# In Vivo Wound Healing and Immune Response Studies of Chitosan Cryogels With Invertebrate Model Organism Galleria mellonella

## TL;DR

This study explores chitosan cryogels as wound dressings using Galleria mellonella larvae, showing improved immune and healing responses when loaded with cinnamic acid.

## Contribution

The first in vivo study using Galleria mellonella to assess chitosan cryogels for wound healing and immune response.

## Key findings

- Cryogels loaded with cinnamic acid significantly improved immune responses in G. mellonella.
- Dual network cryogels showed better mechanical strength and more regular morphology than single network cryogels.
- The use of G. mellonella provided an ethical alternative to mammalian models for preliminary wound healing assessments.

## Abstract

In the present study, it was aimed to prepare single and double network chitosan (Ch) cryogels cross‐linked with glutaraldehyde (G), which can be recommended for use as model wound dressings and hemostatic agents, and to reveal in vivo studies with Galleria mellonella. An in vivo study about Ch cryogels with these larvae was not declared in the literature, so our study is the first of its kind. G. mellonella was used to determine the effects of cryogels on immunity, oxidative stress, and wound healing. Cinnamic acid (CA) was loaded onto the cryogels, and the percent cumulative release data of CA were found to be in the range of 69%–80%. The results show that loading of CA onto [Ch‐3]cry cryogels considerably improved immune responses; the [Ch‐3]cry‐CA group was the most successful in terms of immunological response, oxidative stress balance, and wound healing. In accordance with the 3R principles of ethical animal research, the use of G. mellonella in this study served as a scientifically relevant and ethically responsible alternative model to mammals for preliminary assessment of wound healing potential and innate immune activation. The porous structures, high mechanical strengths, and rapidly swelling‐deswelling abilities of [Ch‐2@Ch]cry and [Ch‐3]cry cryogels indicated that these may be suitable for biomedical applications. Analysis of SEM micrographs indicated that the morphology of dual network cryogels prepared in the form of interpenetrating polymeric networks (IPNs) was more regular and homodispersed with respect to single network cryogels. The compressive elasticity modulus (E) values of IPNs cryogels (0.160 N/mm) is approximately 4.6 times that of Ch cryogels with a single network (0.035 N/mm).

–Comparison of chitosan cryogels: single networks versus full‐IPN structure.–Cryogels displaying high swelling degrees more than 2800%, assigned to the interconnected macroporous structure like sponges.–Cryogels with increased mechanical strength, more homogeneous and smaller pores in vivo wound healing and immune response studies of chitosan cryogels with G. mellonella.–Enhanced the immune system, immunological and antioxidant defenses of G. mellonella via cryogels and their cinnamic acid‐loaded forms.

Comparison of chitosan cryogels: single networks versus full‐IPN structure.

Cryogels displaying high swelling degrees more than 2800%, assigned to the interconnected macroporous structure like sponges.

Cryogels with increased mechanical strength, more homogeneous and smaller pores in vivo wound healing and immune response studies of chitosan cryogels with G. mellonella.

Enhanced the immune system, immunological and antioxidant defenses of G. mellonella via cryogels and their cinnamic acid‐loaded forms.