Chitosan as a potential alternative to collagen for the development of genipin-crosslinked scaffolds

Abstract

Tissue Engineering emerged as a research field to solve problems derived from the loss of tissue and organ failure, specifically through the development of scaffolds. In this work, scaffolds were manufactured using collagen and chitosan as raw materials, evaluating the potential use of the latter as a substitute for collagen. Throughout this work, different proportions of these raw materials were used, and the influence of genipin (crosslinking agent) was evaluated, studying the variations in the mechanical and morphological properties of the scaffolds. The results obtained show that the properties of the scaffolds were strongly dependent on the biopolymer concentration used and on the concentrations of crosslinking agent. According to the results, the best mechanical properties were obtained for hybrid systems with high biopolymer concentrations (2 wt%), especially chitosan. Furthermore, the addition of a small amount of crosslinking agent (genipin) improved low biopolymer concentration systems (1 wt%), showing an improvement in the critical strain.

Introduction

Nowadays, medicine faces problems derived from tissue loss and organ failure, which imply huge costs for healthcare systems. These problems have increased the interest of the scientific community in the field of Tissue Engineering. This is an interdisciplinary field that fundamentally requires: cultured cell technology, the development of 3D structures (scaffolds) that can mimic the structure of an organ, and growth factors, which allow for a precise and continuous control of cell growth and proliferation [1].

The scaffolds are supports that are introduced into damaged tissues to promote the growth and in situ propagation of surrounding healthy resident cells or stem cells and growth factors that can be implanted in this tissue or incorporated into the biomaterial in order to accelerate tissue growth [2].

Recent scientific investigations have obtained multifunctional, biocompatible and biodegradable scaffolds with compatible physical and mechanical properties that promote cell growth [[3], [4], [5]]. The properties that these materials should have are closely linked to their internal structure, porosity, interconnectivity and pore size distribution, which can be modified according to the processing technique and the raw materials used, in both their type and proportion [6]. Raw materials include synthetic polymers and biopolymers, as well as additives or crosslinking agents, which are applied to adjust the physical and mechanical properties of the scaffolds. The use of a specific material is based on the biological relationship that exists between the function, structure and properties of the scaffold. In this sense, biopolymers stand out due to their biodegradability, which removes the need for implant removal surgery. Proteins (fibrinogen, fibronectin, collagen, etc.) and polysaccharides (starch, glucose, chitosan, etc.) are the most suitable biopolymers used.

The main advantage of biopolymers is their biocompatibility, which causes the material to have no adverse effects after implantation or after biodegradation [7]. However, their disadvantage is that they have very poor mechanical properties compared to synthetic polymers. To improve these properties, studies on the crosslinking process (physical and chemical, even enzymatic) with crosslinking agents (glutaraldehyde, genipin, citric acid, glucose, etc.) are being carried out at present [8,9]. However, some studies have shown that physical crosslinking does not meet the mechanical requirements and that chemical crosslinking presents the disadvantage of the cytotoxicity of the agents used in many cases, such as glutaraldehyde [[10], [11], [12]]. Therefore, it is necessary to search for new alternatives that are more biocompatible, such as genipin or citric acid. G is a natural substance which spontaneously crosslinks chitosan, gelatin or collagen and presents a much lower level of cytotoxicity than glutaraldehyde [9]. In addition, it has many medicinal effects, such as anti-inflammatory, anti-cancer and anti-bacterial properties. Besides, it can crosslinks primary amine groups, so its use as a crosslinking agent has recently been evaluated in amine-containing systems for Tissue Engineering [9,13].

Among the different biopolymers that can be used, proteins and polysaccharides have been widely employed. Among them, collagen has been traditionally popular due to its biological properties [[14], [15], [16]]. Collagen is part of the extracellular matrix and is an attractive material for application in this field, as it is easy to modify and process. Furthermore, it is biodegradable, biocompatible, not antigenic and has good mechanical properties, such as ductility. Collagen is mainly extracted from the residues of bovine species and pig skin, bones and cartilage [15,17]. Depending on the origin and way of processing, collagen can have different properties, with biocompatibility standing out, which allows cell proliferation and does not interfere with cell behaviour [17]. However, the main drawbacks of collagen are its high price and its low degradation temperature (37 °C), which can make its properties vary.

On the other hand, among the different polysaccharides, chitosan is very interesting, since it is accessible and an important renewable resource [18]. Chitosan has been widely analyzed in the studies of Prof. Roberts and Prof. Vårum. It is obtained industrially by extensive chitin deacetylation and consists of two types of structural units randomly distributed along the chain [19]. In its crystalline form, it is normally insoluble in neutral aqueous solutions, although its solution is possible in diluted acid solutions [20]. Its biocompatibility and biodegradability have been widely demonstrated as it promotes cell adhesion and reabsorbs through hydrolysis processes by the action of enzymes present in physiological fluids [[21], [22], [23], [24]]. In addition, characteristics such as its molecular weight or its acetylation degree can be adjusted depending on the desired application [[25], [26], [27]]. In this sense, chitosan has a large number of applications, since it is usually employed as a flocculating agent in water treatment, for the production of biofuels and as a thickener in the food industry [21,28]. Among these applications, recent studies have aimed to use chitosan in the field of biomedicine, e.g., for the production of scaffolds [29,30]. However, chitosan as a biopolymer is not bioactive and generally the structures obtained present low mechanical properties [31], thus it is usually combined with other materials for most of these applications. The combination of genipin and chitosan has been previously reported for the fabrication of different structures, from nanofibrous membranes with the studies of Bavariya et al. (2013) [32] to the formation of hydrogels, being especially interesting the studies of Espinosa-Garcia et al. (2007) and Engwer et al. (2017) [33,34]. In addition, other studies produced porous scaffolds with chitosan crosslinked, as the research driven by Gorczyca et al. (2014) and, more recently, Felfel et al. (2019) [35,36].

There are different ways to obtain scaffolds for Tissue Engineering. Freeze-drying is one of the most widely used techniques, as it allows obtaining porous materials by the sublimation of the solvent [37]. Its main advantages are the elimination of the solvent without degrading the polymer and the formation of structures with a high porosity (above 80%) [38]. The type of structures obtained are known as “sponge-like" materials, due to the porous latticework with interconnected macro- and micropores [39]. The use of this technique in the development of biopolymer scaffolds has the added advantage that, by not using high temperatures in the process, it does not denature the biopolymers, with maintain their initial properties [37,[40], [41], [42]].

In this context, our hypothesis is based on the modification of the properties of collagen-based scaffolds by the addition of chitosan in order to adjust the properties of these materials by replacing collagen for chitosan. The main novelty of this work is the improvement of the collagen-based scaffolds properties with the addition of chitosan, obtaining a hybrid scaffold with a reinforced structure due to the synergy produced by the combination and interaction of two different biopolymers. Therefore, combinations of collagen and chitosan are seen as a promising raw material for biomedical purposes.

Thus, the aim of this work was to develop scaffolds with collagen (C) and chitosan (CH) and to evaluate the influence of the total polymer concentration or the incorporation of a crosslinking agent, in this case genipin (G), on the mechanical and morphological properties of the scaffolds. Therefore, systems with a 1 and 2 wt% of total biopolymer concentration (collagen, chitosan or collagen/chitosan mixtures) were prepared with the addition of genipin at two different concentrations (0.03 and 0.05 wt%).