Lysozyme-Induced Degradation of Chitosan: The Characterisation of Degraded Chitosan Scaffolds

Up till now, chitosan has confirmed its versatile application in skin, cartilage and bone tissue engineering, as well as in drug delivery applications. This study is focused on enzymatic degradation of porous chitosan structures usually designed for mentioned purposes. In vitro degradation was monitored during four weeks of incubation at physiological temperature and in two different media, phosphate buffer saline solution and water. The scaffolds were characterised before and after enzymatic degradation using scanning electron microscopy and infrared spectroscopy with Fourier transformations (FTIR). According to the gravimetric analysis, higher weight loss of chitosan scaffolds was observed in buffered medium with respect to the water. The results implied that the total weight loss obtained in buffer involves physical dissolution of chitosan and lysozyme cleavage of glycoside bond. Importantly, FTIR identification of chitosan scaffolds after enzymatic degradation indicated the absence of lysozyme activity in water, indicating that weight loss is a result of the chitosan dissolution. This finding greatly impacts design of degradation experiments and characterisation of degradation behaviour of chitosan-based materials utilised as implants or drug delivery systems. DOI : 10.14302/issn.2640-6403.jtrr-17-1840 Corresponding author: Anamarija Rogina, Faculty of Chemical Engineering and Technology, University of Zagreb, HR-10001 Zagreb, Marulićev trg 19, p.p.177, Croatia. Email: arogina@fkit.hr


Introduction
One of the most important derivatives of chitin is chitosan, poly (D-glucosamine), a product of chitin deacetylation process. Chitosan is a linear polysaccharide, crystalline polymer insoluble at pH greater than 7 [1]. Chitosan has proven to be biodegradable, with antibacterial activity and hydrophilic, possessing functional groups (-OH and -NH 2 ) for secondary bonds formation [2]. Moreover, chitosan possesses high biocompatibility, good miscibility with other polymers and good coating properties for bulk implants. Even during degradation, chitosan-oligomers are found to be bioactive. Besides, chitosan-oligomers can act as probiotics that positively change intestinal microflora balance, inhibit growth of harmful bacteria, promote good digestion and boost immune function [3,4]. Chitosan is nontoxic and has been approved by FDA for would dressing application [5].
In the pharmaceutical field, chitosan is used as a drug delivery system for oral, nasal, parenteral and transdermal applications, as well as implant for gene delivery. High chemical reactivity has also led to several chitosan-drug systems for treating tumours [6][7][8].
Moreover, it is used for encapsulation of living cells as an inner core of composite spheres [9]. In many cases, cross-linked chitosan membranes are used (mostly with glutaraldehyde and genipin) [10][11][12].
Numerous studies are focused on the development of chitosan implants as porous materials for the treatment of tissue defects (including cartilage, skin and bones) or to accelerate the tissue regeneration.
The tissue restoration is a complex process that involves

Characterization of Shitosan Scaffold
The morphology of scaffolds was imaged using scanning electron microscope TESCAN Vega3SEM Easyprobe with electron beam energy of 10 keV.

Statistical Analysis
Statistical comparison of the data was performed using ANOVA one-way test. A p value <0.05 was considered to be statistically significant. All data are presented as a mean value corrected by standard deviation (mean ± SD).

Scaffold's Microstructure
The microstructure of prepared chitosan scaffolds was investigated by SEM imaging. The highly porous structure is clearly observable in figure 1a.    indicating the lack of lysozyme activity in the pH range of 4 -5, and chitosan dissolution process as a mechanism of scaffold's weight loss.

Discussion
Chitosan is one of widely studied implantable materials for regenerative medicine and tissue engineering, mostly for wound and bone tissue restoration [25][26][27]. Among important requirements (biocompatibility, non-immunogenic properties, high porosity, and resistance to different mechanical forces) artificial graft has to be biodegradable with degradation rate that follows the rate of new tissue formation [28].
The degradation process is dictated by the mechanism of molecule cleavage into smaller ones and finally into molecules degradable by the biological processes.  [20].
The weight loss of chitosan scaffolds is generated by both, the lysozyme activity and chitosan dissolution. It is well known that chitosan dissolves when pH is lower than 6.5. However, the dissolution properties depend on molecular weight and MW distribution which is wide for chitosan used in this study. Therefore, chitosan dissolution could be possible even at neutral pH. Degradation behaviour of potential tissue substituent or drug delivery system plays a role in treating the tumour-tissues. The pH of solid tumours is acidic due to increased fermentative metabolism which makes the adjacent normal tissue also acidic, leading to tumour invasion [31]. Chitosan-based nanoparticles showed their potential as drug-delivery carriers for cancer therapy due to the improvement of pharmacological and therapeutic properties of anti-cancer drugs controlling their release rate [32].
Since the peritumoral pH is heterogeneous, its direct monitoring near the treated surface in vivo could elucidate the microenvironmental conditions by pH microelectrodes [33]. The findings brought up by this study could help to determine if the rate of drug release and implant weight loss in similar applications is governed by the chitosan dissolution or biodegradation process.

Conclusions
The