Study of locust bean gum reinforced cyst-chitosan and oxidized dextran based semi-IPN cryogel dressing for hemostatic application

Abstract

Severe blood loss due to traumatic injuries remains one of the leading causes of death in emergency settings. Chitosan continues to be the candidate material for hemostatic applications due to its inherent hemostatic properties. However, available chitosan-based dressings have been reported to have an acidic odor at the wound site due to the incorporation of acid based solvents for their fabrication and deformation under compression owing to low mechanical strength limiting its usability. In the present study semi-IPN cryogel was fabricated via Schiff's base cross-linking between the polyaldehyde groups of oxidized dextran and thiolated chitosan in presence of locust bean gum (LBG) known for its hydrophilicity. Polymerization at -12 °C yielded macroporous semi-IPN cryogels with an average pore size of 124.57 ± 20.31 μm and 85.46% porosity. The hydrophobicity index of LBG reinforced semi-IPN cryogel was reduced 2.42 times whereas the swelling ratio was increased by 156.08% compare to control cryogel. The increased hydrophilicity and swelling ratio inflated the compressive modulus from 28.1 kPa to 33.85 for LBG reinforced semi-IPN cryogel. The structural stability and constant degradation medium pH were also recorded over a period of 12 weeks. The cryogels demonstrated lower adsorption affinity towards BSA. The cytotoxicity assays (direct, indirect) with 3T3-L1 fibroblast cells confirmed the cytocompatibility of the cryogels. The hemolysis assay showed <5% hemolysis confirming blood compatibility of the fabricated cryogel, while whole blood clotting and platelet adhesion assays confirmed the hemostatic potential of semi-IPN cryogel.

Keywords: Chitosan; Cryogel; Hemostasis; Locust bean gum; Oxidized dextran.

Graphical abstract

Fig. 1

Schematic illustration of (A) chitosan modification with cysteine; (B) oxidation of dextran; (C) chemical structure of locust bean gum; (D) semi-IPN network and (E) cryogel fabrication scheme.

Fig. 2

FT-IR spectra of (A) chitosan powder (Black) and cysteine modified chitosan (Red); (B) dextran powder (Black) and polyaldehyde oxidized dextran (Red); (C) ¹H-NMR spectra of pure dextran-70 powder (Red) and polyaldehyde oxidized dextran (Blue).

Fig. 3

(A, B) SEM micrographs and pore size distribution histograms of semi-IPN and (C, D) control cryogel (Scale bar 100 μm); (E) FTIR spectra of LBG powder (Blue), semi-IPN (Red) and control cryogel (Black).

Fig. 4

Swelling study of (A) semi-IPN and control cryogel; (B) inset shows initial fast swelling; (C) Mechanical strength analysis of semi-IPN and control cryogel.

Fig. 5

(A) In vitro degradation pattern of semi-IPN and control cryogel (n = 3) and (B) pH change of degradation medium during degradation study; (C at 0 days and D at 12th week) SEM micrographs of semi-IPN and (E at 0 days and F at 12th week) control cryogel of degradation.

Fig. 6

Cytotoxicity by (A) extract and (B) indirect contact methods through alamar blue assay at 24 and 48 h (*p < 0.05, **p < 0.005, ***p < 0.0005).

Fig. 7

Cytotoxicity by direct contact method: (A) alamar blue assay at 24 and 48 h; (B, C) SEM micrographs of semi-IPN and (D, E) control cryogel with cultured cells at 48 h; (F–H) Confocal microscope images for semi-IPN and (I–K, S3 supplementary) Control cryogel via Live/dead staining (*p < 0.05).

Fig. 8

Blood hemolysis analysis of semi-IPN and control cryogel by in vitro hemolysis assay (***p < 0.0001).

Fig. 9

In vitro whole blood clotting: (A, B) SEM micrographs of semi-IPN cryogel; (C, D) Control cryogel clotted blood; (E) Quantitative measurement of blood clotting efficiency via hemoglobin detection (Scale bar A, C 20 and B, D 10 μm) (*p < 0.05, **p < 0.001).

Fig. 10

In vitro Platelet adhesion: (A, B) SEM micrographs of semi-IPN cryogel; (C, D) Control cryogel with adhered platelets; (E) Quantitative measurement of adhered platelets on semi-IPN and control cryogel (Scale bar 10 μm).