Branched chitosans II: Effects of branching on degradation, protein adsorption and cell growth properties
Introduction
There is a growing need for advanced biomaterials that are biodegradable, can support tissue generation and have mechanical properties comparable to that of native tissue. Chitosan is a promising implantable material that derives potential from its gel-forming properties and cationic nature that allows it to form insoluble ionic complexes with a variety of anionic polymers. Primary amino groups in the chitosan structure can be easily derivatized with useful biological ligands, or modified with other entities to alter mechanical and degradation properties, as well as protein adsorption properties [1], [2], [3]. In addition, the material has been shown to promote wound healing, and exhibits a minimal foreign body response with accelerated angiogenesis [4], [5], [6], [7], [8]. Chitosan has been used in a variety of biomedical applications including: wound dressings [9], [10], [11], [12], drug delivery systems [13], [14] and tissue engineered implants [15], [16], [17], [18], [19]. In most of these efforts, chitosan has been blended, cross-linked, or grafted with another molecule to bring about changes in properties as required for the specific application. Efforts have been made to enhance chitosan’s mechanical properties by incorporating polymers such as poly(ethylene glycol) [20], alginate [21] and silk fibroin [1]. However, addition of another polymer adds an extra level of complexity to the system and may result in adverse changes to other desirable properties.
Given chitosan’s linear architecture and its semi-crystalline nature, manipulation of its molecular architecture offers another method of altering the material’s physical and biological properties. We previously reported on the synthesis and mechanical characterization of a family of branched chitosan materials [22]. In order to facilitate educated choices of these branched polymers for biomaterial applications, additional characterization is needed.
Literature reports suggest that the low degradation rates of highly deacetylated chitosans are partly due to its semi-crystalline nature. Degradation kinetics (as measured by lysozyme-mediated cleavage) is mainly dependent upon two factors, namely molecular weight (MW) and degree of deacetylation (DD) [23], [24], [25], [26]. However, changes in molecular architecture introduce material structure changes that can mimic changes in both MW and DD.
Protein adsorption is one of the early events during the interaction of an implant material with a biological system. In addition to monomer chemistry, polymer architecture also can strongly influence chain organization and hence surface characteristics. Thus, inducing branching of linear chitosan may substantially alter some surface characteristics, and in doing so change aspects of protein adsorption properties that could substantially change cell and tissue responses to chitosan implants. Furthermore, the ability to effectively tune interactions between proteins and an implant material’s surface can be a useful tool in designing biomaterials specific to particular applications.
In this study, branched chitosans were synthesized as previously reported [22], and various properties relevant to implant performance were evaluated. Specifically, vascular endothelial cell growth kinetics on cast films were characterized along with the adsorption of serum proteins, and lysozyme-mediated degradation kinetics.
Section snippets
Synthesis of branched chitosan
Branched chitosan materials were synthesized as previously described [22] using a two-step procedure. The first step involved synthesis of low MW chitosan polymers by nitrous acid depolymerization [27], [28] and the second step involved grafting the low MW chains (i.e. the branches) to high MW chitosan backbones using reductive amination [29]. Chitosan (90% deacetylated, 600 kDa, Fluka) was dissolved in 1% acetic acid to form a 1.5 wt.% solution. Portions of this solution were depolymerized by
Effects of branching on the degradation properties of chitosan films
Grafting of chitosan branches produces large increases in the chitosan molecular weight. In enzyme studies, substrate molecular weight differences are often addressed by keeping the molar ratio of enzyme to substrate constant. However, in these studies the enzyme to chitosan mass ratio was kept constant. This was justified by the fact that lysozyme recognizes and cleaves trisaccharide motifs, which are randomly distributed at multiple locations within the polymer. As a result, maintenance of a
Discussion
Chitosan has been modified in a number of ways to tune the degradation kinetics. The various factors that have been shown to affect the degradation rate include molecular architecture [30], molecular weight (MW), crystallinity, water absorption [31] and degree of deacetylation (DD). Of these, DD and MW have been shown to be the most important [23], [24], [26]. In the work presented here, synthesis of branched chitosans was accomplished by grafting low MW chitosan chains to high MW chitosan
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