## 4 Discussion  
In the present study, the effect of root surface treatment with doxycycline-loaded NO-releasing nanomatrix gel was evaluated for pulp regeneration in replantation of avulsed rat teeth. Because the purpose of this study was to evaluate regenerative potentials for pulp tissues, only avulsed teeth preserved within 1 h in storage media were included. In spite of the avulsed tooth being preserved in an ideal storage medium, the condition of the periodontal ligament cells may be viable but compromised. In addition, an avulsed tooth with an extra-oral time of longer than 1 h cannot maintain its vitality [4; 30]. The duration of storage time after the avulsion may affect cellular viability. In the present study, the grade of pulp regeneration in Group I of replantation after 60 min storage was significantly increased compared to Group II. Additionally, greater vessel proliferation was observed in Group I of replantation after 60 min storage. However, the grade of pulp regeneration did not show significant differences between the two groups of replantation  
Figure 4: Immunohistochemical (IHC) analysis. (**a–d.i–l**) IHC stains of Group I and (**e–h,m–p**) IHC stains of Group II. (**b,d,f,h,j,l,n,p**) High-magnification images of pulp proper of (**a,c,c,e,j,k,m,o**), respectively. (**a,b**) Healing with calcified tissue is observed. Infiltration of macrophages is hardly visible compared to Group II. (**c,d**) CD31-positive cells (asterisks) are visible around the vessel. Furthermore, an extensive amount of calcified tissue is formed around vessel. (**e,f**) Broad infiltration of macrophages is visible. (**g,h**) CD31-positive cells are visible around the vessel. An extensive amount of calcified tissue is formed around the vessel. (**i,j**) Healing with calcified tissue is observed. Additionally, infiltration of macrophages is hardly visible compared to the control group. (**k,l**) Regenerated pulp tissue similar to normal pulp tissue is visible. Greater vessel proliferation is observed compared to the control group. (**m,n**) Complete destruction of root with severe degree of inflammation (yellow arrows). Infiltration of macrophages in pulp proper is also visible. (**o,p**) Vessel proliferation is observed with mild amount of infiltration of macrophages. Scale 190 bars = 100 \(\mu\)m for (**a,c,e,g,i,k,m,o**) and 10 \(\mu\)m for (**b,d,f,h,j,l,n,p**). CT, calcified tissue; M, macrophage; BV, blood vessel. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Group I** & **Group II** & **\(p\)-Value** \\ \hline Pulp regeneration & 2.57 \(\pm\) 0.85 & 1.57 \(\pm\) 1.02 & 0.012 * \\ Pulp inflammation & 1.00 \(\pm\) 0.68 & 1.57 \(\pm\) 0.51 & 0.025 * \\ \hline \hline \end{tabular}
* = statistically significant (\(p<\) 0.05, Mann–Whitney U test).  
\end{table} 
Table 3: Histomorphometric analysis of the replantation after 60 min storage. There were statistical differences in the areas of pulp regeneration and pulp inflammation in Groups I and II. after a 30 min storage period. A possible explanation may include different estimated viable cell numbers according to storage duration. Pulp cells preserved in a 30 min storage media are highly likely to maintain their viabilities compared to pulp cells in a 60 min storage media. Thus, it was shown that pulp cells preserved in a storage media for 30 min can have greater regenerative potential. Pulp tissues of Group I healed with the following three patterns: normal pulp with disorganization of odontoblastic layer; internal reparative dentin formation with connective tissue, bone-like, and/or cementum-like tissue; and pulp necrosis. Our results are different from those of previous replantation studies of doxycycline, which reported that some replanted teeth treated with doxycycline healed with normal pulp tissue and reestablishment of intact odontoblastic layer [6,31]. This comparison may have been affected by different durations of extra-oral time rather than the effect of PA-YK-NO gel.