Postembedding Decalcification of Mineralized Tissue Sections Preserves the Integrity of Implanted Biomaterials and Minimizes Number of Experimental Animals

Abstract

Bone histology of decalcified or undecalcified samples depends on the investigation. However, in research each method provides different information to answer the scientific question. Decalcification is the first step after sample fixation and governs what analysis is later feasible on the sections. Besides, decalcification is favored for immunostaining and in situ hybridization. Otherwise, sample decalcification can be damaging to bone biomaterials implants that contains calcium or strontium. On the other hand, after decalcification mineralization cannot be assessed using histology or imaging mass spectrometry. The current study provides a solution to the hardship caused by material presence within the bone tissue. The protocol presents a possibility of gaining sequential and alternating decalcified and undecalcified sections from the same bone sample. In this manner, investigations using histology, protein signaling, in situ hybridization, and mass spectrometry on the same sample can better answer the intended research question. Indeed, decalcification of sections and grindings resulted in well-preserved sample and biomaterials integrity. Immunostaining was comparable to that of classically decalcified samples. The study offers a novel approach that incites correlative analysis on the same sample and reduces the number of processed samples whether clinical biopsies or experimental animals.

Confirmation of Calcium Phosphate Cement Biodegradation after Jawbone Augmentation around Dental Implants Using Three-Dimensional Visualization and Segmentation Software

“…Preclinical studies on the Paste-CPC utilized in this study reflected the enhancement of bone healing and increased degradation compared to empty defect in rat model through histological, chemical and mechanical examination [50,57,58]. Those results are in line with this study as reflected in the volume measurements (Figure 8)…”

Optimization of Formic Acid-Formalin-Based Decalcification Protocol for Rat Calvarial Bone Histology

“…Decalcification is performed in bone healing studies to make the bone tissue transparent or softer for better visualization and analysis under a microscope (El Khassawna et al 2017). Bones naturally contain calcium salts, which make them hard and opaque, hindering the examination of cellular structures and detailed analysis of the healing process (Barrère et al 2006).…”

Landscape of Well-Coordinated Fracture Healing in a Mouse Model Using Molecular and Cellular Analysis

“…Left femora were harvested with surrounding muscle tissue after euthanasia and fixed in 4% paraformaldehyde (PFA) for the histological examinations as described previously [ 52 , 89 ]. The bone samples for histology were collected at D7, D10, D14, D21, and D28.…”