## 4 Discussion
The previous studies were designed to reproduce clinical conditions of immediate replantation. Thus, the injured pulp tissues were more likely to regenerate because all replanted teeth in the previous studies were exposed for a few seconds to minutes [7,29]. Even if the injured pulp tissue healed with hard tissue formation, it should not be regarded as a failure of pulp regeneration. Pulp canal obliteration is the compensatory response of vital pulp to a severe injury, and it is generally accepted that the incidence of pulp necrosis following pulp canal obliteration is very low [32]. Dental pulp tissues appeared to be healed with fibrous, osteoid, and cementoid tissue formation in the present study. These results corresponded with other studies, suggesting that newly formed pulp tissues after regenerative endodontic therapy are composed of periodontal-like, bone-like, and cementum-like tissues, rather than dentin [33,34,35]. These findings support that the periodontal ligament, bone, and cementum may grow into the pulp canal during the healing process after replantation [35]. Several studies suggested that this healing response may initiate and progress in the absence of pulp tissue in the pulp canal [36,37]. Moreover, bone-like tissues were deposited in the case of a large amount of external root resorption extending beyond the inner dentin surface (Figure 2c,d). It is possible that the endogenous cells located around the root surface were recruited into the pulp canal and differentiated to bone/cementum-like cells because direct communication into the pulp canal was easily available. Doxycycline has been used in attempts to promote healing of avulsed teeth after replantation, and some authors reported a positive effect of doxycycline on pulp revascularization after replantation [5, 6, 7]. They suggested that this effect is associated with the antimicrobial activity of doxycycline because doxycycline could decrease the number of micro-organisms on the root surface during extra-oral time. However, there is controversy about the effect of doxycycline on pulp regeneration after replantation. Some studies reported that doxycycline had no effect on micro-organisms in necrotic pulp. Doxycycline only had an effect on inflammatory inhibition, such as reducing the occurrence of inflammatory resorption or ankylosis [38]. The direct effect of doxycycline on angiogenic capacity is not fully identified in the pulp tissue of an avulsed tooth [24]. Therefore, angiogenic regenerative potential could be achieved using NO-releasing nanomatrix gel. NO is known for its bio-active functions such as the promotion of angiogenesis and subsequent development of mature blood vessels, along with the release of vascular endothelial growth factor (VEGF) [39]. Furthermore, NO inhibits apoptosis of vascular endothelial cells induced by proinflammatory cytokines and proatherosclerotic factors, including angiotensin II [23]. Apoptosis of endothelial cells may contribute to the development of inflammatory processes. Thus, NO can inhibit inflammatory processes. Our results indicated that NO-releasing nanomatrix gel decreases the degree of pulp inflammation. The study includes several limitations. The sample size was rather small because the study was designed as a pilot study; future studies will be verified further using an increased sample size. The regenerative potential of NO-releasing nanomatrix gel is reported to have therapeutic functions in bacterial infections, would healing, and cardiovascular diseases [21,22,39]. However, the interaction between the NO-releasing nanomatrix gel and doxycyclineine is not reported yet; therefore, future studies will be performed to characterize the conditions in vitro, such as optimal concentrations of NO and doxycycline. It is difficult to predict the effect of doxycycline and NO on pulp regeneration after in vivo replantation and follow-up. Thus, the present study referenced the previous regimen of doxycycline with saline and modified the regimen using NO-releasing nanomatrix gel as a substitute for saline [4].