## 2 Materials and Methods  
In the current research, plastic-embedded undecalcified sections from rabbit tibia were used. The embedding medium was Technovit(r) 9100 NEW, (Heraeus Kulzer GmbH, Wehrheim, Germany), which consists of a monomer (MMA, Basic solution), a polymer (polymethyl methacrylate--PMMA), an initiator (Hardener 1), an activator (Hardener 2), and a regulator. The procedure included several steps: fixation, dehydration, pre-infiltration, infiltration, and polymerization (Table 1). The specimens were obtained from the tibial bones of rabbits aged 5-6 months. The soft tissues were removed, and the bone was sliced into sections with approximate length of 5 mm. Samples were fixed for 3 days in 10% buffered formalin (10% NBF, Biognost, Zagreb, Croatia). After fixation, the bones were dehydrated in a series of alcohol solutions of increasing concentration (70% EtOH, 80% EtOH, 90% EtOH, 96% EtOH, 99.8% EtOH) and cleared in xylene. Dehydration is usually performed with alcohol (ethanol) through a series of increasing concentrations that range from 70% to absolute alcohol. Otherwise, premature immersion in solutions with higher concentrations can cause tissue shrinkage and hardening. The samples were then infiltrated and embedded in the MMA-based resin Technovit(r) 9100 NEW (Heraeus Kulzer GmbH, Wehrheim, Germany), according to the manufacturer's instructions. The medium has a low viscosity that ensures fast infiltration. However, it demonstrates high hardness after the polymerization phase. These features make it suitable for hard-tissue histology, such as compact bone, and specimens containing medical and dental implants [1]. **Preparing the solutions:**  
Pre-infiltration 1: Xylene and stabilized basic solution were mixed in a 1:1 ratio. The product was then transferred to a dark glass bottle and stored in a freezer at a temperature of \(-20\,^{\circ}\)C. \begin{table}
\begin{tabular}{c c c c} \hline
**Stages** & **Solution** & **Time** & **Condition** \\ \hline Fixation & 10\% NBF & 72 & RT \\ \hline \multirow{8}{*}{Dehydration} & 70\% EtOH & 4 & RT—vacuum \\  & 80\% EtOH & 4 & RT—vacuum \\  & 90\% EtOH & 16 & RT \\  & 96\% EtOH & 4 & RT \\  & 96\% EtOH & 4 & RT \\  & 99.8\% EtOH & 16 & RT \\  & 99.8\% EtOH & 4 & RT \\  & 99.8\% EtOH & 4 & RT \\  & & Xylene & 16 & RT \\  & & Xylene & 8 & RT \\ \hline Pre-infiltration 1 & Xylene + stabilized MMA (1:1) & 24 & RT—with agitation \\ \hline Pre-infiltration 2 & 200 mL stabilized MMA + 1 g & 24 & RT—with agitation \\  & & Hardener 1 & 24 & 4 \({}^{\circ}\)C—with agitation \\ \hline Pre-infiltration 3 & 200 mL destabilized MMA + 1 & & \\  & g Hardener 1 & & \\ \hline \multirow{2}{*}{Infiltration} & 250 mL destabilized MMA + 20 g PMMA + 1 g Hardener 1 & & \\ \hline Polymerization & Solution A + Solution B (9:1) & 120 & \(-20\,^{\circ}\)C; \\ \hline \end{tabular} RT—room temperature; EtOH—ethyl alcohol (ethanol); MMA—methyl methacrylate; PMMA—poly(methyl methacrylate).  
\end{table} 
Table 1: Laboratory protocol for processing undecalcified bone specimens. Pre-infiltration 2: One gram of Hardener 1 was added to 200 mL stabilized basic solution and mixed for 1 h using a magnetic stirrer. The product was then transferred to a dark glass bottle and stored in a freezer at a temperature of \(-20\)\({}^{\circ}\)C.  
Destabilization of the basic solution: For this purpose, we used column chromatography. The first step was loading the column with 50 g Al\({}_{2}\)O\({}_{3}\) (Aluminium oxide 90 basic, Adsorbent for Column chromatography, Macherey-Nagel GmbH & Co.KG, Duren, Germany), and then the basic solution was passed slowly through the column. About 300 mL of MMA was destabilized in one hour. The destabilized solution could be divided into portions in dark glass bottles and stored at a temperature of \(-20\)\({}^{\circ}\)C for up to 6 months. Pre-infiltration 3: One gram of Hardener 1 was added to 200 mL destabilized MMA and mixed for 1 h using a magnetic stirrer.