Proteomics of Cervical Mineralized Diaphragm in Molar Root–Incisor Malformation

Molar root–incisor malformation (MRIM) is characterized by abnormalities in the root and pulpal floor, which may lead to dental complications. However, research on MRIM remains limited and is largely confined to case-based observations. Therefore, this study aimed to characterize the morphology and proteomic profile of the cervical mineralized diaphragm (CMD) in MRIM. Extracted MRIM-affected teeth ( n = 11) from 6 patients and extracted third molars as controls ( n = 11) were collected. Two MRIM-affected teeth and two control teeth were subjected to micro–computed tomography and scanning electron microscopy. CMD tissues adjacent to the pulpal floor and control pulpal-floor dentin were harvested for protein extraction and analyzed by liquid chromatography–tandem mass spectrometry. Label-free quantification and bioinformatics analyses (gene set enrichment and protein–protein interaction network analysis) were performed, and proteins with >2-fold change were considered differentially expressed. Micro–computed tomography demonstrated a highly radiopaque CMD at the pulpal floor that occluded pulp–root canal communication, with a radiodensity between that of the enamel and dentin and a dense/porous internal architecture. Scanning electron microscopy revealed columnar and crystal-like structures. Proteomic profiles differed between MRIM and controls, with reduced epithelial–mesenchymal transition signaling in MRIM (normalized enrichment score = 1.47, false discovery rate = 0.116; control vs. MRIM). A total of 116 proteins showed >2-fold change (62 upregulated and 54 downregulated). Upregulated proteins included keratinization-associated proteins (KRT75, KRT82, EVPL, and KRT6B) with enrichment of keratinization- and epidermis-related terms, whereas downregulated proteins included SPP1, AMBN, and ECM1, which were associated with biomineral tissue development. Within the limits of this study, the CMD in MRIM exhibits a distinctive mineralized microarchitecture and a proteomic signature implicating altered epithelial-associated and extracellular matrix/mineralization processes. These findings provide candidate targets for tissue-level validation and mechanistic studies of MRIM.