## 4 Discussion  
With the aim of understanding the differences that occur during the healing process of immediate and delayed implants, we conducted this investigation. The results from the present in vivo experimental investigation demonstrated that during the early healing of immediate implants in fresh extraction sockets, there was a significantly higher vertical dimension of the supracrestal soft tissues compared to implants placed in healed ridges,  
Figure 9: Immunohistochemical analysis of alkaline phosphatase. (**A**) Immediate implant 2 weeks. (**B**) Delayed implant 2 weeks. (**C**) Immediate implant 8 weeks. (**D**) Delayed implant 8 weeks.  
although these differences tended to diminish over time. Conversely, soft tissues were thicker in the delayed implant group, especially in the early healing period. The immunohistochemical analysis revealed a significantly higher expression of the proteins ALP and significantly higher at immediate implants after 2 weeks of healing, which translates to an increased MMP2 expression during the proliferative stage of socket healing [36].  
Another key biomarker in bone metabolism is TRAP, a glycoprotein expressed during bone resorption, which is secreted by mature osteoclasts, activated macrophages and dendritic cells [31; 37]. Our results did not find differences between groups, although there was a trend towards higher TRAP expression in delayed healing implants during the late healing phases. These results are in accordance with the histological outcomes reported in the previous report of this investigation, where the buccal bone resorption was more pronounced in the delayed implant group at 8 weeks [16].  
Overall, these immunohistochemical results highlight a higher expression of bone metabolism biomarkers during the early healing phases, especially on immediate implants. Similarly, [PERSON] et al. [38] observed that after 8 weeks healing, immediate implants achieved higher bone to implant contact when compared with delayed implants. [PERSON] et al. [17] also observed higher bone-to-implant contact in immediate implants than in delayed implants after 4 weeks of healing. Moreover, studies in rodents have shown an increased bone osseointegration in dental implants facing periodontal ligament remnants in postextraction sockets, compared to implants around pristine bone [39; 40].  
The results from this experimental in vivo investigation should be interpreted with caution considering the potential limitations of the experimental design, which may not allow for direct human translation of the outcomes. Furthermore, the use of different surgical approaches when placing the implants (flapped in the delayed implants versus flapless in the immediate implants) may account for different bone resorption patterns [24]. However, we chose these different surgical protocols since these are the ones regularly used in clinical practice. Another limitation is related to the inherent limitations of the histological methods utilized: on one hand the use of decalcified sections for the soft tissue histomorphometry, although providing thinner sections and allowing for more precise location of the limits of the epithelium and the bone to implant contact, are more difficult to orientate and several samples had to be excluded of from the analysis due to the presence of alterations that limited the precise measurement of the tissue dimensions. When comparing measurements from decalcified versus undecalcified sections, however, differences were not statistically significant between methods. Regarding the immunohistochemical analysis, one limitation could be the semiquantitative nature of the results, with the possibility of including false positives. However, to avoid this problem, only positive and highly positive results were included [20]. Also, there was no agreement whether the immunohistochemical analysis should be performed from decalcified or from ground sections, as proposed by some authors [41].