Multimodal analysis of biomineralization within a collagen scaffolding in a rat calvarial defect model by using decalcified and undecalcified specimens

This study histologically evaluated the biomineralization process during bone regeneration and the in vivo behavior of a collagen sheet used as scaffolding in a rat 5-mm calvarial defect model. Two experimental groups were established: a group using collagen sheet and bone substitute (BC group), and a group using bone substitute alone (BO group). Bone regeneration was assessed by computed tomography (CT) and both decalcified and undecalcified sections were analyzed using histological staining (hematoxylin and eosin, Villanueva-Goldner [VG], von Kossa, and Join of the Five dyes Revealing CoLlagenous tissue [JFRL]), immunohistochemistry, polarized light microscopy, and low-vacuum scanning electron microscopy (LV-SEM) combined with energy-dispersive X-ray spectroscopy (EDX). CT revealed time-dependent defect reductions, progressing significantly faster in the BC group. In undecalcified specimens, VG staining demonstrated a thick, red, osteoid layer, and serial sections stained with von Kossa showed granular blackish-brown deposits within this layer. LV-SEM/EDX confirmed localized Ca/P accumulation in these deposits, indicating initial biomineralization foci. In decalcified JFRL-stained sections, JFRL color profiles corresponded to gray-scale contrast in LV-SEM images, reflecting collagen fibril organization and the degree of biomineralization. Polarized observation of undecalcified, VG-stained, polished sections revealed the emergence and temporal expansion of orange birefringence within the transplanted collagen sheet and surrounding connective tissue. Immunohistochemistry demonstrated BrdU-, Runx2-, and osterix-positive cells, and osteopontin localization within newly formed matrix in the defect, indicating active osteoblastogenesis. Collagen sheets appear to function not only as physical scaffolding, but also as a bioactive matrix promoting biomineralization by modulating cellular activity and matrix remodeling.