Background <p>Mineral trioxide aggregate (MTA) is a calcium silicate-based endodontic biomaterial with well-established sealing ability, biocompatibility, and hard-tissue induction potential; however, the extent to which its biological performance can be modulated by nanostructured reinforcements remains insufficiently defined. Hexagonal boron nitride (hBN) is a layered two-dimensional nanomaterial with high chemical stability, mechanical robustness, and growing relevance in regenerative biomaterials. Because no directly comparable in vivo study had evaluated hBN incorporation into an MTA matrix in a standardized rat tibial defect model, the present work was designed as an exploratory physicochemical and histological study.</p> Methods <p>Standardized cylindrical defects (2.5&#xa0;mm diameter, 4&#xa0;mm depth) were created in the right tibial metaphysis of 40 systemically healthy rats randomly allocated to four groups (<i>n</i> = 10/group): empty defect control, pure MTA, MTA + 5 wt% hBN, and MTA + 10 wt% hBN. The study was conducted as a single 8-week in vivo experiment following institutional ethics approval. Pure MTA and hBN-modified composites were prepared under standardized mixing conditions and characterized qualitatively by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). After 8 weeks, tibiae were harvested, decalcified, paraffin-embedded, stained with hematoxylin-eosin, and scored semi-quantitatively for fibrous tissue, new bone formation, and osteoblastic cell presence. Non-parametric comparisons were performed using Kruskal-Wallis and Dunn-Bonferroni tests (α = 0.05).</p> Results <p>XRD and FT-IR analyses showed preservation of the principal calcium silicate phase architecture and hydration-related bonding environment of MTA after hBN incorporation, while SEM-EDX demonstrated a progressively denser and more hBN-rich surface organization in the modified groups, particularly at 10 wt% hBN. Histologically, both hBN-containing groups displayed significantly higher new bone formation and osteoblastic cell presence scores than the empty-defect control. Fibrous tissue scores were also higher in the hBN groups than in the control group, indicating that the tissue response should be interpreted as a modified fibro-osseous reparative profile rather than as simple suppression of fibrosis. Compared with pure MTA, the 10 wt% hBN formulation showed the most favorable overall trend within the tested range, although differences between 5 wt% and 10 wt% hBN were not statistically significant.</p> Conclusions <p>Within the boundaries of this exploratory 8-week rat tibial defect study, hBN could be incorporated into MTA without evidence of major physicochemical destabilization and with a more pronounced bone-related healing response than that observed in untreated control defects. The findings do not support an overextended claim of direct osteoblast activation or definitive concentration optimization; rather, they indicate that hBN-reinforced MTA is a promising proof-of-concept composite whose long-term remodeling behavior, mechanical performance, ion release, systemic safety, and true optimum hBN window require further quantitative investigation.</p> Graphical Abstract <p></p>