 
and soft tissue harmony. 
o All photographs will be evaluated by a single calibrated examiner blinded to treatment 
allocation to minimize assessment bias and ensure consistency across all cases. The 
examiner will undergo a calibration session using reference images prior to study initiation 
to standardize scoring criteria. 
5.2. Study Population 
The patient population targeted by this clinical trial comprises adult patients (≥18 years old) 
presenting with single RT1 or RT2 gingival recessions in maxillary or mandibular teeth, requiring 
surgical root coverage with coronally advanced flap and subepithelial connective tissue graft (CAF 
+ SCTG). 
Patients must meet all inclusion criteria and none of the exclusion criteria listed below. 
5.2.1. Inclusion Criteria 
Patients will be considered eligible for enrolment if they meet all the following criteria: 
• Age ≥ 18 years, in good general health (ASA I or II). 
• Presence of a single gingival recession defect classified as RT1 or RT2 according to the Cairo 
classification, located on a maxillary or mandibular tooth. 
• Recession depth ≥ 2 mm, with a clearly identifiable cemento-enamel junction (CEJ). 
• Presence of at least 1 mm of keratinized tissue at the site of the recession defect. 
• No active periodontal disease (probing depth ≤ 4 mm and no bleeding on probing at the selected 
tooth). 
• Adequate periodontal support of adjacent teeth. 
• Full-Mouth Plaque Score (FMPS ≤ 20%) and Full-Mouth Bleeding Score (FMBS ≤ 20%) at screening 
and immediately prior to surgery. 

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• Tooth vitality confirmed and absence of caries, non-carious cervical lesions, or defective 
restorations in the area to be treated. 
• Patient demonstrates good oral hygiene and is motivated to maintain plaque control throughout 
the study period. 
• Patient is able and willing to comply with all study requirements, including scheduled follow-up 
visits and completion of patient-reported outcome measures (PROMs). 
• Patient has provided written informed consent before any study-related procedure. 
 
5.2.2. Exclusion Criteria 
 
• Patients will be excluded from participation if they meet any of the following criteria: 
• Uncontrolled systemic diseases or conditions that contraindicate periodontal surgery (e.g., 
uncontrolled diabetes mellitus (HbA1c ≥7.0), severe cardiovascular disease, immunodeficiency). 
• Pregnant or breastfeeding women. 
• History of autoimmune disease or current immunosuppressive therapy. 
• Known hypersensitivity or allergy to hyaluronic acid or any component of the investigational 
product. 
• Heavy smokers (>10 cigarettes/day) or users of smokeless tobacco/nicotine products within the 
last 6 months. 
• Untreated periodontal disease or presence of probing depth ≥ 4 mm with bleeding on probing at 
the study tooth. 
• Caries, non-carious cervical lesions, root fractures, defective restorations, or endodontic lesions 
in the study tooth. 
• History of mucogingival surgery or previous root coverage procedure at the study tooth. 
• Use of systemic antibiotics, corticosteroids, or anti-inflammatory drugs within 3 months prior to 
surgery (unless considered safe by the investigator). 
• Coagulation disorders or current use of anticoagulant/antiplatelet medication that cannot be 
discontinued for surgery. 
• Hormonal disorders significantly affecting wound healing. 
• Active acute infections or inflammatory processes in the oral cavity at the time of surgery. 

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• Any medical, psychological, or social condition that, in the opinion of the investigator, could 
interfere with study participation or adherence to the follow-up schedule. 
Patients may be informed about the study by any member of the department. However, after 
verifying the inclusion and exclusion criteria, the principal investigator will ultimately validate the 
patient's inclusion in the study. 
5.2.3. Sample Size 
This randomized, controlled PMCF study will enroll a total of 34 patients, allocated 1:1 to: 
• Test group (n = 17): CAF + SCTG with adjunctive hyaDENT BG® 
• Control group (n = 17): CAF + SCTG without hyaDENT BG® 
The slight increase from the initially planned 30 patients is intended to accommodate an 
approximate 10% drop-out rate, ensuring that about 30 patients (15 per group) complete the study 
as evaluable. The sample size calculation remains based on the primary endpoint (CRC at 12 months) 
with 30 evaluable patients, while the additional 4 subjects provide a margin of safety for potential 
losses to follow-up. 
The sample size was determined to reliably detect a clinically meaningful difference in the primary 
outcome. Previous randomized studies (e.g. Pilloni et al. 2019) have reported CRC rates of roughly 
80% with CAF+HA versus 33% with CAF alone, an absolute difference of ~47 percentage points. 
Using this large effect size (47 pp), a two-sided α = 0.05, and power (1–β) of 80%, a minimum of 15 
patients per group was deemed sufficient to demonstrate a statistically significant difference in CRC. 
By enrolling 17 per group (34 total), the study preserves this power assumption while accounting 
for possible drop-outs, thereby maintaining at least 15 analyzable subjects in each arm. 
Sensitivity Analysis of Power: The sample size is powered to detect a large CRC difference (47 pp), 
based on prior data. However, if the true effect is smaller (e.g. 20–30 pp), the study may be 
underpowered to reach statistical significance. This highlights a potential risk of type II error for 
modest but clinically relevant differences. Exact power estimates for these scenarios will be 
calculated by the statistician using final parameters. Overall, the sample size represents a practical 
balance between feasibility and detecting a meaningful effect 

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20 
 
5.2.4. Early Withdrawal / Removal of Patients from Treatment 
Any patient may withdraw from the study at any time, without prejudice to their ongoing dental 
care. If a patient fails to attend a scheduled follow-up visit, the investigator (or designee) will attempt 
to contact the patient to reschedule the appointment and document the reason for non-attendance. 
If a patient states that he/she no longer wishes to participate, the reason for withdrawal will be 
recorded whenever possible (e.g., lack of interest, relocation, change of dentist, adverse event). 
Patients will be reminded of the importance of attending follow-up visits for their own clinical care 
and to allow collection of safety data. 
Alternative treatment options appropriate for the patient’s condition will be offered in case of 
withdrawal before treatment completion. 
The investigator may withdraw a patient from the study in the following cases: 
• Non-compliance with the protocol requirements. 
• Repeated failure to attend follow-up visits. 
• Development of a medical condition, SAE, or AE which, in the opinion of the investigator, 
contraindicates further participation. 
• Identification of a new exclusion criterion (or one not previously recognized) that precludes 
continued participation. 
• Withdrawal of informed consent. 
The investigator must withdraw any patient in case of tooth loss or if the site becomes unsuitable 
for evaluation of the primary endpoint. 
All patient withdrawals will be documented in the study termination case report form (eCRF), 
including the reason for withdrawal. Whenever possible, the investigator will make every effort to 
collect the primary endpoint data (CRC at 12 months) prior to final withdrawal. 
In case of premature study withdrawal after surgery, the patient will continue to receive follow-up 
care according to current clinical practice. 
5.2.4.1. Procedures for Replacement of Patients 

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Patients who withdraw from the study after randomization will not be re-enrolled. Their data will be 
included in the analysis following the intention-to-treat (ITT) principle whenever possible. 
If a patient withdraws during the enrolment phase and prior to surgery, the investigator will make 
all reasonable efforts to recruit a new eligible patient in order to maintain the planned sample size 
(30 patients, 15 per group). This approach is intended to preserve the statistical power of the study 
and the validity of the comparative analysis between the test and control groups. 
5.2.5. Screening Failures 
 
Any patient that has signed the informed consent form and does not receive the study product is 
considered a screening failure. In the event of a screening failure, the eCRF page should be 
completed up to the visit when the patient was determined to be a screening failure. The study 
termination eCRF page should also be completed. 
5.3. Study treatment 
5.3.1. Concomitant Interventions 
 
Any concomitant Interventions at the intervention area of the study are allowed and must be 
recorded (date and specify intervention). 
 
5.4. Study Duration 
The total study duration is expected to be approximately 2 years, calculated from the inclusion of 
the first patient to the completion of the 12-month follow-up visit of the last enrolled patient. 
For each patient, the individual study participation will last approximately 13 months, including the 
baseline screening, surgery, and all scheduled follow-up visits up to 12 months post-surgery. 
For the purpose of study planning, monitoring, and reporting, the following definitions are applied: 
• First Patient First Visit (FPFV): the date on which the first eligible patient undergoes the baseline 
visit and is enrolled in the study. 
• Last Patient First Visit (LPFV): the date on which the last eligible patient undergoes the baseline 
visit and is enrolled in the study. 

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22 
 
• Last Patient Last Visit (LPLV): the date on which the last enrolled patient completes the final (12-
month) follow-up visit. 
• Final report / evaluation: the period following LPLV during which data cleaning, statistical analysis, 
and preparation of the final study report are performed. 
 
5.5. Discussion of Study Design 
This investigation is designed as a post-market clinical follow-up (PMCF) study, since both the 
test and control interventions (CAF + SCTG with or without adjunctive hyaDENT BG®) are 
established techniques in periodontal plastic surgery. A prospective, randomized, controlled 
parallel-arm design was chosen to generate high-level clinical evidence on the adjunctive use 
of cross-linked hyaluronic acid versus the conventional gold-standard alone. To minimize bias 
and account for baseline variability, the randomization will be stratified by recession 
type (Cairo RT1 vs RT2 classification), ensuring that defects of differing initial severity are 
evenly distributed between the test and control groups. Allocation concealment is maintained 
via sealed, sequentially numbered opaque envelopes, and blinding is upheld for outcome 
examiners, which together strengthen the internal validity of the trial. 
The sample size and power considerations have been tailored to balance scientific rigor with 
practical feasibility. The trial plans to recruit 34 patients to allow for a ~10% drop-out rate, 
aiming for 30 patients to complete the 12-month follow-up. This enrollment strategy 
preserves the statistical power of the study by compensating for potential attrition. The 
sample size is driven by the primary outcome (complete root coverage at 12 months), which 
is a critical clinical and esthetic benchmark in mucogingival surgery. Detecting a significant 
difference in the proportion of sites with complete root coverage between groups will directly 
address the primary objective and could validate the added value of hyaluronic acid in 
enhancing surgical outcomes. 
In addition to the primary endpoint, the study will evaluate multiple secondary endpoints 
covering clinical efficacy (such as mean root coverage, CAL gain, and keratinized tissue gain), 
patient-centered outcomes (pain/discomfort levels and oral health-related quality of life), and 
esthetic results (RES score). A hierarchical statistical testing plan has been incorporated for 
these secondary endpoints to allow a confirmatory interpretation of the most relevant 

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23 
 
outcomes while controlling the overall Type I error. In practice, this means the analysis will 
formally test secondary outcomes in a prioritized sequence, enabling the trial to make 
stronger evidence-based claims about secondary benefits (e.g., improvements in mean root 
coverage or patient-reported pain) if the data support them. By pre-specifying this hierarchy, 
the study design addresses multiplicity and increases the reliability of conclusions drawn from 
secondary analyses. 
Overall, the chosen design – including stratified randomization, an adequate sample with 
drop-out accommodation, and a controlled approach to multiple endpoints – is intended to 
ensure that the findings are both scientifically robust and applicable to real-world clinical 
practice. The results of this study will provide valuable clinical data on the potential benefits 
of adjunctive hyaDENT BG® in periodontal surgery, which can be compared against published 
outcomes in the literature and used to guide evidence-based decision-making in periodontal 
therapy. 
6. MATERIALS AND METHODS 
 
6.1. Study outline 
A prospective, experimental, controlled, randomized study will be performed on 30. patients divided 
into two groups. In the test group, patients will undergo coronally advanced flap surgery combined 
with connective tissue graft with application of hyaluronic acid. In the control group, patients will 
undergo the same surgery without the application of hyaluronic acid. 
The study will be complemented by an in vitro component in which a sample of the connective 
tissue graft will be taken from each patient to isolate fibroblast cells, culture them, and evaluate 
their viability, proliferation, differentiation, and mediation in cell signaling in the presence or 
absence of hyaluronic acid.  
7. CLINICAL TRIAL 
7.1. Sample Size and Randomization 
Each patient will be randomly assigned to one of the two study groups (test or control) in a 1:1 
ratio. The randomization sequence will be generated using a computer program (e.g., IBM 
SPSS) with stratification by recession type (Cairo classification RT1 vs RT2) and using permuted 

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blocks of random sizes. This stratified, blocked randomization ensures that patients with RT1 
and RT2 recession defects are equally balanced across the two groups, preventing any 
imbalance in baseline recession severity. Allocation concealment will be maintained through 
the use of opaque, sealed, sequentially numbered envelopes. An independent investigator (not 
involved in the treatment) will open the next envelope in sequence immediately after flap 
elevation, thereby revealing the group assignment. The treating surgeon will thus be unblinded 
at the point of graft placement (since they must know whether to apply the HA gel), but the 
patients will not be explicitly informed of their group allocation. (Patients might potentially 
guess their group based on postoperative symptoms or the presence of the HA application, so 
complete patient blinding cannot be guaranteed; however, all outcome assessors remain fully 
blinded to group assignment.) 
Based on the sample size calculations and previous study data, approximately 17 patients per 
group (34 total) will be enrolled in this trial. This enrollment target includes a buffer for 
potential drop-outs, with the goal of ending up with at least 15 evaluable patients in each arm 
at study completion. The groups are defined as follows: 
• 
Test group (n ≈ 17): CAF + SCTG with adjunctive hyaDENT BG® (HA applied at the 
recipient site). 
• 
Control group (n ≈ 17): CAF + SCTG without the adjunctive hyaDENT BG® (no HA 
application). 
By employing stratified randomization and concealing the allocation, the study design mitigates 
selection bias and ensures that both groups are comparable with respect to key prognostic 
factors (notably the recession type). The 1:1 allocation with an effective sample of ~30 
completers (15 per group) provides sufficient power to detect the expected difference in the 
primary endpoint, while the slight over-enrollment to 34 accounts for attrition and preserves 
statistical integrity. 
7.2. Study population  
All patients undergoing coronally advanced flap combined with a subepithelial connective tissue 
graft, at the clinical centre CEPI, Porto, will be screened for this study.  

25 
 
25 
 
7.3. Ethical considerations  
The research project will be submitted to the Ethics Committee. All patients signed a written 
informed consent before participation. The identification of all data is replaced by a code in order 
to ensure complete confidentiality and anonymity. 
7.4. Surgical procedure  
All patients receive basic periodontal therapy and oral hygiene instructions prior to treatment. The 
periodontal surgery is performed by a single experienced periodontist. Hyaluronic acid (HA) 
application is performed by a third examiner who is not involved in the surgical treatment sequence. 
Surgical chair time was measured using a chronometer from the first incision to the last suture in 
both groups. Coronally Advanced Flap Combined with a subepithelial connective tissue graft was 
performed in both patient groups to accomplish root coverage. 
Surgical technique:  
• 
Local infiltrative anesthesia (2% lidocain with epinephrine at a concentration of 1:100,000) 
• 
Incision design is performed with a microblade (USM-6700) or with a 15c blade 
• 
After flap preparation, a gentle root planing is performed using gracey curettes /mini-five  
• 
Coronal advanced flap is obtained by cutting the superficial muscle fibers using a blade 
parallel to the external surface of the mucosa. 
• 
Anatomical papillae are de-epithelialized with the scalpel blade and microsurgery scissors. 
• 
The width of the graft was chosen according to the amount of tissue required to cover the 
exposed root and 3 mm of connective tissue mesial and distal to it. 
• 
Immediately after harvesting, the subepithelial connective tissue graft (SCTG) is placed in a 
sterile dish 
• 
In the test group, the SCTG is completely immersed (fully soaked) in sterile hyaluronic acid 
gel 
• 
The graft remains immersed for approximately 1–3 minutes to ensure full surface contact 
and to prevent dehydration 
• 
Excess hyaluronic acid is allowed to drain passively without compression prior to graft 
placement 

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26 
 
• 
In the control group, the SCTG is maintained under sterile conditions and hydrated with 
sterile saline solution for a comparable time interval, without contact with hyaluronic acid 
Suture: 
• 
Suture for connective grafts should be resorbable Vicryl 7-0 with a 6,6 mm P1 needle (3/8 of 
circumference).  
• 
Suture for the flap is 6-0 resorbable PGA with 11 mm C3 (3/8 circumference) cutting needle 
or 6-0 resorbable Vicryl with 13 mm C3 (3/8 circumference) cutting needle.  
 
7.5. Postoperative protocol 
Post-operative pain and edema were controlled with ibuprofen. Patients received 600 mg at the 
beginning of the surgical procedure. Subsequent doses were taken only if necessary to control pain. 
Patients had to record the quantity of analgesics taken during the first week post-surgery.  
All patients were instructed to intermittently apply an ice bag on the operated area. Patients were 
instructed to discontinue tooth brushing and avoid any trauma at the surgical site. A 60-second rinse 
with 0.12% chlorhexidine digluconate was prescribed twice daily, starting on the third day after 
surgery, and continued for the first two weeks. 
Fourteen days after the surgical treatment, the sutures were removed. Plaque control in the 
surgically treated area was maintained by chlorhexidine rinsing for an additional 1 week after suture 
removal. After this period, patients were again instructed in mechanical tooth cleaning of the 
treated tooth using an ultra-soft toothbrush and a roll technique for 1 month. During this month, 
chlorhexidine rinsing was used twice a day. Then the patient started to use a soft- toothbrush and 
chlorhexidine once a day for another month. All patients were recalled for prophylaxis 2 and 4 weeks 
after suture removal and, subsequently, once every 2 months until the final examination (12 
months).   
Patients were recalled for follow-up (and professional oral hygiene/maintenance procedures, clinical 
measurements as needed and photographs) on days 4, 8, 15 and every 2 months until 12 months. 
7.6. Clinical parameters 

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The following clinical measurements are taken at baseline and at 12 months after surgery for each 
tooth by a blinded calibrated examiner: full-mouth plaque score (FMPS), full-mouth bleeding score 
(FMBS), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL), using a 
CP-15 mm graduated periodontal probe.  
Photographic recording is done at every visit in order to compare the healing process between the 
two groups. Photographic record: before treatment, during surgery and follow up at days 4, 8, 15 
and every 2 months until 12 months.  
Clinical measurements will be performed under standardized clinical conditions (adequate lighting, 
gentle probing force, and dry field). To ensure measurement standardization and reproducibility, an 
individualized acrylic stent will be fabricated for each participant to serve as a fixed reference point 
for all clinical measurements, as described by Zucchelli et al. 2010 [35]. The stent will be positioned 
on the occlusal surfaces of adjacent teeth and will include a vertical guiding groove aligned with the 
mid-buccal aspect of the treated tooth, ensuring consistent probe angulation and insertion point at 
each timepoint. All measurements will be performed by a single calibrated examiner to eliminate 
inter-examiner variability and ensure consistency across all study visits. 
Prior to the beginning of the study, an intra-examiner calibration process will be performed in order 
to assess measurement reproducibility. For this purpose, clinical measurements will be recorded in 
a 
sample 
of 
10 
patients 
not 
included 
in 
the 
study 
population. 
The same clinical parameters will be reassessed by the same examiner after an interval of 
approximately 15 days. Intra-examiner reliability will be evaluated by calculating the Cohen’s kappa 
coefficient (κ) for categorical variables and/or the intraclass correlation coefficient (ICC) for 
continuous variables, as appropriate. A kappa or ICC value ≥ 0.75 will be considered indicative of 
acceptable intra-examiner agreement. Only after achieving adequate intra-examiner calibration will 
patient recruitment and data collection for the study commence. 
 
7.6.1. Full-mouth plaque score (FMPS)  
A simplified variant of the Plaque score (Visible Plaque score), proposed by Ainamo and Bay (1975), 
is used. In this case, the classification criterion is binominal and is based on the presence/absence 
of plaque on tooth surfaces: 0 - Absence of plaque and 1 - Presence of visible plaque  

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28 
 
Calculation of the Full-mouth plaque score:  
FMPS(%) = no. of surfaces with plaque × 100 
                       nº of surfaces evaluated 
 
 
 
7.6.2. Full-mouth bleeding score (FMBS) 
 
For the evaluation of gingival inflammation, a simplified variant, also proposed by Ainamo and Bay 
(1975), is used. In this case the classification criterion is also binominal: 0 - Absence of bleeding 
and 1 - Presence of bleeding from the gingival margin up to 10s after probing  
Calculation of the Full-mouth bleeding score:  
FMBS (%) = no. of surfaces with bleeding x 100 
 
 
no. of evaluated surfaces  
7.6.3. Gingival recession depth (RD) 
Measured from the CEJ to the most apical extension of the gingival margin using a calibrated UNC-
15 periodontal probe, with readings rounded to the nearest 0.5 mm.  
7.6.4. Height of keratinized tissue (HKT) 
Measured as the distance (in millimeters) between the gingival margin and the mucogingival 
junction (MGJ) using a calibrated UNC-15 periodontal probe, with readings rounded to the nearest 
0.5 mm.  
7.6.5. Probing Depth 
 
The probing depth is defined as the distance, in millimeters, from the gingival margin to the bottom 
of the gingival sulcus/pocket. All tooth surfaces (mesio-buccal, centro-buccal, disto-buccal, disto-
lingual, centro-lingual and mesio-lingual) are evaluated with a periodontal probe, which is graded 
every 3 mm. The measurements are recorded to the nearest milimeter. By summing all the PD values 
it is possible to calculate the average PD value for each patient at the various evaluation times.  
7.6.6. Bleeding on probing (BOP) 
 

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29 
 
Bleeding after probing is a common means of assessing the presence of subgingival inflammation, 
although its negative predictive value is higher, i.e. when there is no bleeding. In this way, it is 
possible to check whether a pocket is in its active or inactive state. The measurement is made 
concurrently with the evaluation of the PD. A simplified variant of the bleeding on probing was 
proposed by Mühlemann & Son, (1971). 
In this case the classification criterion is binominal:  
0- Absence of bleeding within 15s;  
1- Presence of bleeding from the gingival margin within 15s.  
The calculation is obtained by dividing the areas where there was bleeding on probing by the total 
number of areas evaluated. 
7.6.7. Clinical attachment level (CAL)  
 
The clinical attachment level is defined as the distance in millimeters from the cementoenamel 
junction (CEJ) to the bottom of the gingival sulcus or pocket. All tooth surfaces (mesio-buccal, centro-
vestibular, disto-buccal, disto-lingual, centro-lingual and mesio-lingual) are evaluated with a 
periodontal probe, which is graded every 3 mm. The measurements are recorded to the nearest 
milimeter. By summing all the PD values, it is possible to calculate the average value for each patient 
at the various evaluation times. The clinical evaluation requires measuring the distance from the free 
gingival margin to the CEJ.  
Thus, the level of insertion can be calculated as follows:  
In the presence of gingival recession: Pocket Depth + distance from the gingival margin to the CEJ. 
7.6.8. Patient related outcome measures 
 
Postoperative pain, number of analgesic pills taken, and number of days the pills were taken were 
assessed using questionnaires.  
Postoperative questionnaires evaluated the subjects' postoperative pain, interference with daily life, 
and the patient's perception of swelling using the 100-mm visual analog scale (VAS). 

30 
 
30 
 
The questionnaire was given to the patients 1 week after surgery. The subjects were also asked to 
indicate the location of the pain: donor site, recipient site, or elsewhere in the mouth. 
8. IN VITRO  
 
8.1 Cell culture and HA preparations  
 
8.1.1 Hyaluronic acid formula 
 
HA (hyaDENT BG) is a formula containing 1000 kDa monomers of HA cross-linked to butanediol 
diglycidyl ether (BDDE) complexes and natural HA. (1.6% cross-linked hyaluronic acid and 0.2% natural 
hyaluronic acid). 
 
8.1.2. Concentration of the hyaluronic acid preparations 
 
HA will be used at a final concentration of 4 mg/ml in DMEM medium supplemented with 0.3% FBS, 
as described in the literature. The final concentration of 4 mg/mL was selected to balance biological 
relevance, experimental feasibility, and clinical translatability. First, previous experiments 
demonstrated that HA diluted to 4 mg/mL elicited biological responses comparable to those observed 
with undiluted commercial HA preparations, while allowing improved handling and homogeneous 
distribution in cell culture conditions [14]. Second, concentrations in the range of 1–5 mg/mL are 
commonly reported in the literature for in vitro studies evaluating the effects of hyaluronic acid on 
periodontal and oral cells, particularly under low-serum conditions, supporting the biological validity 
of this concentration range [36-38]. Third, from a clinical perspective, commercially available HA 
formulations applied during periodontal surgery are rapidly diluted in situ by blood, gingival crevicular 
fluid, and saliva. Therefore, the use of a 4 mg/mL concentration in vitro more closely reflects the 
effective HA concentration at the surgical site after application, rather than the nominal 
concentration of the undiluted product. 
Taken together, these considerations support the use of 4 mg/mL as a biologically active, clinically 
relevant, and methodologically justified concentration for the present study, with this concentration 

31 
 
31 
 
range being widely supported by the existing literature on in vitro and translational periodontal 
models involving hyaluronic acid. 
 
8.1.3. Cell culture 
 
Primary human connective tissue fibroblasts are obtained from each patient undergoing clinical 
treatment. Tissue samples will be collected from subepithelial palatal connective tissue grafts 
obtained from systemically and periodontally healthy individuals undergoing periodontal root 
coverage surgery. Each donor tissue sample will be minced into 1 mm pieces and then pipetted into 
a 25 cm3 tissue culture flask and cultured in Dulbecco's modified Eagle's medium (DMEM) 
supplemented with 10% fetal bovine serum (FBS). 
 
8.2. Cell viability assay  
 
Cell viability can be assessed by CellTiter-Blue viability assay (e.g.) Procedure: After 24 hours of 
starvation, cells are seeded in triplicate at 5 × 103 cells/well in 96-well plates coated with HA at the 
indicated concentrations (between 0 and 4 mg/mL) prepared in 0.3% FCS/DMEM. 2 hours after 
seeding, CellTiter-Blue® Reagent (20 μL/well) is added to the cells for 4 hours before recording 
fluorescence using a luminometer. Experimental values are normalized to the values for untreated 
cells. 
 
8.3. Cell proliferation assay  
 
The proliferation rates of HA-treated cells are determined using the 5-bromo-2'- deoxyuridine (BrdU) 
chemiluminescent ELISA (enzyme-linked immunosorbent assay). Procedure: After 24 h of starvation, 
cells are seeded in triplicate at 2 × 103 cells/well in 3% FBS/DMEM in black 96-well microtiter plates 
coated with HA at a final concentration of 4 mg/ml. Cells were allowed to proliferate for 0, 24, 48, 72 
and 96 h before labeling with BrdU for 2 h. The BrdU uptake in the newly synthesized DNA is 
determined according to the manufacturer's instructions using a luminometer. Experimental values 
are normalized to the values of untreated control cells at time point 0. 
 

32 
 
32 
 
8.4. Cell migration assay  
 
Cell migration is tested using Transwell® Polycarbonate Membrane Inserts. Procedure: After 24 hours 
of starvation, 5 × 104 cells are seeded in the upper insert chamber in serum-free DMEM. The lower 
chamber is coated with HA at a final concentration of 4 mg/mL in 10% FCS/DMEM. Cells are allowed 
to migrate through the filter for 18 hours at 37°C before fixation and staining with crystal violet. 
Images of duplicate inserts are acquired using a microscope. Migration is quantified using ImageJ's 
Fiji distribution. 
 
8.5. RNA analysis by qRT-PCR 
 
For oral fibroblast cells quantitative RT-PCR is used to investigate the expression of COL1A1, COL3A1, 
TGFB1, TGFB3, PDGFB, FGF2, EGF, IL1A, IL1B, TNF, MMP1, MMP2, MMP3 and MMP8 genes. 
Procedure: Total RNA from cells treated with HA or growth factor is isolated using the RNeasy Mini 
Kit. RNA is reverse transcribed and the relative transcripts of the listed genes, normalized to GAPDH, 
are measured using FastStart Universal SYBR Green Master ROX and primer sequences are shown in 
the table. Real-time PCR is performed using a 7500 Real-Time PCR system. Data are analyzed using 
the ∆∆Ct efficiency method. All samples are run in duplicate.  
 
8.6. Protein analysis by Western Blot  
 
Complete cell extracts of HA-treated oral fibroblasts are prepared by lysis in RIPA buffer. Proteins of 
interest are visualized using anti-phospho-akt, anti-akt, anti-phospho-erk1/2, anti-erk, anti-phospho-
p38, anti-p38 and anti-vinculin antibodies followed by horseradish peroxidase-conjugated secondary 
antibodies for detection with the SuperSignalTM West Dura Substrate protein immunodetection 
reagent. The expression of phospho-Akt, phospho-Erk1/2 or phospho-p38 protein relative to the 
respective total protein control is quantified by densitometry. 
 
9. RESULTS  
 
9.1. Clinical Trial 

33 
 
33 
 
 
We intend to evaluate the effect of HA, compared to the control group, on clinical parameters:  
1. Full-mouth plaque score (FMPS) 
2. Full-mouth bleeding score (FMBS) 
3. Bleeding on probing (BOP) 
4. Probing Depth (PD)  
5. Clinical attachment level (CAL) 
 
Evaluation times: before treatment, days 4, 8, 15 and every 2 months until 12 months 
 
9.2 In-vitro study 
 
It is intended to evaluate the effect of HA preparation, compared to control cells, on oral fibroblast 
cells (obtained from gingival grafts) regarding the:  
 
1. Viability of primary cells. Ability to adhere and assume a fibroblast-specific fusiform 
morphology (Fig.1);  
2. Proliferative and migratory capacity of the cells; 
3. Expression of COL3A1 and TGFB3 genes (characterize wound healing without scarring); 
4. Expression of genes encoding growth factors and cytokines involved in wound healing; 
5. Expression of MMP2 and MMP3 (matrix metalloproteinases) genes 
6. Phosphorylation of Akt, Erk1/2 and p38 kinases. 
 
 
Fig. 3: Scanning electron microscope images (SEM) of primary human PDL cells seeded 
onto dentin discs in control, HA and HA cl samples. PDL cells seeded in cross-linked HA 

34 
 
34 
 
demonstrated qualitatively more elongated cell morphology when compared to control 
and HA.  
 
 
 
Fig 4: Left image: Neutrophils in humans are recruited by IL-8 to injury sites where they 
release IL-1α and TNF-α and activate fibroblasts and keratinocytes to facilitate wound 
healing. Right image: Cellular and molecular interactions at the wound site. Fibroblast 
proliferation and differentiation into myofibroblasts are essential to produce ECM, 
collagen and α-SMA.Macrophages release mediators that facilitate angiogenesis in order 
to nourish infiltrated and newly proliferated cells at the wound site (Komi et al).  

35 
 
35 
 
Table 1: General properties and effects of mast cell-derived mediators in the wound 
healing process (Komi et al.). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

36 
 
36 
 
10. BIBLIOGRAPHY  
1. Cortellini P, Bissada NF. Mucogingival conditions in the natural dentition: narrative 
review, case definitions, and diagnostic considerations. J Periodontol. 2018;89 Suppl 
1:S204–S213. doi:10.1002/JPER.16-0671 
2. Cairo F. Periodontal plastic surgery of gingival recessions at single and multiple 
teeth. Periodontol 2000. 2017;75(1):296–316. doi:10.1111/prd.12184 
3. Tonetti MS, Jepsen S, Cortellini P, et al. Principles in prevention of periodontal 
diseases. J Clin Periodontol. 2015;42 Suppl 16:S5–S11. doi:10.1111/jcpe.12368 
4. Trombelli L, Farina R. Epidemiology and risk factors for gingival recession. Periodontol 
2000. 2017;75(1):132–51. doi:10.1111/prd.12164 
5. Kassab MM, Cohen RE. The etiology and prevalence of gingival recession. J Am Dent 
Assoc. 2003;134(2):220–5. doi:10.14219/jada.archive.2003.0137 
6. Chambrone L, Tatakis DN. Periodontal soft tissue root coverage procedures: a 
systematic review from the AAP regeneration workshop. J Periodontol. 2015;86(2 
Suppl):S8–S51. doi:10.1902/jop.2015.130674 
7. Zucchelli 
G, 
Mounssif 
I. 
Periodontal 
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surgery. Periodontol 
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