Clinical Efficacy of Adjunctive Photodynamic Therapy with Periodontal Regenerative Surgery in Severe Periodontitis: A Randomized Controlled Trial on Gingival Thickness Remodeling and Inflammatory Cytokine Modulation

Abstract

Objective:

Assess clinical efficacy of photodynamic therapy (PDT) combined with periodontal regenerative surgery (PTR) in severe periodontitis, focusing on gingival thickness (GT), inflammatory markers, and bone repair.

Methods:

Sixty patients were randomized to Combination (PDT + PTR) or Control (PTR alone) groups. PDT utilized 660 nm laser with methylene blue. Outcomes included probing depth (PD), clinical attachment loss (CAL), bleeding on probing (BOP), keratinized tissue width (KTW), GT, cone-beam computed tomography bone parameters, and gingival crevicular fluid cytokines (IL-1β, TNF-α, PGE2, MMP-8) at 3/6/12 months.

Results:

At 12 months, Combination group showed superior PD reduction (p < 0.05), CAL gain (p < 0.05), lower BOP (16.67% vs. 43.33%, p < 0.05), increased KTW/GT, and enhanced bone density/defect filling versus Control. Inflammatory markers were significantly reduced at 3 months (p < 0.05). Three-wall defects exhibited better CAL/GT outcomes than two-wall defects (p < 0.05).

Conclusions:

The combined PDT and PTR therapy demonstrated significant improvements in key clinical parameters, reduced inflammatory biomarkers, and enhanced radiographic bone fill in severe periodontitis patients.

Keywords

photodynamic therapy periodontal tissue regeneration severe periodontitis periodontal state inflammatory cytokines

Introduction

Periodontitis is characterized by progressive destruction of periodontal supporting tissues, including the gingiva, alveolar ridge, and jawbone. Severe periodontitis (stage IV) represents the most advanced stage of gum disease, often leading to tooth mobility and eventual loss, significantly impacting oral health and quality of life.^1,2 Traditional periodontal therapy includes subgingival scaling and root planning (SRP) and alveolar ridge smoothing, which effectively control inflammation but often fail to achieve complete regeneration and functional recovery of periodontal tissues in patients with severe periodontitis.^3,4

Recent advancements in regenerative medicine and photodynamic therapy (PDT) have significantly improved periodontal treatment outcomes. Periodontal tissue regeneration surgery (PTR) uses guided tissue regeneration (GTR) techniques and bone graft materials to aim for the reconstruction of damaged periodontal structures.⁵ However, the efficacy of PTR is influenced by various factors, including surgical technique, patient individual differences, and postsurgical inflammatory responses. Therefore, identifying an adjunctive therapy that enhances PTR effectiveness while minimizing adverse effects holds significant clinical value.

PDT is a targeted cell destruction or tissue modification method based on the principle of generating photodynamic reactions using photosensitizing agents or specific light sources.^6,7 Its applications in periodontology focus on its antimicrobial and anti-inflammatory properties, capable of effectively eliminating pathogens in periodontal pockets while modulating local immune responses.^8,9 Studies have shown that PDT not only reduces the number of pathogenic bacteria in periodontal pockets but also decreases inflammatory cytokine levels in gingival crevicular fluid (GCF), thereby improving the periodontal microenvironment and promoting tissue repair.^10,11 It is important to note that the therapeutic action of antimicrobial PDT (aPDT) may extend beyond the targeted photochemical destruction of pathogens. As evidenced in recent spectrophotometric studies, a significant portion of the incident light may transmit beyond the photosensitizer. This transmitted light can exert a secondary, beneficial effect known as photobiomodulation (PBM), which is known to modulate inflammation and stimulate cellular processes critical for tissue healing and regeneration.^12–14 Therefore, the observed clinical outcomes in this trial may result from the combined effects of aPDT and PBM.

The primary objective of this study was to assess the clinical efficacy of adjunctive aPDT combined with PTR in patients with stage IV severe periodontitis, with a focus on gingival thickness (GT) remodeling, modulation of key inflammatory cytokines, and radiographic bone regeneration.

Materials and Methods

Study design and ethical approval

This prospective clinical study was approved by the Institutional Ethics Committee of Yunnan University Affiliated Hospital (No. 2022-LL-325) and conducted in accordance with the Declaration of Helsinki. A total of 60 patients (986 affected teeth) diagnosed with stage IV periodontitis based on the 2018 classification of periodontal diseases were enrolled from the Department of Stomatology between August 2022 and September 2023. Participants were randomly assigned to either the Combination group or the Control group (n = 30 per group) using computer-generated randomization codes. The flowchart of this study is shown in Figure 1 The sample size was determined based on a power analysis (α = 0.05, power = 80%) to detect a 20% intergroup difference in clinical attachment loss (CAL). Baseline demographic and clinical characteristics showed no statistically significant intergroup differences (p > 0.05, Table 1).

Table 1.

Baseline Characteristics of Two Groups

Characteristics	Combination group	Control group	t/x²	p
N	n = 30	n = 30
Gender	18	19	0.071	0.791
	12	11
Age (year)	41.28 ± 10.25	42.20 ± 11.33	0.329	0.743
Number of affected teeth	491	495	—	—
Disease duration (year)	1.05 ± 0.32	1.06 ± 0.28	0.129	0.898
Smoking history	8 (26.67)	7 (23.33)	0.023	0.878
Diabetes history	5 (16.67)	6 (20.00)	0.111	0.739
OHI-S	4.01 ± 0.56	4.03 ± 0.49	0.147	0.883

OHI-S, Oral Hygiene Index–Simplified.

Inclusion and exclusion criteria

Inclusion criteria

(1) Stage IV periodontitis was defined according to the 2017 American Academy of Periodontology classification, characterized by ≥6 mm CAL at ≥3 teeth per site and radiographic bone loss extending into the furcation area or vertical defects >5 mm.¹⁵ Only patients with moderate to severe forms were included in this trial; (2) age 18–60 years with ≥20 remaining teeth; (3) no periodontal therapy within the preceding 6 months or antibiotic use within 1 month; (4) written informed consent obtained from all participants and their legal guardians.

Exclusion criteria

(1) Systemic immune disorders, coagulation abnormalities, or malignancies; (2) severe cardiopulmonary, hepatic, or renal dysfunction; (3) history of hypersensitivity to study medications or biological agents; (4) poor treatment compliance or inability to complete follow-up; (5) pregnancy, lactation, or presence of severe systemic conditions (such as uncontrolled cardiovascular disease, advanced liver/kidney dysfunction, or active malignancy) requiring long-term pharmacological treatment. Patients with well-controlled chronic diseases—including type 2 diabetes mellitus and tobacco use—were included if clinically stable and not on medications likely to affect inflammatory response or wound healing.

Treatment protocols

Initial phase therapy

All patients underwent standardized subgingival SRP using Gracey curettes (Hu-Friedy, USA) and ultrasonic scalers (EMS, Switzerland) prior to PDT or PTR. The periodontal pockets were irrigated with sterile saline to remove debris, followed by meticulous mechanical debridement to achieve complete root surface cleaning and smoothing. Patients received individualized oral hygiene instructions and education on plaque control to optimize baseline periodontal health. During the surgical phase, after flap elevation and debridement, rubber dam isolation was applied to the target teeth to maintain a contamination-free field. Polishing instruments were strictly prohibited in the operative area to prevent particulate contamination during the entire regenerative procedure. Target sites were selected based on pre-SRP assessment of CAL ≥6 mm and probing depth (PD) >5 mm. Inclusion criteria remained unchanged until surgery, with final site selection confirmed during the surgical procedure.

SRP was performed in two to four sessions at 1- to 2-week intervals, tailored to individual periodontal severity (assessed by baseline CAL and PD). Reevaluation occurred at 4–6 weeks post-treatment. Clinical end-points for phase completion: (a) absence of bleeding on probing (BOP−); (b) reduction in PD ≥2 mm; (c) smooth, calculus-free root surfaces confirmed by explorer examination.

Prophylactic systemic antibiotics were not administered to any patient. Instead, a single application of 1% minocycline hydrochloride (Sunstar INC) gel was locally delivered into the periodontal pocket during surgical flap elevation and placement of the regenerative membrane. The use of local antimicrobial therapy aimed to minimize systemic antibiotic exposure while effectively targeting subgingival pathogens.

Periodontal tissue regeneration

Preoperative evaluation included clinical assessment of PD, gingival inflammation severity, tooth mobility, and radiographic/3D imaging to characterize alveolar bone loss patterns. Systemic health screening was performed to exclude surgical contraindications. After SRP, localized anesthesia was administered, and an intrasulcular incision was made along with the gingival margin to elevate a full-thickness mucoperiosteal flap via the modified Widman technique. The root surface and osseous defects were exposed, followed by thorough removal of residual calculus, plaque, and infected granulation tissue. Root surfaces were polished, and bone defects were grafted with deproteinized bovine bone matrix (DBBM; Bio-Oss^®, Geistlich Pharma) to restore alveolar architecture. A resorbable collagen membrane (GTR membrane; Bio-Gide^®, Geistlich Pharma) was positioned to cover the grafted site, ensuring adaptation to the root surface to prevent epithelial downgrowth and facilitate periodontal ligament regeneration. The flap was repositioned and sutured using non-resorbable sutures. Postoperative care included prophylactic antibiotics, analgesics, and instructions to avoid mechanical trauma to the surgical site. Healing progress was monitored through scheduled follow-ups. The schematic illustration of guided tissue regeneration approach in periodontal therapy is shown in Figure 2.

Photodynamic therapy

Adjunctive PDT was performed using a diode laser system (Zhengzhou Jiatai Biotechnology Co., China) with a wavelength of 660 nm in continuous wave mode. The laser energy was delivered subgingivally via a sterile optical fiber with a tip diameter of 0.9 mm. The power density was set at 100 mW/cm². Each periodontal site was irradiated for 60 sec. For sites with a PD ≥7 mm, the irradiation procedure (60 sec) was repeated once, resulting in two passes per site: first in a buccolingual direction along with the root surface, and second in a mesiodistal direction after a 30-sec interval. Subsequent DBBM grafting and GTR membrane placement followed the standard PTR protocol. Based on previous studies, 0.01% methylene blue (MB) is commonly used in PDT applications due to its optimal balance between phototoxicity and cell viability.¹⁶ Our selection of this concentration was guided by the finding that MB at concentrations above 50 μg/mL can effectively disrupt biofilm structures while maintaining acceptable cytocompatibility.¹⁷ Regarding irradiation time, a 60-sec exposure duration has been shown to achieve maximal therapeutic effect without excessive phototoxicity in similar PDT protocols. Although this study did not include exhaustive dose–response experiments, preliminary data from preclinical trials showed that MB concentrations above 1% significantly increased phototoxicity while only marginal improvements in biofilm eradication were observed.¹⁸ This suggests the chosen concentration represents an optimal trade-off between efficacy and safety.

Group allocation

The Combination group received PTR with adjunctive PDT, while the Control group underwent PTR alone.

Follow-up and outcome assessment

Clinical assessments were performed at four time points: (1) baseline (i.e., prior to nonsurgical SRP); (2) 3 months after SRP; (3) 6 months post-treatment; and (4) 12 months post-treatment. Periodontal parameters, including PD, CAL, gingival recession, and GT, were recorded at each visit. All periodontal probing was performed using a standardized manual periodontal probe (Hu-Friedy, Chicago, IL), calibrated to 0.25 mm increments, with consistent pressure of 0.2 N applied. GCF inflammatory cytokine levels were assessed at baseline and 3 months post-treatment. Missing data (n = 3) were handled using last observation carried forward (LOCF), as per the CONSORT guidelines for randomized controlled trials (RCTs) with chronic conditions. An intention-to-treat (ITT) analysis was performed to account for protocol deviations or dropouts.

Periodontal parameter evaluation

The periodontal probe is employed to measure PD, CAL, BOP, and keratinized tissue width (KTW) by using the Florida Electronic Probe to Assess Periodontal Indices. Each assessment method is carefully defined as follows: PD¹⁹: Using the Hu-Friedy UNC-15 probe, the probe tip is inserted along the tooth’s long axis with a force of 20–25 g until resistance is encountered. The probe is then moved gently within the periodontal pocket to measure the distance from the gingival margin to the bottom of the sulcus (in millimeters). This measurement is repeated three times, and the mean value is recorded. CAL²⁰ is calculated by subtracting the distance from the gingival margin to the cementoenamel junction (CEJ) from the PD measurement. If the result is 0 mm or if the CEJ cannot be reached, there is no CAL. When the gingival margin lies apically relative to the CEJ, the two distances are summed for calculation. This process is performed three times, and the mean value is reported. BOP²¹: During PD measurement with the probe, blood flow at the gum pocket is observed 10 sec after probing. If bleeding is observed at this stage, it is recorded as a positive BOP; otherwise, it is noted as negative. This simple yet crucial evaluation provides important insights into periodontal health status. KTW: Using a periodontal probe, the KTW is measured at the buccal and lingual sides of the affected tooth. Three measurements are taken consecutively, and the average value represents the KTW around the treated tooth. All periodontal measurements were performed by a single calibrated examiner (trained in the CPITN and EPT criteria) using the same protocol across all visits. Intra-examiner reproducibility was assessed with 20% of sites remeasured at baseline, yielding an intraclass correlation coefficient (ICC) of 0.94 for CAL. Two examiners independently measured all sites; discrepancies were resolved by consensus in the presence of a third blinded investigator.

Alveolar bone defect assessment

Cone-beam computed tomography (CBCT; Hyperion X9 Pro, MyRay, Anhui Meiya Optoelectronics, China) was performed preoperatively and at 6-month follow-up to quantitatively evaluate alveolar bone remodeling, including bone defect fill (%) and trabecular bone density changes. Scans were acquired using a standardized low-dose protocol: 85 kV, 4 mA, pulsed exposure (20 sec), 0.15 mm voxel size, and 5 × 5 cm field of view centered on target teeth, with patients seated (Frankfort plane parallel to ground) and stabilized using a chin rest. To ensure measurement validity and reliability, calibrated examiners, two independent periodontists (ICC >0.92 for all measurements), performed analyses after training on 10 non-study CBCT volumes. Validated Software: Sagittal reconstructions perpendicular to the tooth axis were analyzed using NNT Viewer v5.0 (MyRay), employing multiplanar reformation to measure bone height (CEJ to bone crest, mm), defect volume (mm³), and trabecular density [Hounsfield units (HU)] at identical coordinates pre-/post-treatment. Reference Methodology: Measurements followed established protocols for periodontal CBCT analysis, with proven accuracy for defect detection (sensitivity >0.89) and submillimeter reproducibility in alveolar bone quantification.²² Importantly, all CBCT scans in this trial were clinically indicated for the diagnosis and surgical planning of severe periodontitis cases and for standard postoperative follow-up. The imaging protocol was strictly optimized according to the ALARA principle, utilizing the smallest field of view and the lowest possible exposure settings. The schematic illustration of guided tissue regeneration approach in periodontal therapy is shown in Figure 3.

Bone fill rate was calculated as:

Bone Fill Rate (%) = \frac{Post - treatment Bone Height - Pre - treatment Bone Height}{Pre - treatment Bone Height} \times 100

GCF analysis

GCF samples were collected from four sites (buccal, lingual, mesial, distal) using 2 × 10 mm sterile filter strips (Periopaper, Oraflow, USA). After air-drying, strips were inserted into pockets until mild resistance was felt, retained for 30 sec, and stored at −70°C. Contaminated samples were discarded. Thawed strips were eluted with phosphate-buffered saline and centrifuged at 1704g for 12 minutes, and supernatants analyzed via Enzyme-Linked Immunosorbent Assay (R&D Systems, USA) for IL-1β, TNF-α, PGE2, and MMP-8 levels.

Microbiological and clinical plaque assessment

Subgingival plaque samples prospectively stored at baseline and 12-month follow-up were analyzed. Clinical plaque accumulation was assessed using the Silness-Löe Index (0–3 scale) at both time points. For microbial quantification, quantitative PCR (qPCR) targeting Porphyromonas gingivalis and Tannerella forsythia (T. forsythia) was performed. DNA extraction used QIAamp DNA Mini Kit (Qiagen, Germany). SYBR Green assays with validated primers:

P. gingivalis:

F-5′-AGGCAGCTTGCCATACTGCG-3′, R-5′-ACTGTTAGCAACTACCGATGT-3′;

T. forsythia:

F-5′-GCGTATGTAACCTGCCCGCA-3′, R-5′-TGCTTCAGTGTCAGTTATACCT-3′.

American Type Culture Collection strains (Pg 33277, Tf 43037) provided standard curves. All reactions included negative controls and technical triplicates.

Statistical analysis

All statistical analyses were performed using SPSS 25.0 and GraphPad Prism 9.0. Continuous variables with normal distribution were expressed as mean ± standard deviation, and intergroup comparisons were conducted via independent t-tests after confirming homogeneity of variances (Levene’s test). For longitudinal intragroup changes, repeated-measures Analysis of Variance was employed to evaluate time-dependent effects across multiple time points. When the interaction between group and time reached significance (p < 0.05), post hoc comparisons were performed using Bonferroni-corrected paired t-tests to control for type I error inflation due to multiple comparisons.

To address missing data, an LOCF approach was applied, as recommended in the CONSORT statement for RCTs with chronic conditions requiring long-term follow-up. Additionally, ITT analysis was conducted by including all randomized participants regardless of protocol deviations or dropouts. This approach ensures that the results reflect real-world clinical scenarios and minimizes bias due to missing data.

Categorical variables were analyzed with chi-square tests or Fisher’s exact tests (when expected frequencies were ≤5). Ordinal data were evaluated using the Mann–Whitney U test. To address potential confounding factors, multivariate analysis of covariance was applied where appropriate. A two-tailed p < 0.05 was considered statistically significant for all analyses.

Results

Periodontal health indices at pre- and post-treatment evaluation

After SRP, both groups showed significant improvement in periodontal parameters. At 4–6 weeks post-SRP, mean PD was 5.71 ± 0.51 mm in the Combination group versus 5.82 ± 0.59 mm in the Control group (p > 0.05). Mean CAL improved to 5.25 ± 0.60 mm and 5.18 ± 0.54 mm, respectively (p > 0.05).

The average improvement in PD was 1.07 ± 0.25 mm (Combination group) versus 1.06 ± 0.27 mm (control; p > 0.05), and the corresponding improvement in CAL was 1.13 ± 0.29 mm versus 1.07 ± 0.27 mm (p > 0.05). These findings indicate comparable initial response to SRP between groups.

After 12 months of therapy, the Combination group exhibited significant improvements in periodontal health compared with the Control group: The PD was notably lower in the Combined group than in the Control group (2.64 ± 0.37 mm vs. 3.85 ± 0.34 mm, p < 0.05), the CAL also improved significantly (3.42 ± 0.44 mm vs. 3.91 ± 0.48 mm, p < 0.05), and the BOP positivity rate was lower in the Combined group compared with the Control group (16.67% vs. 43.33%, p < 0.05). These findings are summarized in Figures 4 and 5.

Comparison of bone healing outcomes between the two groups

The KTWs of Combination group showed significant superiority over the Control group in terms of bone regeneration volume (2.03 ± 0.22 mm vs. 1.57 ± 0.30 mm; p < 0.05), similar advantages were observed for GT (1.61 ± 0.22 mm vs. 1.38 ± 0.23 mm; p < 0.05), and higher bone mineral density in newly formed bones was also noted (115.58 ± 9.79 HU vs. 104.03 ± 8.78 HU; p < 0.05). Further, the bone defect filling rate was significantly higher in the treatment group compared with the Control group (73.35 ± 8.92% vs. 61.31 ± 7.59%; p < 0.05), as shown in Figure 6.

Comparison of GCF inflammatory biomarkers between the two groups

After 3 months of treatment, levels of four inflammatory biomarkers—IL-1β, MMP-8, PGE2, and TNF-α—in GCF were significantly lower in the Combination group than in the Control group (p < 0.05). Details are presented in Figure 7.

Plaque control and pathogen reduction

Clinical plaque scores revealed superior control in the Combination group versus the Control group at 12 months. While baseline scores were comparable (Combination: 1.38 ± 0.33; Control: 1.41 ± 0.30, p = 0.71), the Combination group achieved significantly lower plaque accumulation post-intervention (12-month: Combination 0.82 ± 0.18 vs. Control 0.95 ± 0.21, p = 0.008).

qPCR analysis demonstrated synergistic microbial benefits: The Combination group showed markedly greater reductions in P. gingivalis (ΔCt: −4.5 ± 1.2 vs. −1.7 ± 0.8, <0.001) and T. forsythia (ΔCt: −4.1 ± 0.9 vs. −1.5 ± 0.6, p = 0.003) versus Control.

Discussion

PTR has long been a cornerstone in restoring the structural and functional integrity of periodontal tissues through GTR and bone grafting materials. However, its efficacy is often compromised by postoperative inflammation and bacterial colonization, which hinder optimal healing. PDT, with its dual antimicrobial and anti-inflammatory properties, offers a promising adjunct to PTR by reducing pathogenic microbial load in periodontal pockets and mitigating infection risks, thereby enhancing regenerative outcomes.²³ The therapeutic mechanism of PDT relies on the photochemical interaction among a photosensitizer, light of a specific wavelength, and molecular oxygen. Upon irradiation, the photosensitizer generates reactive oxygen species (ROS), including singlet oxygen, which disrupts bacterial cell membranes, proteins, and DNA, effectively eliminating periodontal pathogens.^24,25 This study evaluated the synergistic effects of PDT combined with PTR in stage IV periodontitis, focusing on clinical outcomes, GT restoration, and modulation of inflammatory mediators.

In this trial, 0.01% MB was selected as the photosensitizer due to its peak absorption at 660 nm, enabling deep tissue penetration and robust ROS generation. As a phenothiazine derivative, MB exhibits potent bactericidal activity against periodontal pathogens such as P. gingivalis while modulating host-derived inflammatory cytokines such as TNF-α and IL-1β.^26,27 Our findings demonstrated that the Combination group (PTR + PDT) achieved significantly greater reductions in PD, CAL, and BOP compared with PTR alone. These improvements were paralleled by increased GT and KTW, suggesting enhanced soft tissue regeneration—a critical factor in long-term periodontal stability. These results align with previous studies highlighting PDT’s capacity to reduce microbial burden and suppress inflammation, thereby fostering a conducive microenvironment for tissue repair.^28–30

The pathogenesis of periodontitis is closely linked to the dysregulated expression of inflammatory mediators. Pro-inflammatory cytokines such as IL-1β and TNF-α drive tissue destruction by recruiting inflammatory cells and activating osteoclasts, while PGE2 amplifies vascular permeability and inflammatory cascades.^31,32 Concurrently, MMP-8 degrades collagen fibers, destabilizing the periodontal attachment apparatus. In our cohort, adjunctive PDT significantly attenuated GCF levels of IL-1β, TNF-α, PGE2, and MMP-8 at 3 months, underscoring its role in rebalancing the inflammatory milieu. This aligns with evidence that PDT modulates local immune responses, suppresses cytokine overproduction, and promotes resolution of inflammation—key prerequisites for tissue regeneration.^33,34

The efficacy of MB-mediated PDT is further supported by its unique photochemical properties. At 660 nm, MB achieves optimal tissue penetration, enabling ROS generation even in deep periodontal pockets. A flexible fiberoptic tip was used to deliver 660 nm laser light directly within the periodontal pocket, ensuring precise activation of the MB photosensitizer and avoiding light attenuation associated with external irradiation. Upon activation, MB generates ROS and subsequently returns to its ground state in a self-renewing cycle. The MB solution was freshly prepared to minimize molecular aggregation, thereby preserving its optimal photodynamic activity in the aqueous pocket environment.¹⁴ Studies have shown that a 60-sec irradiation with MB reduces bacterial viability in plaque biofilms by 10⁴–10⁶-fold while concurrently inhibiting MMP-8 activity and downregulating pro-inflammatory cytokine expression.^35–37 Our protocol utilized a narrow-diameter laser tip to deliver light precisely to the base of deep pockets, ensuring effective ROS generation and microbial eradication—a critical advantage over conventional mechanical debridement.^38,39

Alveolar bone regeneration remains a major challenge in advanced periodontitis. While traditional therapies often yield suboptimal bone fill, emerging adjunctive strategies aim to modulate the local microenvironment to favor osteogenesis.⁴⁰ Our study demonstrated that PDT combined with regenerative surgery resulted in superior trabecular bone density and bone fill rates compared with surgery alone. This positive outcome aligns with the findings of Cetiner et al.,²³ who reported enhanced clinical attachment gain in deep pockets and elevated osterix expression after adjunctive aPDT in regenerative surgery for Stage III/IV grade C periodontitis, suggesting a pro-osteogenic modulation. Conversely, a recent RCT found that low-level laser application-transgingival as an adjunct to nonsurgical therapy did not confer additional radiographic bone density benefits.⁴¹ This discrepancy may underscore the critical importance of the treatment context; the synergistic effect of PDT with surgical bone grafting and membrane placement, as employed in our and Cetiner’s protocol, may be essential for realizing its full regenerative potential. Mechanistically, beyond its known antimicrobial and anti-inflammatory effects, PDT may create a favorable microenvironment for bone healing by mitigating inflammation-induced impairment of stem cell function. This concept is supported by a novel study showing that an adhesive hydrogel rescued the osteogenic capacity of periodontal ligament stem cells by inhibiting inflammation-induced senescence via the NRF2 pathway, highlighting a promising target for microenvironment modulation.⁴² Future studies should investigate whether PDT exerts similar effects on cellular senescence and key pathways like NRF2 to promote osteogenesis in the human periodontium.

The significantly lower plaque scores in the Combination group at 12 months, coupled with superior reductions in keystone pathogens (P. gingivalis and T. forsythia), indicate a dual mechanism of adjunctive PDT: (1) direct antimicrobial action against anaerobic bacteria and (2) disruption of biofilm reformation. Unlike conventional surgery, PDT’s photoactivated oxidants penetrate biofilm matrices, impairing bacterial adhesion and reducing plaque accumulation. This synergistic effect explains the enhanced gingival remodeling observed clinically—reduced pathogen load and improved plaque control collectively mitigate inflammation, facilitating tissue regeneration.^43,44

Despite these advances, this study has several limitations. Outcome variability may be attributed to heterogeneous factors, including photosensitizer concentration, light dosage, and anatomical variations. Further, light attenuation within deep periodontal pockets could compromise PDT efficacy. Notably, the use of a standardized irradiation time per site did not account for variations in pocket surface area related to tooth type and morphology, which may influence the delivered energy density. While we assessed plaque samples at baseline and follow-up, and quantified the copy numbers of two key periodontal pathogens, a more comprehensive microbial analysis was lacking. The absence of high-throughput sequencing data limits our understanding of the broader shifts in the subgingival microbiome after PDT. Although clinical end-points (PD, CAL, BOP) serve as established proxies for biofilm control, direct quantification of subgingival biofilm parameters was not performed; integrating such microbiological data would significantly enhance mechanistic insights into PDT’s mode of action. Future research should focus on developing personalized PDT protocols (e.g., algorithm-based irradiation dosing according to pocket topography), standardizing other parameters, developing novel photosensitizers with improved tissue penetration, incorporating robust biofilm quantification methods, and investigating the effects of PDT on periodontal stem cells.

Conclusions

The combination of adjunctive PDT and PTR significantly improved GT remodeling, modulated key inflammatory cytokines (IL-1β, TNF-α, IL-6), and enhanced radiographic bone regeneration in patients with stage IV periodontitis. These findings demonstrate that PDT serves as an effective adjunct to conventional regenerative therapy, offering a complementary strategy for tissue repair and inflammation control. The observed outcomes support the clinical relevance of this combined approach in managing severe periodontal defects.

Authors’ Contributions

D.H.: Conceptualization, formal analysis, investigation, visualization, writing—original draft, and writing—review and editing. L.W.: Conceptualization, data curation, formal analysis, writing—original draft, and writing—review and editing. X.W.: Conceptualization, data curation, formal analysis, visualization, writing—original draft, and writing—review and editing.

Footnotes

Author Disclosure Statement

The authors declare that they have no competing interests.

Funding Information

No funding was received for this article.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

The study was approved by the Ethics Committee of Yunnan University Affiliated Hospital (No. 2022-LL-325). Patients who participated in this research signed the informed consent and had complete clinical data. Signed written informed consents were obtained from the patients and/or guardians.

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