Characterization of Pulp Inflammation: an Observational Study Using Biological Samples Collected During Routine Dental Care

Vital pulp therapy (VPT) is a key component of minimally invasive dentistry and aims to preserve pulp vitality whenever clinically feasible. Teeth with vital pulp have been shown to exhibit better long-term survival than endodontically treated teeth. Consequently, VPT procedures-including indirect pulp capping, direct pulp capping, partial pulpotomy, and full pulpotomy-should be preferred over root canal treatment when the biological status of the pulp allows it. The success of VPT relies on two essential factors: the use of bioactive materials capable of promoting pulpal healing, and an accurate assessment of the severity and extent of pulpal inflammation. However, complications related to inappropriate case selection remain frequent and often occur within the first year following treatment. These failures are largely attributed to the difficulty of precisely evaluating the inflammatory status of the pulp, highlighting a major unmet need in endodontic diagnosis, particularly in the context of emergency care. Pulpal inflammation is a defense response of the dental pulp to external aggression such as carious lesions, fractures, periodontal disease, or restorative procedures. This biological process is dynamic, progressive, and spatially heterogeneous. Inflammation initially develops locally in the pulp tissue adjacent to the injury and may subsequently extend to involve the entire coronal pulp and, in more advanced stages, the radicular pulp. When inflammation remains confined to the coronal pulp, removal of the inflamed tissue through partial or total pulpotomy followed by the application of a bioactive material may be sufficient to achieve healing and long-term success. At the clinical level, however, the assessment of pulpal inflammation remains challenging. The most widely used diagnostic classification of pulpal diseases is mainly based on patient-reported pain characteristics and responses to thermal sensibility tests. This classification distinguishes healthy pulp, reversible pulpitis, irreversible pulpitis (acute or chronic), and pulp necrosis. Intraoperative criteria, such as the evaluation of bleeding and hemostasis, are sometimes used to estimate pulpal status, but these criteria remain subjective and poorly standardized. Several studies have demonstrated discrepancies between clinical diagnosis and the actual histological condition of the pulp tissue. Moreover, the current diagnostic framework does not allow a clear correspondence between clinical diagnosis and the indication for specific VPT procedures. While four VPT techniques are available, only three inflammatory diagnostic categories are recognized, which contributes to uncertainty in therapeutic decision-making and significant variability in clinical practice. In 2017, a new diagnostic classification known as the Endolight classification was proposed. Based on histological observations of inflamed pulp tissue and prognostic data from VPT outcomes, this classification distinguishes between initial, mild, moderate, and severe pulpitis. It aims to better guide treatment selection and reduce variability in clinical practice. However, this classification has not yet been validated through studies directly comparing clinical diagnosis with in situ biological analyses of human pulp tissue. At the tissue and molecular levels, several studies have explored the mechanisms underlying pulpal inflammation. Histologically, reversible forms of pulpitis are characterized by preserved odontoblastic layer continuity and absence of necrotic tissue, whereas irreversible forms are associated with disruption of the odontoblastic layer, inflammatory infiltrates, necrosis, or abscess formation. Molecular studies have identified several inflammatory mediators whose expression increases with the severity of pulpal inflammation, including interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor alpha (TNF-α). The identification of reliable molecular and cellular biomarkers remains a major challenge but represents a critical step toward the development of more accurate diagnostic and prognostic tools. The present study is designed to characterize the immuno-inflammatory profiles of human dental pulp across different clinical forms of pulpal inflammation by integrating clinical, transcriptomic, and proteomic data. This is a prospective, monocentric observational study conducted at the Department of Dental Medicine of Pitié-Salpêtrière Hospital. Patients are recruited during routine dental care, and pulp tissue samples or whole pulp-containing teeth are collected as operative waste during endodontic treatment or tooth extraction. No additional procedures are performed, and patient care is not modified by participation in the study. Adult patients (≥18 years) whose pulp tissue is removed as part of standard care and who do not express opposition to participation are eligible for inclusion. Patients who have taken anti-inflammatory medication within 12 hours prior to the procedure or who are under legal protection are not included. Each participant is included for a single visit corresponding to the dental procedure during which the sample is collected, with no subsequent clinical follow-up. Clinical and demographic data are extracted from medical records and include patient-related variables (age, sex, medical history, medication, pain intensity assessed using a visual analog scale) and tooth-related variables (clinical pulpal diagnosis according to both the current classification and the Endolight classification, tooth type and number, oral pathology affecting the tooth and its severity, restorative status and quality, and radiographic parameters). Biological samples are pseudonymized at the time of collection and processed according to the planned analyses. Transcriptomic profiling is performed using RNA sequencing, with validation by real-time quantitative PCR and in situ hybridization. Proteomic analyses, including degradomic approaches, are performed using mass spectrometry; this component of the study is conducted within a specific project funded by the French National Research Agency (ANR), whose objectives are fully integrated and compatible with the present protocol. Protein expression is further assessed using ELISA assays and immunohistochemical or immunofluorescence analyses. The primary outcome of the study is the identification and characterization of immuno-inflammatory gene and protein expression profiles in human dental pulp tissue according to the clinical diagnosis of pulpal inflammation. Secondary outcomes include the assessment of concordance between clinical diagnostic categories-particularly those of the Endolight classification-and biological inflammatory profiles, as well as the analysis of associations between inflammatory profiles and patient-related or tooth-related factors. Biological samples are stored only for the duration of the research and are used exclusively for the analyses described in this protocol. All samples are destroyed at the end of the study, and no long-term biological collection is constituted. By providing an integrated clinical and biological characterization of pulpal inflammation, this study aims to support more precise, biology-driven diagnostic and therapeutic decision-making, improve the indication of vital pulp therapies, and ultimately optimize the management of endodontic emergencies.