Comparative Assessment of Five Decalcification Methods for Human Dental Tissue: An Ex Vivo Laboratory Study

Histological examination of human teeth is essential for the diagnosis of developmental anomalies, odontogenic lesions, and other dental pathologies. However it remains technically challenging because highly mineralized tissues require extensive processing before thin sections suitable for microscopic evaluation can be obtained. This study aimed to identify a decalcification protocol suitable for routine dental pathology by balancing rapid processing with high-quality tissue preservation. To achieve this, we compared five commonly used decalcification protocols using complementary histological and spectroscopic analyses.

Forty-nine human teeth were decalcified using one of the five investigated agents: HNO₃, HCOOH, DC1, Microdecfast, or Löwy's solution. Decalcification speed, sectioning quality, and histological staining characteristics (H&E, PAS, Gömöri, and von Kossa) were evaluated. Laser-induced breakdown spectroscopy (LIBS) was used for spectral analysis, 2D elemental mapping, and semi-quantitative assessment of Ca, Mg, P, and C, enabling direct comparison of demineralization efficiency and spatial homogeneity across agents.

Decalcification efficiency differed markedly among the tested agents. HNO₃ provided the fastest and most consistent tissue softening, whereas Löwy's solution was the slowest and often failed to achieve complete decalcification. LIBS demonstrated that HNO₃ and DC1 produced the most homogeneous depletion of Ca, Mg, and P, while Microdecfast and HCOOH resulted in less uniform mineral removal. All decalcifying agents depleted mineral elements beyond calcium, indicating that some protocols (HNO₃ and DC1) may be unsuitable for studies requiring preservation of the native elemental composition. Less homogeneous demineralization was associated with harder tissues, inferior sectioning quality, and increased sample fragmentation.

By integrating histology with LIBS analysis, this study demonstrates that decalcifying agents differ substantially in their decalcification speed, tissue preservation, and mineral removal profiles. Notably, 5% HNO₃ emerged as the most efficient agent, enabling rapid decalcification with preserved staining quality and homogeneous mineral depletion. These findings provide practical guidance for diagnostic histopathology laboratories by facilitating faster processing of dental specimens while maintaining the tissue quality required for accurate microscopic diagnosis.