3D Printable Dynamic Hydrogel: As Simple as it Gets!

3D printing technology offers a vast range of applications for tissue engineering applications. Over the past decade a vast range of new equipment has been developed; while, 3D printable biomaterials, especially hydrogels, are investigated to fit the printability requirements. The current candidates for bioprinting often require post-printing cross-linking to maintain their shape. On the other hand, dynamic hydrogels are considered as the most promising candidate for this application with their extrudability and self-healing properties. However, it proves to be very difficult to match the required rheological in a simple material. Here, this study presents for the first time the simplest formulation of a dynamic hydrogel based on thiol-functionalized hyaluronic acid formulated with gold ions that fulfill all the requirements to be printed without the use of external stimuli, as judged by the rheological studies. The printability is also demonstrated with a 3D printer allowing for the printing of the dynamic hydrogel as it is, achieving 3D construct with a relatively good precision and up to 24 layers, corresponding to 10 mm high. This material is the simplest 3D printable hydrogel and its mixture with cells and biological compounds is expected to open a new era in 3D bioprinting.

Application of Extrusion-Based Hydrogel Bioprinting for Cartilage Tissue Engineering.

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed.

Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells.

Effective insertion of vertically aligned nanowires (NWs) into cells is critical for bioelectrical and biochemical devices, biological delivery systems, and photosynthetic bioenergy harvesting. However, accurate insertion of NWs into living cells using scalable processes has not yet been achieved. Here, NWs are inserted into living Chlamydomonas reinhardtii cells (Chlamy cells) via inkjet printing of the Chlamy cells, representing a low-cost and large-scale method for inserting NWs into living cells. Jetting conditions and printable bioink composed of living Chlamy cells are optimized to achieve stable jetting and precise ink deposition of bioink for indentation of NWs into Chlamy cells. Fluorescence confocal microscopy is used to verify the viability of Chlamy cells after inkjet printing. Simple mechanical considerations of the cell membrane and droplet kinetics are developed to control the jetting force to allow penetration of the NWs into cells. The results suggest that inkjet printing is an effective, controllable tool for stable insertion of NWs into cells with economic and scale-related advantages.

REVIEW ARTICLE Engineering bio-inks for 3D bioprinting cell mechanical microenvironment.

144Three-dimensional (3D) bioprinting has become a promising approach to constructing functional biomimetic tissues for tissue engineering and regenerative medicine. In 3D bioprinting, bio-inks are essential for the construction of cell microenvironment, thus affecting the biomimetic design and regenerative efficiency. Mechanical properties are one of the essential aspects of microenvironment, which can be characterized by matrix stiffness, viscoelasticity, topography, and dynamic mechanical stimulation. With the recent advances in functional biomaterials, various engineered bio-inks have realized the possibility of engineering cell mechanical microenvironment in vivo. In this review, we summarize the critical mechanical cues of cell microenvironments, review the engineered bio-inks while focusing on the selection principles for constructing cell mechanical microenvironments, and discuss the challenges facing this field and the possible solutions for them.

Advances in 3D Bioprinting: Techniques, Applications, and Future Directions for Cardiac Tissue Engineering.

Cardiovascular diseases are the leading cause of morbidity and mortality in the United States. Cardiac tissue engineering is a direction in regenerative medicine that aims to repair various heart defects with the long-term goal of artificially rebuilding a full-scale organ that matches its native structure and function. Three-dimensional (3D) bioprinting offers promising applications through its layer-by-layer biomaterial deposition using different techniques and bio-inks. In this review, we will introduce cardiac tissue engineering, 3D bioprinting processes, bioprinting techniques, bio-ink materials, areas of limitation, and the latest applications of this technology, alongside its future directions for further innovation.

Rheological and Biological Impact of Printable PCL-Fibers as Reinforcing Fillers in Cell-Laden Spider-Silk Bio-Inks.

The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.

Simple and robust 3D bioprinting of full-thickness human skin tissue.

Artificial skins have been used as skin substitutes for wound healing in the clinic, and as in vitro models for safety assessment in cosmetic and pharmaceutical industries. The three-dimensional (3D) bioprinting technique provides a promising strategy in the fabrication of artificial skins. Despite the technological advances, many challenges remain to be conquered, such as the complicated preparation conditions for bio-printed skin and the unavailability of stability and robustness of skin bioprinting. Here, we formulated a novel bio-ink composed of gelatin, sodium alginate and fibrinogen. By optimizing the ratio of components in the bio-ink, the design of the 3D model and the printing conditions, a fibroblasts-containing dermal layer construct was firstly fabricated, on the top of which laminin and keratinocytes were sequentially placed. Through air-liquid interface (ALI) culture by virtue of sterile wire mesh, a full-thickness skin tissue was thus prepared. HE and immunofluorescence staining showed that the bio-printed skin was not only morphologically representative of the human skin, but also expressed the specific markers related to epidermal differentiation and stratum corneum formation. The presented easy and robust preparation of full-thickness skin constructs provides a powerful tool for the establishment of artificial skins, holding critical academic significance and application value.

3D-Printed Pectin/Carboxymethyl Cellulose/ZnO Bio-Inks: Comparative Analysis with the Solution Casting Method.

Bio-inks consisting of pectin (Pec), carboxymethyl cellulose (CMC), and ZnO nanoparticles (ZnO) were used to prepare films by solution casting and 3D-printing methods. Field emission scanning electron microscopy (FE-SEM) was conducted to observe that the surface of samples made by 3D bioprinter was denser and more compact than the solution cast samples. In addition, Pec/CMC/ZnO made by 3D-bioprinter (Pec/CMC/ZnO-3D) revealed enhanced water vapor barrier, hydrophobicity, and mechanical properties. Pec/CMC/ZnO-3D also showed strong antimicrobial activity within 12 h against S. aureus and E. coli O157: H7 bacterial strains compared to the solution cast films. Further, the nanocomposite bio-inks used for 3D printing did not show cytotoxicity towards normal human dermal fibroblast (NDFB) cells but enhanced the fibroblast proliferation with increasing exposure concentration of the sample. The study provided two important inferences. Firstly, the 3D bioprinting method can be an alternative, better, and more practical method for fabricating biopolymer film instead of solution casting, which is the main finding of this work defining its novelty. Secondly, the Pec/CMC/ZnO can potentially be used as 3D bio-inks to fabricate functional films or scaffolds and biomedical applications.

3D Bio-printing For Skin Tissue Regeneration: Hopes and Hurdles.

For many years, discovering the appropriate methods for the treatment of skin irritation has been challenging for specialists and researchers. Bio-printing can be extensively applied to address the demand for proper skin substitutes to improve skin damage. Nowadays, to make more effective biomimicry of natural skin, many research teams have developed cell-seeded bio-inks for bioprinting of skin substitutes. These loaded cells can be single or co-cultured in these structures. The present review gives a comprehensive overview of the methods, substantial parameters of skin bioprinting, examples of in vitro and in vivo studies, and current advances and challenges in skin tissue engineering.

Development of Bio-Active Patches Based on Pectin for the Treatment of Ulcers and Wounds Using 3D-Bioprinting Technology.

Biodegradable 3D-printable inks based on pectin have been developed as a system for direct and indirect wound-dressing applications, suitable for 3D printing technologies. The 3D-printable inks formed free-standing transparent films upon drying, with the latter exhibiting fast disintegration upon contact with aqueous media. The antimicrobial and wound-healing activities of the inks have been successfully enhanced by the addition of particles, comprised of chitosan and cyclodextrin inclusion complexes with propolis extract. Response Surface Methodology (RSM) was applied for the optimization of the inks (extrusion-printing pressure, shrinkage minimization over-drying, increased water uptake and minimization of the disintegration of the dry patches upon contact with aqueous media). Particles comprised of chitosan and cyclodextrin/propolis extract inclusion complexes (CCP), bearing antimicrobial properties, were optimized and integrated with the produced inks. The bioprinted patches were assessed for their cytocompatibility, antimicrobial activity and in vitro wound-healing properties. These studies were complemented with ex vivo skin adhesion measurements, a relative surface hydrophobicity and opacity measurement, mechanical properties, visualization, and spectroscopic techniques. The in vitro wound-healing studies revealed that the 3D-bioprinted patches enhanced the in vitro wound-healing process, while the incorporation of CCP further enhanced wound-healing, as well as the antimicrobial activity of the patches.

Instant Gelation System as Self-Healable and Printable 3D Cell Culture Bioink Based on Dynamic Covalent Chemistry.

The rapid development of additive manufacturing techniques in the field of tissue regeneration offers unprecedented success for artificial tissue and organ fabrication. However, some limitations still remain for current bioinks, such as the compromised cell viability after printing, the low cross-linking efficiency leading to poor printing resolution and speed due to the relatively slow gelation rate, and the requirement of external stimuli for gelation. To address these problems, herein, a biocompatible and printable instant gelation hydrogel system has been developed based on a designed hyperbranched poly(ethylene glycol) (PEG)-based multihydrazide macro-cross-linker (HB-PEG-HDZ) and an aldehyde-functionalized hyaluronic acid (HA-CHO). HB-PEG-HDZ is prepared by the postfunctionalization of hyperbranched PEG-based multivinyl macromer via thiol-ene chemistry. Owing to the high functional group density of HB-PEG-HDZ, the hydrogel can be formed instantly upon mixing the solutions of two components. The reversible cross-linking mechanism between the hydrazide and aldehyde groups endows the hydrogel with shear-thinning and self-healing properties. The minimally toxic components and cross-linking chemistry allow the resulting hydrogel to be a biocompatible niche. Moreover, the fast sol-to-gel transition of the hydrogel, combining all of the advanced characteristics of this platform, protects the cells during the printing procedure, avoids their damage during extrusion, and improves the transplanted cell survival.

Three-Dimensional Printing Constructs Based on the Chitosan for Tissue Regeneration: State of the Art, Developing Directions and Prospect Trends.

Chitosan (CS) has gained particular attention in biomedical applications due to its biocompatibility, antibacterial feature, and biodegradability. Hence, many studies have focused on the manufacturing of CS films, scaffolds, particulate, and inks via different production methods. Nowadays, with the possibility of the precise adjustment of porosity size and shape, fiber size, suitable interconnectivity of pores, and creation of patient-specific constructs, 3D printing has overcome the limitations of many traditional manufacturing methods. Therefore, the fabrication of 3D printed CS scaffolds can lead to promising advances in tissue engineering and regenerative medicine. A review of additive manufacturing types, CS-based printed constructs, their usages as biomaterials, advantages, and drawbacks can open doors to optimize CS-based constructions for biomedical applications. The latest technological issues and upcoming capabilities of 3D printing with CS-based biopolymers for different applications are also discussed. This review article will act as a roadmap aiming to investigate chitosan as a new feedstock concerning various 3D printing approaches which may be employed in biomedical fields. In fact, the combination of 3D printing and CS-based biopolymers is extremely appealing particularly with regard to certain clinical purposes. Complications of 3D printing coupled with the challenges associated with materials should be recognized to help make this method feasible for wider clinical requirements. This strategy is currently gaining substantial attention in terms of several industrial biomedical products. In this review, the key 3D printing approaches along with revealing historical background are initially presented, and ultimately, the applications of different 3D printing techniques for fabricating chitosan constructs will be discussed. The recognition of essential complications and technical problems related to numerous 3D printing techniques and CS-based biopolymer choices according to clinical requirements is crucial. A comprehensive investigation will be required to encounter those challenges and to completely understand the possibilities of 3D printing in the foreseeable future.

3D biofabrication for tubular tissue engineering.

The therapeutic replacement of diseased tubular tissue is hindered by the availability and suitability of current donor, autologous and synthetically derived protheses. Artificially created, tissue engineered, constructs have the potential to alleviate these concerns with reduced autoimmune response, high anatomical accuracy, long-term patency and growth potential. The advent of 3D bioprinting technology has further supplemented the technological toolbox, opening up new biofabrication research opportunities and expanding the therapeutic potential of the field. In this review, we highlight the challenges facing those seeking to create artificial tubular tissue with its associated complex macro- and microscopic architecture. Current biofabrication approaches, including 3D printing techniques, are reviewed and future directions suggested.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Three-Dimensional Printing of Tissue/Organ Analogues Containing Living Cells.

The technical advances of three-dimensional (3D) printing in the field of tissue engineering have enabled the creation of 3D living tissue/organ analogues. Diverse 3D tissue/organ printing techniques with computer-aided systems have been developed and used to dispose living cells together with biomaterials and supporting biochemicals as pre-designed 3D tissue/organ models. Furthermore, recent advances in bio-inks, which are printable hydrogels with living cell encapsulation, have greatly enhanced the versatility of 3D tissue/organ printing. Here, we introduce 3D tissue/organ printing techniques and biomaterials that have been developed and widely used thus far. We also review a variety of applications in an attempt to repair or replace the damaged or defective tissue/organ, and develop the in vitro tissue/organ models. The potential challenges are finally discussed from the technical perspective of 3D tissue/organ printing.