Abstract
Recent advances in nanocellulose technology have revealed the potential of crystalline cellulose nanofibers to reinforce materials which are useful for tissue engineering, among other functions. However, the low biodegradability of nanocellulose can possess some problems in biomedical applications. In this work, alginate particles with encapsulated enzyme cellulase extracted from Trichoderma reesei were prepared for the biodegradation of crystalline cellulose nanofibers, which carrier system could be incorporated in tissue engineering biomaterials to degrade the crystalline cellulose nanoreinforcement in situ and on-demand during tissue regeneration. Both alginate beads and microparticles were processed by extrusion-dropping and inkjet-based methods, respectively. Processing parameters like the alginate concentration, concentration of ionic crosslinker Ca2+, hardening time, and ionic strength of the medium were varied. The hydrolytic activity of the free and encapsulated enzyme was evaluated for unmodified (CNFs) and TEMPO-oxidized cellulose nanofibers (TOCNFs) in suspension (heterogeneous conditions); in comparison to solubilized cellulose derivatives (homogeneous conditions). The enzymatic activity was evaluated for temperatures between 25–75 °C, pH range from 3.5 to 8.0 and incubation times until 21 d. Encapsulated cellulase in general displayed higher activity compared to the free enzyme over wider temperature and pH ranges and for longer incubation times. A statistical design allowed optimizing the processing parameters for the preparation of enzyme-encapsulated alginate particles presenting the highest enzymatic activity and sphericity. The statistical analysis yielded the optimum particles characteristics and properties by using a formulation of 2% (w/v) alginate, a coagulation bath of 0.2 M CaCl2 and a hardening time of 1 h. In homogeneous conditions the highest catalytic activity was obtained at 55 °C and pH 4.8. These temperature and pH values were considered to study the biodegradation of the crystalline cellulose nanofibers in suspension. The encapsulated cellulase preserved its activity for several weeks over that of the free enzyme, which latter considerably decreased and practically showed deactivation after just 10 d. The alginate microparticles with their high surface area-to-volume ratio effectively allowed the controlled release of the encapsulated enzyme and thereby the sustained hydrolysis of the cellulose nanofibers. The relative activity of cellulase encapsulated in the microparticles leveled-off at around 60% after one day and practically remained at that value for three weeks.
Highlights
The outstanding mechanical properties of crystalline cellulose nanofibers, the high availability of cellulose in nature, and the renewability and proved cytocompatibility of the nanofibers make them an excellent candidate for the reinforcement of biomaterials in tissue engineering applications [1,2,3,4,5,6,7]
To make use of the excellent mechanical properties and biocompatibility of cellulose nanofibers, it would be advantageous to achieve their biodegradability in vivo, since biomaterial functionalities can depend on the length of time for which they are needed to remain in the body [30,31]
The maximum catalytic activity of the enzyme was achieved at pH 4.8 and 55 ◦ C for studies performed with cellulase encapsulated in alginate particles
Summary
The outstanding mechanical properties of crystalline cellulose nanofibers, the high availability of cellulose in nature, and the renewability and proved cytocompatibility of the nanofibers make them an excellent candidate for the reinforcement of biomaterials in tissue engineering applications [1,2,3,4,5,6,7]. The integration of enzyme carrier systems within cellulose nanofiber-filled tissue engineering biomaterials could be the solution to enable the controlled biodegradation of these crystalline nanofibers over the long term, as required for the specific tissue repairing/regeneration. Cellulose-nanofiber reinforced chitosan biomaterials were applied in the repairing of the mechanically-demanding tissue intervertebral disc with promising results achieved in ex vivo investigations with pig animal models [14]. A strategy would be the incorporation of enzyme delivery systems within the cellulose-nanofiber biomaterials to allow sustained and on-demand degradation of nanocellulose while the biomaterial plays a bioactive and mechanical role during tissue regeneration
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