This study is a clinical investigation designed to evaluate the efficacy of curcumin-mediated photodynamic therapy (PDT) on tissue repair of oral vesiculobullous lesions in patients with epidermolysis bullosa (EB), as well as its impact on quality of life and the identification of salivary biomarkers associated with disease status and healing processes. EB is a rare disorder characterized by extreme fragility of the skin and mucosa, leading to painful blister formation and chronic lesions that significantly impair oral function, nutrition, and overall quality of life. Given the absence of curative treatments, therapeutic strategies that promote tissue repair and reduce symptom burden are of high clinical relevance.
Participants will include children, adolescents, and adults diagnosed with EB who are able to feed orally, as well as a control group without EB matched by age and sex. Recruitment will take place at a specialized reference center for neurodevelopment and rehabilitation, with additional control sampling conducted at a university-based clinical facility. All participants or their legal guardians will provide informed consent prior to enrollment, and assent will be obtained from minors when applicable.
The study protocol includes clinical evaluation, questionnaire-based assessments, salivary sample collection, and application of photodynamic therapy. Clinical oral examination will assess dental caries using the International Caries Detection and Assessment System (ICDAS), molar-incisor hypomineralization using the SES-HMI index, and soft tissue lesions characterized by type, color, and anatomical location. Pain intensity associated with oral lesions will be measured using the Wong-Baker Faces Pain Rating Scale at baseline and for seven consecutive days following treatment.
Photodynamic therapy will be performed using a 0.1% curcumin gel applied topically to oral lesions for five minutes, followed by irradiation with a blue LED light source (wavelength 440-480 nm) for approximately 90 seconds, delivering an estimated energy dose of 9 J. The procedure will be repeated over three consecutive days. The primary outcome is tissue repair, assessed clinically, while secondary outcomes include pain reduction and improvement in oral health-related quality of life.
Quality of life will be evaluated using validated instruments appropriate to age group. For children, the Parental-Caregiver Perceptions Questionnaire (P-CPQ) will be administered to caregivers, while adolescents and adults will complete the Oral Health Impact Profile (OHIP-14). These instruments will be applied prior to each PDT session to assess changes over time. Dietary intake will also be assessed using a validated food frequency questionnaire to account for potential nutritional influences on oral health and healing.
Saliva will be collected as a non-invasive biological fluid for biomarker discovery. Unstimulated saliva samples will be obtained in the morning using standardized procedures, stored under controlled conditions, and analyzed using advanced omics approaches, including mass spectrometry (ESI-MS and LC-MS), Fourier-transform infrared spectroscopy (ATR-FTIR), and chemometric analysis. Data processing will include normalization, spectral correction, and multivariate analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Machine learning algorithms-including logistic regression, linear discriminant analysis, random forest, and support vector machines-will be applied to classify and identify relevant biomarker patterns. Model validation will be conducted using repeated stratified cross-validation procedures.
Quality assurance procedures will be implemented to ensure data integrity and protocol adherence. A standardized data collection framework will be used, with all study personnel trained and calibrated prior to data acquisition. Data entry will follow predefined validation rules, including range checks, logical consistency checks, and automated verification procedures to identify discrepancies or missing values. Source data verification will be conducted through comparison with original clinical records and case report forms to ensure accuracy and completeness.
A comprehensive data dictionary will be developed, defining all variables collected in the study, including their sources, coding structures, permissible values, and reference ranges where applicable. Standardized terminologies and classifications will be applied when relevant to ensure interoperability and reproducibility. Standard Operating Procedures (SOPs) will guide all stages of the study, including participant recruitment, clinical examination, sample collection and handling, data management, statistical analysis, adverse event reporting, and protocol deviations.
Monitoring procedures will include periodic internal review of study conduct, adherence to protocol, and data quality. Although this is a single-center study, audit readiness will be ensured through proper documentation and traceability of all procedures. Any protocol amendments or deviations will be documented and managed وفق established change control processes.
The sample size is defined based on the available population of EB patients treated at the recruitment center, given the rarity of the condition. While formal power calculation is limited by feasibility constraints, the study is designed to generate exploratory and hypothesis-generating data regarding treatment efficacy and biomarker identification.
Missing data will be handled using predefined strategies depending on the nature and extent of missingness. Data will be categorized as missing, not applicable, or not interpretable, and appropriate statistical techniques such as imputation or sensitivity analyses will be considered where necessary to minimize bias.
Statistical analysis will include descriptive statistics for all variables, with qualitative data expressed as frequencies and percentages and quantitative data summarized using means, standard deviations, medians, and interquartile ranges. Inferential analyses will be conducted to compare outcomes within groups (pre- and post-treatment) and between groups (EB versus controls). Parametric or non-parametric tests will be applied בהתאם data distribution, with a significance level set at 5%. Receiver Operating Characteristic (ROC) curve analysis will be used to evaluate the diagnostic performance of identified biomarkers, and correlation analyses will assess associations between clinical findings and molecular data.
All analyses will be performed using validated statistical software. The study follows recognized clinical research guidelines and ethical standards, ensuring participant safety, confidentiality, and scientific rigor. The results are expected to contribute to the development of innovative, non-invasive therapeutic and diagnostic strategies for patients with epidermolysis bullosa.
