# Biohydrogel Based on Dynamic Covalent Bonds for Wound Healing Applications
## 3 Results and Discussion
### Formulation and Relevant Characterization of Hydrogels
#### 3.1.5 Blood Clotting Time  
Clotting time is the time required for a sample of blood to coagulate in vitro under standard conditions; the normal clotting time in humans is approximately 2-8 min [25]. Clotting times are presented in Table 1. Mean clotting times for the 3%, 4% hydrogels and 3% hydrogel/Savlon mixture were 9.1, 9.3, and 10.7 min, respectively. Test blood dropped on an untreated Petri dish (no hydrogel) coagulated in 4.2 min. These results indicate that the hydrogels prolonged clotting time by around 5-6 min.  
Notably, the natural clotting process requires calcium ions [26]. The alginate in the hydrogel may be bonding with the calcium ions in the blood, thereby inhibiting clotting and prolonging clotting time; the cross-linking of alginate with calcium ions is well established [27]. Biomolecules such as fibrin, which are necessary in clot formation [28], might have been affected. This clotting time prolongation may be appropriate or desirable in thrombobolic conditions, where blood coagulation can result in disastrous blocking of tiny blood vessels, as well as other biomedical applications.  
Nanocomposite hydrogels containing 20 mg magnesium aluminometasilicate caused blood clotting within 4 min, which is comparable to the untreated (control) blood. However, the nanocomposite hydrogel with a higher concentration of the silicate (200 mg) showed a very high anti-coagulant property with blood clotting time taking as long as 180 min (3 h) (Table 1). In this case, clotting factors may have been disrupted. Conversely, the lower silicate concentrations showed shorter clotting times compared to the non-composite PBA-Alg hydrogels, which may indicate that the low content silicates competitively bound to the PBA-Alg, reducing the effect of the latter on blood clotting. Nevertheless, as the silicate concentration and surface area increased, it caused extensive adsorption onto clotting factors which resulted in extended clotting time. The high adsorption capacity of these magnesium aluminosilicates may have influenced the result. In terms of applications, these results show that low silicate-based PBA-Alg may be favored if normal blood clotting is desired, whereas high silicate-based PBA-Alg would be preferred if an anti-coagulant effect is desired. A one-way analysis of variance (ANOVA) was used to analyze blood clotting data with statistical significance at \(p<0.05\).  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Treatment** & **Clotting Time (min)** \\ \hline
3\% PBA-Alg & 9.09 \(\pm\) 0.04 \({}^{*}\) \\
4\% PBA-Alg & 9.34 \(\pm\) 0.03 \({}^{*}\) \\
3\% PBA-Alg + Savlon & 10.75 \(\pm\) 0.04 \({}^{*}\) \\ none (negative control) & 4.20 \(\pm\) 0.02 \\ nanocomposite (20 mg silicate) & 4.08 \(\pm\) 0.02 \\ nanocomposite (80 mg silicate) & 5.55 \(\pm\) 0.03 \({}^{*}\) \\ nanocomposite (140 mg silicate) & 11.50 \(\pm\) 0.05 \({}^{*}\) \\ nanocomposite (200 mg silicate) & 180.00 \(\pm\) 0.12 \({}^{**}\) \\ \hline \hline \end{tabular} \({}^{*}\), \({}^{**}\) indicate statistical significance at \(p<0.05\), \(p<0.01\), respectively, compared to control.  
\end{table}
Table 1: In vitro blood clotting time.