## 3 Results
### Antibacterial Properties of CHX and TCS Loaded Micromolded Pieces  
The results of agar tests for representative Gram-negative (_E. coli_) and Gram-positive (_S. epidermidis_) bacteria are shown in Figure 13. The presence of an inhibition halo in the loaded samples, not observed around the neat PLA, reflects the drug antibacterial activity. Therefore, the ultrasound heating source does not have a negative effect in the antibacterial activity of the evaluated drugs. These results show that the drugs maintain their molecular structure intact and consequently their activity during  
Figure 12: Release profile of PLA samples loaded with 3 wt-% of TCS (blue) and CHX (black) in absolute ethanol (solid lines) and PBS-EtOH 70% (dashed lines). the micromolding process. The sizes of the inhibition halo are directly linked with antibacterial activity level. Accordingly, the activity of both drugs against _S. epidermidis_ was much higher than for _E. coli_. Comparing the CHX and TCS response, a greater effect was observed for TCS loaded samples despite its smaller release rate, its greater inherent bactericidal activity demonstrated as discussed below. Differences in the size of the inhibition halo were nevertheless minimized when high drug concentrations were used. The previously indicated small release rate, which is derived from the high specimen compaction, suggests that the initial bactericide effect was caused by the drug located at the specimen surface, which needs a minimum loading concentration to be significant. These results shown in Figure 13 can be explained taking into account the different antibacterial mechanisms of both drugs. TCS diffuses through the bacterial cytoplasmic membrane and interferes with its lipid metabolism, leading to clear inhibition halos for both bacteria. By contrast, CHX salts are dissociated at physiologic pH and consequently positively charged CHX cations become the delivered agents. The bactericidal effect of CHX is a result of the binding of this cationic molecule with the negatively charged bacterial cell walls. Responses are consequently dependent on the structure of the bacterial walls, which are only constituted by peptidoglycan (PGs) in the case of Gram-positive bacteria, while these PGs are coated by proteins, phospholipids and lipopolysaccharide in Gram-negative bacteria. Coatings provide a protective effect to the action of CHX cationic molecules. Therefore, halos produced by TCS (large-medium sizes) and CHX (reduced size) can be well-differentiated in an _E. coli_ medium. Both TCS and CHX act as biocides at high concentrations and at lower concentrations have a bacteriostatic effect. Drug effects can be analyzed through the broth culture quantification assay. Figure 14 compares the bacterial growth curves of unloaded (solid lines) and loaded (dashed and dotted lines for CHX and TCS, respectively) PLA processed specimens. Both drugs had a clear bactericidal and bacteriostatic effect in the two studied bacterium media. Thus, bacterial growth was reduced to close to 20%, related to the 100% of neat PLA. PLA loaded with 3 wt-% of CHX firstly had a high bactericidal effect, suppressing the bacterial growth up to 24 h, and, subsequently, a bacteriostatic activity that kept the total bacterial growth under 20%. For the samples loaded with TCS, the bactericidal effect was a little less effective since a delay of the growth around 10-15 h was observed with respect to CHX. Nevertheless, the bacteriostatic effect was active for the whole assay, allowing  
Figure 13: Agar tests performed with PLA samples loaded with 1.5/3/6 wt-% of each drug (TCS or CHX) for _E. coli_ and _S. epidermidis_. For the sake of completeness, PLA unloaded samples are also evaluated. for reaching similar growth levels in both bacterium media than those observed for CHX loaded specimens.