Introduction: Recently, several research groups have demonstrated biomaterials for potential use in hemostatic applications. Among these biomaterials, chitosan and gelatin have attracted considerable interest owing to their advantageous biological properties. Chitosan has been found to promote tissue growth and wound healing because of its excellent biocompatibility, biodegradation, and non-toxic properties. Gelatin is known for its wound-healing properties because it prevents fluid loss due to exudation in a wound. In this study, we investigated the potential of chitosan nanofiber mats containing gelatin (CS-Gel) for use as wound dressings with enhanced hemostatic functions. We also investigated the potential of synergy between the hydrophilic gelatin and sonicated nanofiber mats to improve hemostatic function.
Materials and Methods: Chitosan (Mw: ~370 kDa) was purchased from Sigma. Gelatin type B from bovine skin (approximately 225 bloom), trifluoroacetic acid, and dichloromethane were purchased from Sigma. NaOH and methanol for neutralization of electrospun nanofiber mats were obtained from Duksan and Merck, respectively. Nanofiber mats were sonicated for 1 minute at a power of 225 W using a VCX 750 ultra-sonicator, a process that increases porosity and mat thickness. The surface morphology, mechanical properties, and initial contact angle analysis of CS-Gel nanofibers mat were evaluated. Moreover, CS-Gel nanofiber mats with enlarged porosity, produced by ultra-sonication, showed improved blood clotting efficiency, cell viability and cell infiltration compared with non-sonicated Chi-Gel nanofiber mats.
Results and Discussion: Figure 1 shows the FE-SEM image and mechanical properties of the CS-Gel nanofiber mats before and after ultra-sonication treatment. FE-SEM images from figure 1 shows the differences in surface and cross section morphology of the CS-Gel nanofiber mats before (Figure 1a) and after (Figure 1b) ultra-sonication, respectively. The morphology of the mat surface was roughed after ultra-sonication (increased pore size). The mechanical properties of the all nanofibers mat using universal testing machine (UTM) calculated from the stress-strain curves are shown in figure 1c. We found that the CS-Gel and sonicated CS-Gel nanofiber mats had a tensile strength of 4.6 and 3.0 MPa, an elastic modulus of 1.5 and 0.7 MPa, and an elongation at break of 9.2 and 9.6%, respectively.
Figure 1. (a, b) FE-SEM images of CS-Gel and sonicated CS-Gel nanofiber mat (scale bar: 50 um), and (c) mechanical properties
We quantitatively compared the platelet absorption properties and cell infiltration of CS-Gel and sonicated CS-Gel nanofiber mats using blood-clotting studies (Figure 2). In this figure, more platelets adhered to the sonicated nanofiber mat than to the non-sonicated mat, and the clot was substantially increased by infiltrated blood in the inner part of the CS-Gel nanofiber mats produced by sonication. These results further support the potential of sonicated CS-Gel nanofiber mats as a hemostatic agent.
Figure 2. FE-SEM images of platelets adhesion on the (a) CS-Gel and (b) sonicated CS-Gel nanofiber mats (scale bar: 20 um). Confocal z-stack images of the (c) CS-Gel and (d) sonicated CS-Gel nanofiber mats.
A comparison of cell infiltration on CS-Gel and sonicated CS-Gel nanofiber mats after 7 days of culture showed that cells on the non-sonicated nanofiber mats did not infiltrate below the most superficial layer, whereas cells on the sonicated CS-Gel nanofiber mats gradually infiltrated deep into the nanofiber mats.
Conclusions: To improve the hemostatic function of chitosan nanofiber mats, we studied the synergetic effects of gelatin blending and porosity control. We demonstrated a synergistic effect between the hydrophilic gelatin and sonication treatment that improved hemostatic function, cell viability, and cell infiltration. Collectively, our findings indicate that sonicated CS-Gel blended nanofiber mats are promising candidates for use as 3-dimensional tissue engineering scaffolds as well as hemostatic wound dressings.