## 4 Discussion  
In recent years, there has been an increased utilization of orthopedic and dental implants and methods for guided bone regeneration. This has attracted the attention of re-searchers and led to a comprehensive histological evaluation of the bone--implant and bone--soft-tissue interfaces. The conventional techniques for histological observation have been insufficient for studying undecalcified bone specimens and biomaterials. This has necessitated the use of embedding media in which the integrity of both tissues and im-plants is preserved. An MMA-based medium meets these criteria. It allows a distinction \begin{table}
\begin{tabular}{c c} \hline
**Solution** & **Immersion Time** \\ \hline
70\% EtOH & 20 min \\ dH\({}_{2}\)O & 2 min \\ Toludiine blue & 30 min \\ dH\({}_{2}\)O & Quick rinse \\ Air dry & 24 h \\ Xylene & 1 min \\ \hline \end{tabular}
\end{table} 
Table 6: A modified protocol for staining with toluidine blue. Figure 4: The stages of the suggested protocol for staining with toluidine blue. Figure 3: Photomicrographs of cross-sections through rabbit tibia—magnification 4\(\times\) (**a**) and 20\(\times\) (**b**). Toludiine blue staining demonstrates osteons (Haversin systems), cement lines at their boundaries, and interstitial and circumferential lamellae. between the mineralized and unmineralized phases of bone and detailed visualization of cellular components [1]. The staining of resin-embedded samples requires the use of stains with a molecular weight that allows their passage through the resin. In this research, we used toluidine blue staining, which meets the above-mentioned requirement. It demonstrates excellent properties in the detection of cell nuclei and bone cells (osteoblast, osteocytes, and osteoclasts), as well as the non-mineralized osteoid areas. Modifications of the staining protocols for resin-embedded sections are often necessary, since these techniques are commonly performed without the removal of the plastic. Therefore, different regimens, durations, and adjustments of the conditions could be applied [19]. The optimal staining protocol offers simplicity of execution, repeatability, cost-effectiveness, and the clear identification of different structural and cellular components and the distinctions between them. A modified protocol for staining undecalcified bone specimens using toluidine blue is introduced in this research after qualitative evaluation of various staining techniques. The conducted experiments in rabbits aimed to validate the modified staining protocol for histological visualization of bone microstructure (osteons, cement lines, etc.) and, thus, to minimize the risk of unsatisfactory staining of human bone specimens used for research in the field of implant dentistry. Our findings are consistent with [PERSON], [PERSON], and [PERSON] [43], who also suggested a staining protocol using toluidine blue solution without acid pre-etching. However, [PERSON] and [PERSON] [33] and [PERSON] and [PERSON] [30] reported that the acid-etching stage provides bone decalcification and is necessary for optimal staining results. This study heterogeneity could be due to differences in the pH of the applied toluidine blue solutions. While [PERSON] and [PERSON] [33] and [PERSON] and [PERSON] [30] used toluidine blue with pH 7.0 to 9.0, the pH of the solution used by [PERSON], [PERSON], and [PERSON] [43] was 3.5 and, in our research, it was 2.0. Moreover, it has been demonstrated that immersion in alcoholic solutions has etching properties. It softens the embedding media and allows for easier penetration of the dye [30]. These findings imply that the staining protocol can be significantly simplified by the application of toluidine blue with a lower pH. The suggested protocol allows for the clear identification of bone matrix and cellular components using undecalcified bone slices with a thickness of 20-40 \(\upmu\)m. Studies on implant osseointegration often use bone specimens with a thickness in this range [37; 38; 40].