The development of non-cellularized composites of chitosan (CHI) hydrogels, filled with cellulose nanofibers (CNFs) of the type nanofibrillated cellulose, was proposed for the repair and regeneration of the intervertebral disc (IVD) annulus fibrosus (AF) tissue. With the achievement of CNF-filled CHI hydrogels, biomaterial-based implants were designed to restore damaged/degenerated discs. The structural, mechanical and biological properties of the developed hydrogel composites were investigated. The neutralization of weakly acidic aqueous CNF/CHI viscous suspensions in NaOH yielded composites of physical hydrogels in which the cellulose nanofibers reinforced the CHI matrix, as investigated by means of microtensile testing under controlled humidity. We assessed the suitability of the achieved biomaterials for intervertebral disc tissue engineering in ex vivo experiments using spine pig models. Cellulose nanofiber-filled chitosan hydrogels can be used as implants in AF tissue defects to restore IVD biomechanics and constitute contention patches against disc nucleus protrusion while serving as support for IVD regeneration.

The development of non-cellularized injectable suspensions of viscous chitosan (CHI) solutions (1.7–3.3% (w/w)), filled with cellulose nanofibers (CNF) (0.02–0.6% (w/w)) of the type nanofibrillated cellulose, was proposed for viscosupplementation of the intervertebral disc nucleus pulposus tissue. The achievement of CNF/CHI formulations which can gel in situ at the disc injection site constitutes a minimally-invasive approach to restore damaged/degenerated discs. We studied physico-chemical aspects of the sol and gel states of the CNF/CHI formulations, including the rheological behavior in relation to injectability (sol state) and fiber mechanical reinforcement (gel state). CNF-CHI interactions could be evidenced by a double flow behavior due to the relaxation of the CHI polymer chains and those interacting with the CNFs. At high shear rates resembling the injection conditions with needles commonly used in surgical treatments, both the reference CHI viscous solutions and those filled with CNFs exhibited similar rheological behavior. The neutralization of the flowing and weakly acidic CNF/CHI suspensions yielded composite hydrogels in which the nanofibers reinforced the CHI matrix. We performed evaluations in relation to the biomedical application, such as the effect of the intradiscal injection of the CNF/CHI formulation in pig and rabbit spine models on disc biomechanics. We showed that the injectable formulations became hydrogels in situ after intradiscal gelation, due to CHI neutralization occurring in contact with the body fluids. No leakage of the injectate through the injection canal was observed and the gelled formulation restored the disc height and loss of mechanical properties, which is commonly related to disc degeneration.

Soft tissues are commonly fiber-reinforced hydrogel composite structures, distinguishable from hard tissues by their low mineral and high water content. In this work, we proposed the development of 3D printed hydrogel constructs of the biopolymers chitosan (CHI) and cellulose nanofibers (CNFs), both without any chemical modification, which processing did not incorporate any chemical crosslinking. The unique mechanical properties of native cellulose nanofibers offer new strategies for the design of environmentally friendly high mechanical performance composites. In the here proposed 3D printed bioinspired CNF-filled CHI hydrogel biomaterials, the chitosan serves as a biocompatible matrix promoting cell growth with balanced hydrophilic properties, while the CNFs provide mechanical reinforcement to the CHI-based hydrogel. By means of extrusion-based printing (EBB), the design and development of 3D functional hydrogel scaffolds was achieved by using low concentrations of chitosan (2.0–3.0% (w/v)) and cellulose nanofibers (0.2–0.4% (w/v)). CHI/CNF printed hydrogels with good mechanical performance (Young’s modulus 3.0 MPa, stress at break 1.5 MPa, and strain at break 75%), anisotropic microstructure and suitable biological response, were achieved. The CHI/CNF composition and processing parameters were optimized in terms of 3D printability, resolution, and quality of the constructs (microstructure and mechanical properties), resulting in good cell viability. This work allows expanding the library of the so far used biopolymer compositions for 3D printing of mechanically performant hydrogel constructs, purely based in the natural polymers chitosan and cellulose, offering new perspectives in the engineering of mechanically demanding hydrogel tissues like intervertebral disc (IVD), cartilage, meniscus, among others.

Chitosan–caseinate nanoparticles were synthesized by polyelectrolyte complex (PEC) formation. Caseinate is an anionic micellar nanocolloid in aqueous solutions, which association with the polycationic chitosan yielded polyelectrolyte complexes with caseinate cores surrounded by a chitosan corona. The pre-structuration of caseinate micelles facilitates the formation of natural polyelectrolyte nanoparticles with good stability and sizes around 200 nm. Such natural nanoparticles can be loaded with molecules for applications in drug-controlled release. In the nanoparticles processing, parameters such as the chitosan degree of acetylation (DA) and molecular weight, order of addition of the polyelectrolytes chitosan (polycation) and caseinate (polyanion), and added weight ratio of polycation:polyanion were varied, which were shown to influence the structure of the polyelectrolyte association, the nanoparticle size and zeta potential. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) analyses revealed the chemical structure of hydrogel colloidal systems consisting of nanoparticles that contain chitosan and caseinate. Transmission electron microscopy (TEM) allowed further characterization of the spherical morphology of the nanoparticles. Furtherly, insulin was chosen as a model drug to study the application of the nanoparticles as a safe biodegradable nanocarrier system for drug-controlled release. An insulin entrapment efficiency of 75% was achieved in the chitosan-caseinate nanoparticles.

The 3D printing of a multifunctional hydrogel biomaterial with bioactivity for tissue engineering, good mechanical properties and a biodegradability mediated by free and encapsulated cellulase was proposed. Bioinks of cellulase-laden and cellulose nanofiber filled chitosan viscous suspensions were used to 3D print enzymatic biodegradable and biocompatible cellulose nanofiber (CNF) reinforced chitosan (CHI) hydrogels. The study of the kinetics of CNF enzymatic degradation was studied in situ in fibroblast cell culture. To preserve enzyme stability as well as to guarantee its sustained release, the cellulase was preliminarily encapsulated in chitosan–caseinate nanoparticles, which were further incorporated in the CNF/CHI viscous suspension before the 3D printing of the ink. The incorporation of the enzyme within the CHI/CNF hydrogel contributed to control the decrease of the CNF mechanical reinforcement in the long term while keeping the cell growth-promoting property of chitosan. The hydrolysis kinetics of cellulose in the 3D printed scaffolds showed a slow but sustained degradation of the CNFs with enzyme, with approximately 65% and 55% relative activities still obtained after 14 days of incubation for the encapsulated and free enzyme, respectively. The 3D printed composite hydrogels showed excellent cytocompatibility supporting fibroblast cell attachment, proliferation and growth. Ultimately, the concomitant cell growth and biodegradation of CNFs within the 3D printed CHI/CNF scaffolds highlights the remarkable potential of CHI/CNF composites in the design of tissue models for the development of 3D constructs with tailored in vitro/in vivo degradability for biomedical applications.

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.