Air-loaded Gas Vesicle Nanoparticles Promote Cell Growth in Three-dimensional Bioprinted Tissue Constructs.

Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea. These hollow nanostructures are composed of a protein shell that allows gases to permeate freely while excluding the water flow. After printing cells with gas vesicles of various concentrations, the cells were observed to have increased activity and proliferation. These results suggest that air-filled gas vesicles can help overcome the diffusion barrier throughout the 3D bioprinted constructs by increasing oxygen availability to cells within the center of the construct. The biodegradable nature of the gas vesicle proteins combined with our promising results encourage their potential use as oxygen-promoting materials in biological samples.

Ultrashort Peptide Bioinks Support Automated Printing of Large-Scale Constructs Assuring Long-Term Survival of Printed Tissue Constructs.

We report about rationally designed ultrashort peptide bioinks, overcoming severe limitations in current bioprinting procedures. Bioprinting is increasingly relevant in tissue engineering, regenerative and personalized medicine due to its ability to fabricate complex tissue scaffolds through an automated deposition process. Printing stable large-scale constructs with high shape fidelity and enabling long-term cell survival are major challenges that most existing bioinks are unable to solve. Additionally, they require chemical or UV-cross-linking for the structure-solidifying process which compromises the encapsulated cells, resulting in restricted structure complexity and low cell viability. Using ultrashort peptide bioinks as ideal bodylike but synthetic material, we demonstrate an instant solidifying cell-embedding printing process via a sophisticated extrusion procedure under true physiological conditions and at cost-effective low bioink concentrations. Our printed large-scale cell constructs and the chondrogenic differentiation of printed mesenchymal stem cells point to the strong potential of the peptide bioinks for automated complex tissue fabrication.

Self-assembling tetrameric peptides allow in situ 3D bioprinting under physiological conditions.

We have developed an in situ bioprinting method that allows the printing of cells under true physiological conditions by applying self-assembling ultrashort peptides as bioinks. This method avoids cell stressing methods, such as UV-treatment, chemical crosslinking and viscous bioink printing methods. We further demonstrate that different nanomaterials can easily be synthesized or incorporated in the 3D bioprinted peptide scaffolds which opens up the possibility of functionalized 3D scaffolds.

Exploring nanofibrous self-assembling peptide hydrogels using mouse myoblast cells for three-dimensional bioprinting and tissue engineering applications.

Injured skeletal muscles which lose more than 20% of their volume, known as volumetric muscle loss, can no longer regenerate cells through self-healing. The traditional solution for recovery is through regenerative therapy. As the technology of three-dimensional (3D) bioprinting continues to advance, a new approach for tissue transplantation is using biocompatible materials arranged in 3D scaffolds for muscle repair. Ultrashort self-assembling peptide hydrogels compete as a potential biomaterial for muscle tissue formation due to their biocompatibility. In this study, two sequences of ultrashort peptides were analyzed with muscle myoblast cells (C2C12) for cell viability, cell proliferation, and differentiation in 3D cell culture. The peptides were then extruded through a custom-designed robotic 3D bioprinter to create cell-laden 3D structures. These constructs were also analyzed for cell viability through live/dead assay. Results showed that 3D bioprinted structures of peptide hydrogels could be used as tissue platforms for myotube formation - a process necessary for muscle repair.

Preparation and printability of ultrashort self-assembling peptide nanoparticles.

Nanoparticles (NPs) have left their mark on the field of bioengineering. Fabricated from metallic, magnetic, and metal oxide materials, their applications include drug delivery, bioimaging, and cell labeling. However, as they enter the body, the question remains - where do they go after fulfilling their designated function? As most materials used to produce NPs are not naturally found in the body, they are not biodegradable and may accumulate overtime. There is a lack of comprehensive, long-term studies assessing the biodistribution of non-biodegradable NPs for even the most widely studied NPs. There is a clear need for NPs produced from natural materials capable of degradation in vivo. As peptides exist naturally within the human body, their non-toxic and biocompatible nature comes as no surprise. Ultrashort peptides are aliphatic peptides designed with three to seven amino acids capable of self-assembling into helical fibers within macromolecular structures. Using a microfluidics flow-focusing approach, we produced different peptide-based NPs that were then three-dimensional (3D) printed with our novel printer setup. Herein, we describe the preparation method of NPs from ultrashort self-assembling peptides and their morphology in both manual and 3D-printed hydrogels, thus suggesting that peptide NPs are capable of withstanding the stresses involved in the printing process.

Optimization of a 3D bioprinting process using ultrashort peptide bioinks.

The field of three-dimensional (3D) bioprinting is rapidly emerging as an additive manufacturing method for tissue and organ fabrication. The demand for tissues and organ transplants is ever increasing, although donors are not as readily available. Consequently, tissue engineering is gaining much attention to alleviate this problem. The process of achieving well-structured 3D bioprinted constructs using hydrogel bioinks depends on symmetrical precision, regulated flow rates, and viability of cells. Even with the mentioned parameters optimized, the printed structures need additional refining by removing excessive liquids, as peptide hydrogel bioprints encapsulate water. However, it is challenging to eliminate the confined fluids without compromising the printing process. In this paper, we introduced a vacuum system to our 3D bioprinting robotic arm and thus optimized the printing quality for complex and refined 3D scaffolds. Moreover, the proposed vacuum system supports printing with cells. Our results show improved printing resolution which facilitates the printing of higher and more stable structures.