Embedded 3D Printing of Cryogel-Based Scaffolds.

Cryogel-based scaffolds have attracted great attention in tissue engineering due to their interconnected macroporous structures. However, three-dimensional (3D) printing of cryogels with a high degree of precision and complexity is a challenge, since the synthesis of cryogels occurs under cryogenic conditions. In this study, we demonstrated the fabrication of cryogel-based scaffolds for the first time by using an embedded printing technique. A photo-cross-linkable gelatin methacryloyl (GelMA)-based ink composition, including alginate and photoinitiator, was printed into a nanoclay-based support bath. The layer-by-layer extruded ink was held in complex and overhanging structures with the help of pre-cross-linking of alginate with Ca2+ present in the support bath. The printed 3D structures in the support bath were frozen, and then GelMA was cross-linked at a subzero temperature under UV light. The printed and cross-linked structures were successfully recovered from the support bath with an integrated shape complexity. SEM images showed the formation of a 3D printed scaffold where porous GelMA cryogel was integrated between the cross-linked alginate hydrogels. In addition, they showed excellent shape recovery under uniaxial compression cycles of up to 80% strain. In vitro studies showed that the human fibroblast cells attached to the 3D printed scaffold and displayed spread morphology with a high proliferation rate. The results revealed that the embedded 3D printing technique enables the fabrication of cytocompatible cryogel based scaffolds with desired morphology and mechanical behavior using photo-cross-linkable bioink composition. The properties of the cryogels can be modified by varying the GelMA concentration, whereby various shapes of scaffolds can be fabricated to meet the specific requirements of tissue engineering applications.

Design and bioprinting for tissue interfaces.

Tissue interfaces include complex gradient structures formed by transitioning of biochemical and mechanical properties in micro-scale. This characteristic allows the communication and synchronistic functioning of two adjacent but distinct tissues. It is particularly challenging to restore the function of these complex structures by transplantation of scaffolds exclusively produced by conventional tissue engineering methods. Three-dimensional (3D) bioprinting technology has opened an unprecedented approach for precise and graded patterning of chemical, biological and mechanical cues in a single construct mimicking natural tissue interfaces. This paper reviews and highlights biochemical and biomechanical design for 3D bioprinting of various tissue interfaces, including cartilage-bone, muscle-tendon, tendon/ligament-bone, skin, and neuro-vascular/muscular interfaces. Future directions and translational challenges are also provided at the end of the paper.

3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface.

Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional (3D) tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, anin situcrosslinked hybrid, multi-material 3D bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow mesenchymal stem cells-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing anin situcrosslink approach with ruthenium (Ru) and sodium persulfate photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial.

Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.

3D Fiber Reinforced Hydrogel Scaffolds by Melt Electrowriting and Gel Casting as a Hybrid Design for Wound Healing.

Emerging biomanufacturing technologies have revolutionized the field of tissue engineering by offering unprecedented possibilities. Over the past few years, new opportunities arose by combining traditional and novel fabrication techniques, shaping the hybrid designs in biofabrication. One of the potential application fields is skin tissue engineering, in which a combination of traditional principles of wound dressing with advanced biofabrication methods could yield more efficient therapies. In this study, a hybrid design of fiber-reinforced scaffolds combined with gel casting is developed and the efficiency for in vivo wound healing applications is assessed. For this purpose, 3D fiber meshes produced by melt electrowriting are selectively filled with photocrosslinkable gelatin hydrogel matrices loaded with different growth factor carrier microspheres. Additionally, the influence of the inclusion of inorganic bioactive glass particles within the composite fibrous mesh is evaluated. Qualitative evaluation of secondary wound healing criteria and histological analysis shows that hybrid scaffolds containing growth factors and bioactive glass enhances the healing process significantly, compared to the designs merely providing a fiber-reinforced bioactive hydrogel matrix as the wound dressing. This study aims to explore a new application area for melt electrowriting as a powerful tool in fabricating hybrid therapeutic designs for skin tissue engineering.

3D bioprinting of molecularly engineered PEG-based hydrogels utilizing gelatin fragments.

Three-dimensional (3D) bioprinting is an additive manufacturing process in which the combination of biomaterials and living cells, referred to as a bioink, is deposited layer-by-layer to form biologically active 3D tissue constructs. Recent advancements in the field show that the success of this technology requires the development of novel biomaterials or the improvement of existing bioinks. Polyethylene glycol (PEG) is one of the well-known synthetic biomaterials and has been commonly used as a photocrosslinkable bioink for bioprinting; however, other types of cell-friendly crosslinking mechanisms to form PEG hydrogels need to be explored for bioprinting and tissue engineering. In this work, we proposed micro-capillary based bioprinting of a novel molecularly engineered PEG-based bioink that transiently incorporates low molecular weight gelatin (LMWG) fragments. The rheological properties and release profile of the LMWG fragments were characterized, and their presence during hydrogel formation had no effect on the swelling ratio or sol fraction when compared to PEG hydrogels formed without the LMWG fragments. For bioprinting, PEG was first functionalized with cell-adhesive RGD ligands and was then crosslinked using protease-sensitive peptides via a Michael-type addition reaction inside the micro-capillary. The printability was assessed by the analysis of extrudability, shape fidelity, and printing accuracy of the hydrogel filaments after the optimization of the gelation conditions of the PEG-based bioink. The LMWG fragments supplemented into the bioink allowed the extrusion of smooth and uniform cylindrical strands of the hydrogel and improved shape fidelity and printing accuracy. Encapsulated cells in both bioprinted and non-bioprinted PEG-based hydrogels showed high viability and continued to proliferate over time in culture with a well-defined cell morphology depending on the presence of the cell adhesive peptide RGD. The presented micro-capillary based bioprinting process for a novel PEG-based bioink can be promising to construct complex 3D structures with micro-scale range and spatiotemporal variations without using any cytotoxic photoinitiator, UV light, or polymer support.

Exploiting Urazole's Acidity for Fabrication of Hydrogels and Ion-Exchange Materials.

In this study, the acidity of urazole (pKa 5-6) was exploited to fabricate a hydrogel in two simple and scalable steps. Commercially available poly(hexamethylene)diisocyanate was used as a precursor to synthesize an urazole containing gel. The formation of urazole was confirmed by FT-IR and 1H-NMR spectroscopy. The hydrogel was characterized by microscopy imaging as well as spectroscopic and thermo-gravimetric analyses. Mechanical analysis and cell viability tests were performed for its initial biocompatibility evaluation. The prepared hydrogel is a highly porous hydrogel with a Young's modulus of 0.91 MPa, has a swelling ratio of 87%, and is capable of exchanging ions in a medium. Finally, a general strategy was demonstrated to embed urazole groups directly into a crosslinked material.

Preparation and characterization of nanoclay-hydrogel composite support-bath for bioprinting of complex structures.

Three-dimensional bioprinting of cell-laden hydrogels in a sacrificial support-bath has recently emerged as a potential solution for fabricating complex biological structures. Physical properties of the support-bath strongly influence the bioprinting process and the outcome of the fabricated constructs. In this study, we reported the application of a composite Pluronic-nanoclay support-bath including calcium ions as the crosslinking agent for bioprinting of cell-laden alginate-based hydrogels. By tuning the rheological properties, a shear-thinning composite support-bath with fast self-recovery behavior was yielded, which allowed continuous printing of complex and large-scale structures. The printed structures were easily and efficiently harvested from the support-bath without disturbing their shape fidelity. Moreover, the results showed that support-bath assisted bioprinting process did not influence the viability of cells encapsulated within hydrogel. This study demonstrates that Pluronic-nanoclay support-bath can be utilized for bioprinting of complex, cell-laden constructs for vascular and other tissue engineering applications.

3D printing of silver-doped polycaprolactone-poly(propylene succinate) composite scaffolds for skin tissue engineering.

Scaffold-based tissue engineering approaches have been commonly used for skin regeneration or wound healings caused by diseases or trauma. For an ideal complete healing process, scaffold structures need to meet the criteria of biocompatibility, biodegradability, and antimicrobial properties, as well as to provide geometrical necessities for the regeneration of damaged tissue. In this study, design, synthesis and characterization of a three dimensional (3D) printable copolymer based on polycaprolactone-block-poly(1,3-propylene succinate) (PCL-PPSu) including anti-microbial silver particles is presented. 3D printing of PCL-PPSu copolymers provided a lower processing temperature compared to neat PCL, hence, inclusion of temperature-sensitive bioactive reagents into the developed copolymer could be realized. In addition, 3D printed block copolymer showed an enhanced hydrolytic and enzymatic degradation behavior. Cell viability and cytotoxicity of the developed copolymer were evaluated by using human dermal fibroblast (HDF) cells. The addition of silver nitrate within the polymer matrix resulted in a significant decrease in the adhesion of different types of microorganisms on the scaffold without inducing any cytotoxicity on HDF cells in vitro. The results suggested that 3D printed PCL-PPSu scaffolds containing anti-microbial silver particles could be considered as a promising biomaterial for emerging skin regenerative therapies, in the light of its adaptability to 3D printing technology, low-processing temperature, enhanced degradation behavior and antimicrobial properties.

Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting.

Three-dimensional (3D) bioprinting enables the controlled fabrication of complex constructs for tissue engineering applications and has been actively explored in recent years. However, its progress has been limited by the existing difficulties in the development of bioinks with suitable biocompatibility and mechanical properties and at the same time adaptability to the process. Herein, we describe the engineering of a nanocomposite agarose bioink with tailored properties using 2D nanosilicate additives. The suitability of agarose for 3D bioprinting has been debated due to its bioinert nature and compatibility with extrusion-based bioprinting. Nanosilicates were used to tailor the flow behavior of agarose solutions, and detailed rheological characterization of different bioink formulations was performed to elucidate the effect of nanosilicates on the flow behavior and gelation of agarose bioinks. The proper selection of nanosilicate concentration resulted in extrusion 3D printed structures with high shape fidelity and structural integrity. Moreover, the influence of addition of nanosilicates on the bioactivity of agarose was studied, and nanocomposite bioinks showed significant improvement in metabolic activity of encapsulated cells. The bioactivity of the nanocomposite bioinks was also evaluated through a cell spreading assay; the charged surfaces of nanosilicates resulted in full spreading and elongation of fibroblasts, and the extent of change in morphology of cells was found to be directly correlated with the nanosilicate concentration. Our findings suggested that engineered agarose-nanosilicate bioinks can be exploited as a new generation of hydrogel bioinks for extrusion 3D bioprinting with tunable flow properties and bioactivity.