## 4 Percentage of _P. gingivalis_ Inhibition with D-CTL Inside the Gel
Differences of antimicrobial action between L-CTL and D-CTL could be explained by the opposite chiral conformation of the peptides [49]. Cleavages are usually performed on the weakest peptide bond [37] (Figure 4) and we found cleavages of peptidic bonds involving S, G, and A. All these experiments should be carried out by changing pure water that degrades the peptide [59] with human saliva that represents a better physiological condition. Moreover, the resistance of D-CTL to human endoproteases strengthens the interest of this AMP [49]. Figure 3: Determination of the MIC of D-CTL against _P. gingivalis_. (**a**) The MIC of D-CTL in water was estimated to be 30 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) (no recontamination); (**b**) percentage of _P. gingivalis_ (_P.g_) inhibition when bacteria is in contact with gel. The efficient concentration of D-CTL inside gel is 470 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) at more than 24 h (3 mg\(\cdot\)mL\({}^{-1}\) from the stock solution). Microscopic views (optical microscopy and SEM) show that the surface of the gel without AMP is completely colonized by _P. gigivalis_ at 24 h (Figures 5a and 6c,d), and TEM confirms these results (Appendix A Figure A3). Bacteria grow on the whole surface of the gel but do not grow inside the bulk, this was certainly because of the strengthened framework. Adjunction of D-CTL inside the gel impedes bacterial colonization (Figures 5b and 6e,f) after 24 h. Hence, it is a candidate for early healing inside implant cavities but also for other medical applications. A longer time of in vitro evaluation is needed to determinate the maximum time of antibacterial action at the surface and if bacteria grow inside the gel. In this way, we could see if a part of CTL, which certainly participates in the structure of the gel, is released during aging in a wet environment. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Main** **Peptides** & **Experimental** **Molecular** **Weight (_m/z_)** & **Theoretical Molecular** **Weight (_m/z_)** & **Theoretical Sequences of Peptide (with/without Oxygen)** \\ \hline CTL & 1859.96 & 186.17 & RSMRLSFRERGYGFR \\ \hline A & 1620.83 & 1620.81 & RSMRLSFRERGY + 4 \\ \hline B & 1563.84 * & 1563.78 & -SMRLSFRERGYGFR + 1 oxydation \\ \hline C & 1448.78 & 1448.62 & -SMRLSFRERGY + 3 oxydations \\ \hline D & 1279.64 & 1279.71 & —RSMRLSFR \\ \hline E & 1052.61 & 1052.57 & —RSMRLSFR \\ \hline F & 989.59 * & 989.49 & \
\begin{tabular}{c} FRARYGF + 1 \\ oxydation \\ \end{tabular} \\ \hline G & 769.44 & 769.41 &
\begin{tabular}{c} FRARYG \\ \end{tabular} \\ \hline \hline \end{tabular}  
* peptide recovered in other samples (data not shown).  
\end{table} 
Table 1: Molecular weight as determined by MALDI-TOF mass spectrometry and sequence of the recovered peptides. Figure 4: Peptides derived from CTL. sequence of L/D-CTL, and specific cleavage sites producing released fragments. Figure 5: Alg-Cat 1% without/with antimicrobial agent and _P. gingivalis_ (optical microscope view). (**a**) Gel without antibacterial agent is recovered by bacteria at the surface at 24 h (**b**) when D-CTL is added inside the gel, no bacteria are observed, showing an antibacterial effect. Figure 6: Alg-Cat 1% with/without antimicrobial agent and _P. gingivalis_ (MEB view). (**a,b**) Gel presents a porous structure that could be layered by biofilm; (**c,d**) _P. gingivalis_ colonize the surface and pores when an antibacterial agent is not present; (**e,f**) when charged with D-CTL, no bacteria were found at 24 h.