From Aggregates to Porous Three-Dimensional Scaffolds through a Mechanochemical Approach to Design Photosensitive Chitosan Derivatives

Abstract

The crustacean processing industry produces large quantities of waste by-products (up to 70%). Such wastes could be used as raw materials for producing chitosan, a polysaccharide with a unique set of biochemical properties. However, the preparation methods and the long-term stability of chitosan-based products limit their application in biomedicine. In this study, different scale structures, such as aggregates, photo-crosslinked films, and 3D scaffolds based on mechanochemically-modified chitosan derivatives, were successfully formed. Dynamic light scattering revealed that aggregation of chitosan derivatives becomes more pronounced with an increase in the number of hydrophobic substituents. Although the results of the mechanical testing revealed that the plasticity of photo-crosslinked films was 5⁻8% higher than that for the initial chitosan films, their tensile strength remained unchanged. Different types of polymer scaffolds, such as flexible and porous ones, were developed by laser stereolithography. In vivo studies of the formed structures showed no dystrophic and necrobiotic changes, which proves their biocompatibility. Moreover, the wavelet analysis was used to show that the areas of chitosan film degradation were periodic. Comparing the results of the wavelet analysis and X-ray diffraction data, we have concluded that degradation occurs within less ordered amorphous regions in the polymer bulk.

Keywords: chitosan; laser stereolithography; long-term stability; mechanochemical synthesis; scaffold; tissue reaction.

Figure 1

Scheme of the experimental work.

Figure 2

The relation between the hydrodynamic diameter and the degree of substitution of chitosan amino groups with allyl fragments. Solutions were prepared with acetic acid (2%) at a final concentration of 0.02 g/dL.

Figure 3

XRD analysis of allylchitosan film (sample AC2) before (spectrum 1) and after (spectrum 2) UV cross-linking.

Figure 4

Three-dimensional scaffold after laser stereolithography (a) and freeze drying (b).

Figure 5

Surface SEM images of the 3D scaffold (a) and freeze-dried matrix (b), bar = 100 μm.

Figure 6

Tissue reaction to the films and the porous 3D scaffolds based on allylchitosans: the connective tissue formed the capsule (CAP) with blood vessels (V) around the implanted chitosan films (CHF) and the chitosan sponges (CHS); CAP consisted of two layers: the inner layer (IL) was an immature connective tissue (granulation tissue) with macrophages (MPH) and giant cells (GC), while the outer layer (OL) consisted of a more mature connective tissue; IL grew into the film fractures (F) and CHS pores forming connective tissue septa (S); some MPH and GC adhered to the surface of scaffolds, hematoxylin and eosin staining, simple microscopy (a) CHF (AC2) implantation (30 days): the CHF material was oxyphilic, 100×; (b–d)—3D scaffold implantation: (b) 30 days: the CHS material was oxyphilic, 100×; (c) a basophilic foci (yellow arrows) in the surface septa of the CHS material, 60 days, 200×; and (d) deep CHS sections: most of the scaffold septa were moderately basophilic, 90 days, 400×.

Figure 7

A histological section of chitosan film (a), a waveletgram (b), and the integral spatial spectrum of the structural optical inhomogeneities along the film section in the direction of its surface (c).

Figure 8

The structure of the synthesized allylchitosans.