3D double-reinforced graphene oxide - nanocellulose biomaterial inks for tissue engineered constructs.

The advent of improved fabrication technologies, particularly 3D printing, has enabled the engineering of bone tissue for patient-specific healing and the fabrication of in vitro tissue models for ex vivo testing. However, inks made from natural polymers often fall short in terms of mechanical strength, stability, and the induction of osteogenesis. Our research focused on developing novel printable formulations using a gelatin/pectin polymeric matrix that integrate synergistic reinforcement components i.e. graphene oxide (GO) and oxidized nanocellulose fibers (CNF). Using 3D printing technology and the aforementioned biomaterial composite inks, bone-like scaffolds were created. To simulate critical-sized flaws and demonstrate scaffold fidelity, 3D scaffolds were successfully printed using formulations with varied GO concentrations (0.25, 0.5, and 1% wt with respect to polymer content). The addition of GO to hydrogel inks enhanced not only the compressive modulus but also the printability and scaffold fidelity compared to the pure colloid-gelatin/pectin system. Due to its strong potential for 3D bioprinting, the sample containing 0.5% GO is shown to have the greatest perspectives for bone tissue models and tissue engineering applications.

The current state of the art in gellan-based printing inks in tissue engineering.

With the advancement of enhanced fabrication technologies, specifically 3D printing, it is now possible to build artificial tissue for personalized healing. However, inks developed from polymers often fail to meet expectations in terms of mechanical strength, scaffold integrity, and the stimulation of tissue formation. Developing new printable formulations as well as adapting existing printing methods is an essential aspect of contemporary biofabrication research. In order to push the boundaries of the printability window, various strategies have been developed employing gellan gum. This has resulted in major breakthroughs in the development of 3D hydrogels scaffolds that exhibit significant resemblance to genuine tissues and enables the fabrication of more complex systems. In light of the many uses of gellan gum, the purpose of this paper is to provide a synopsis of the printable ink designs drawing attention to the various compositions and fabrication approaches that may be used for tuning the properties of 3D printed hydrogels for tissue engineering applications. The purpose of this article is to outline the development of gellan-based 3D printing inks and to encourage research by highlighting the possible applications of gellan gum.

Inorganic/Biopolymers Hybrid Hydrogels Dual Cross-Linked for Bone Tissue Regeneration.

In tissue engineering, the potential of re-growing new tissue has been considered, however, developments towards such clinical and commercial outcomes have been modest. One of the most important elements here is the selection of a biomaterial that serves as a "scaffold" for the regeneration process. Herein, we designed hydrogels composed of two biocompatible natural polymers, namely gelatin with photopolymerizable functionalities and a pectin derivative amenable to direct protein conjugation. Aiming to design biomimetic hydrogels for bone regeneration, this study proposes double-reinforcement by way of inorganic/biopolymer hybrid filling composed of Si-based compounds and cellulose nanofibers. To attain networks with high flexibility and elastic modulus, a double-crosslinking strategy was envisioned-photochemical and enzyme-mediated conjugation reactions. The dual cross-linked procedure will generate intra- and intermolecular interactions between the protein and polysaccharide and might be a resourceful strategy to develop innovative scaffolding materials.

Nanoengineered biomimetic hydrogels: A major advancement to fabricate 3D-printed constructs for regenerative medicine.

Nanostructured compounds already validated as performant reinforcements for biomedical applications together with different fabrication strategies have been often used to channel the biophysical and biochemical features of hydrogel networks. Ergo, a wide array of nanostructured compounds has been employed as additive materials integrated with hydrophilic networks based on naturally-derived polymers to produce promising scaffolding materials for specific fields of regenerative medicine. To date, nanoengineered hydrogels are extensively explored in (bio)printing formulations, representing the most advanced designs of hydrogel (bio)inks able to fabricate structures with improved mechanical properties and high print fidelity along with a cell-interactive environment. The development of printing inks comprising organic-inorganic hybrid nanocomposites is in full ascent as the impact of a small amount of nanoscale additive does not translate only in improved physicochemical and biomechanical properties of bioink. The biopolymeric nanocomposites may even exhibit additional particular properties engendered by nano-scale reinforcement such as electrical conductivity, magnetic responsiveness, antibacterial or antioxidation properties. The present review focus on hydrogels nanoengineered for 3D printing of biomimetic constructs, with particular emphasis on the impact of the spatial distribution of reinforcing agents (0D, 1D, 2D). Here, a systematic analysis of the naturally-derived nanostructured inks is presented highlighting the relationship between relevant length scales and size effects that influence the final properties of the hydrogels designed for regenerative medicine.

3D Bioprinting of Biosynthetic Nanocellulose-Filled GelMA Inks Highly Reliable for Soft Tissue-Oriented Constructs.

Bioink-formulations based on gelatin methacrylate combined with oxidized cellulose nanofibrils are employed in the present study. The parallel investigation of the printing performance, morphological, swelling, and biological properties of the newly developed hydrogels was performed, with inks prepared using methacrylamide-modified gelatins of fish or bovine origin. Scaffolds with versatile and well-defined internal structure and high shape fidelity were successfully printed due to the high viscosity and shear-thinning behavior of formulated inks and then photo-crosslinked. The biocompatibility of 3D-scaffolds was surveyed using human adipose stem cells (hASCs) and high viability and proliferation rates were obtained when in contact with the biomaterial. Furthermore, bioprinting tests were performed with hASCs embedded in the developed formulations. The results demonstrated that the designed inks are a versatile toolkit for 3D bioprinting and further show the benefits of using fish-derived gelatin for biofabrication.

Cellulose Nanofiber-Based Hydrogels Embedding 5-FU Promote Pyroptosis Activation in Breast Cancer Cells and Support Human Adipose-Derived Stem Cell Proliferation, Opening New Perspectives for Breast Tissue Engineering.

The structure and biocompatibility analysis of a hydrogel based on cellulose nanofibers (CNFs) combined with alginate/pectin (A.CNF or P.CNF) and enriched with 1% or 5% 5-FU revealed more favorable properties for the cellular component when pectin was dispersed within CNFs. 5-Fluorouracil (5-FU) is an antimetabolite fluoropyrimidine used as antineoplastic drug for the treatment of multiple solid tumors. 5-FU activity leads to caspase-1 activation, secretion and maturation of interleukins (IL)-1, IL-18 and reactive oxygen species (ROS) generation. Furthermore, the effects of embedding 5-FU in P.CNF were explored in order to suppress breast tumor cell growth and induce inflammasome complex activation together with extra- and intracellular ROS generation. Exposure of tumor cells to P.CNF/5-FU resulted in a strong cytotoxic effect, an increased level of caspase-1 released in the culture media and ROS production-the latter directly proportional to the concentration of anti-tumor agent embedded in the scaffolds. Simultaneously, 5-FU determined the increase of p53 and caspase-1 expressions, both at gene and protein levels. In conclusion, P.CNF/5-FU scaffolds proved to be efficient against breast tumor cells growth due to pyroptosis induction. Furthermore, biocompatibility and the potential to support human adipose-derived stem cell growth were demonstrated, suggesting that these 3D systems could be used in soft tissue reconstruction post-mastectomy.

Bioinspired 3D printable pectin-nanocellulose ink formulations.

The assessment of several ink formulations for 3D printing based on two natural macromolecular compounds is presented. In the current research we have exploited the fast crosslinking potential of pectin and the remarkable shear-thinning properties of carboxylated cellulose nanofibrils, which is known to induce a desired viscoelastic behavior. Prior to 3D printing, the viscoelastic properties of the polysaccharide inks were evaluated by rheological measurements and injectability tests. The reliance of the printing parameters on the ink composition was established through one-dimensional lines printing, the base units of 3D-structures. The performance of the 3D-printed structures after ionic cross-linking was evaluated in terms of mechanical properties and rehydration behavior. MicroCT was also used to evaluate the morphology of the 3D-printed objects regarding the effect of pectin/nanocellulose ratio on the geometrical features of scaffolds. The proportionality between the two polymers proved to be the determining factor for the firmness and strength of the printed objects.