Two experiments were carried out in order to determine the best procedure to prepare the biopolymer that will serve as a reservoir for the whole patch.
Experiment 1: Without HCl
PVA (0.5 g) was dissolved in 5 mL of distilled water by stirring and heating at 60°C for 40 minutes (flask was covered to avoid water evaporation). Mixture consistency was verified at 20, 30 and 40 minutes by spreading a little sample over a glass slide, which was put over a heating plate having a 40 °C temperature and it was kept in this way for 15 min. Besides of this, the mixture was expanded on a metal plate and it was let to dry for approximately 24 hours at room temperature.
Experiment 2: With HCl
Same conditions from experiment 1 were followed, but 100 μL of HCl 1M were added to the mixture after 13 min of heating (a sample was collected to test consistency) and at 27 min another 100 μL of HCl were added.
Selection of nutritive agar medium concentration
To select the concentration, we considered to analyze motility, appearance and consistence of agar concentrations of 2.5, 3, 3.5, 4 and 4.5 g/L.
Selection of PVA concentration into the medium
Polyvinyl alcohol (PVA) is a water soluble synthetic polymer. To select the ideal concentration, different concentrations of polymer were added to the medium (stored at room temperature) and heights were monitored every three days for 12 days, as a measure of medium preservation. PVA concentrations used were 5, 10 and 15 % (v/v), using a stock solution (5 g of polymer in 5 mL of distilled water, and heated as previously described).
Membrane permeability for microorganisms
Two experiments were performed in order to demonstrate that bacteria cannot flow through the nitrocellulose membrane that separates the biopatch from the burn injury.
Nutrient agar (3.5 g/L) was added to three Petri dishes, 7 mL each, and afterwards inoculated with a wild strain of E. coli through three punctures made with an inoculation loop. A sterile nitrocellulose membrane was added to each Petri dish, fully covering the inoculation sites, followed by two sterile round filter paper pieces, the one on the top (number 2) being smaller than the one underneath (number 1) (Figure 1). Petri dishes were incubated 24 h at 30°C. Thereafter, filter paper pieces were removed in sterile conditions and each one transferred to a tube filled with sterile nutrient broth. The filter paper piece number 1 (the one right above the membrane) was previously put in contact with a Petri dish filled with nutrient agar for some seconds. These Petri dishes were incubated 24 h at 30°C and the tubes with filter paper pieces 24 h at 37°C. Each one of the initial Petri dishes had a negative control (no microorganism inoculated at the beginning). The same protocol was followed.
We followed the same methodology as in experiment 1, with the only exception that the inoculated Petri dishes were incubated 24 h at 30°C and then the membranes and filter paper pieces were added.
A positive control for both experiments was performed (no nitrocellulose membrane was put into the system). These experiment control was realized in triplicate.
Figure 1. System for membrane permeability for microorganisms experiment. A) System diagram. B) Real system. NM: nitrocellulose membrane. FP1: filter paper 1, FP2: filter paper 2.