Bioprinting Of Hydrogels Research Articles

A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K/AKT signaling pathway.

Development of nano-laponite as bioinks based on cell-loaded hydrogels has recently attracted significant attention for promoting bone defect repairs and regeneration. However, the underlying mechanisms of the positive function of laponite in hydrogel was not fully explored. In this study, the effect of 3D bioprinted nano-laponite hydrogel construct on bone regeneration and the potential mechanism was explored in vitro and in vivo. In vitro analyses showed that the 3D construct protected encapsulated cells from shear stresses during bioprinting, promoted cell growth and cell spreading, and BMSCs at a density of 107/mL exhibited an optimal osteogenesis potential. Osteogenic differentiation and ectopic bone formation of BMSCs encapsulated inside the 3D construct were explored by determination of calcium deposition and x-ray, micro-CT analysis, respectively. RNA sequencing revealed that activation of PI3K/AKT signaling pathway of BMSCs inside the laponite hydrogel significantly upregulated expression of osteogenic related proteins. Expression of osteogenic proteins was significantly downregulated when the PI3K/AKT pathway was inhibited. The 3D bioprinted nano-laponite hydrogel construct exhibited a superior ability for bone regeneration in rat bones with defects compared with groups without laponite as shown by micro-CT and histological examination, while the osteogenesis activity was weakened by applications of a PI3K inhibitor. In summary, the 3D bioprinted nano-laponite hydrogel construct promoted bone osteogenesis by promoting cell proliferation, differentiation through activation of the PI3K/AKT signaling pathway.

Photo-Crosslinkable Hydrogels for 3D Bioprinting in the Repair of Osteochondral Defects: A Review of Present Applications and Future Perspectives.

An osteochondral defect is a common and frequent disease in orthopedics and treatment effects are not good, which can be harmful to patients. Hydrogels have been applied in the repair of cartilage defects. Many studies have reported that hydrogels can effectively repair osteochondral defects through loaded cells or non-loaded cells. As a new type of hydrogel, photo-crosslinked hydrogel has been widely applied in more and more fields. Meanwhile, 3D bioprinting serves as an attractive platform to fabricate customized tissue-engineered substitutes from biomaterials and cells for the repair or replacement of injured tissues and organs. Although photo-crosslinkable hydrogel-based 3D bioprinting has some advantages for repairing bone cartilage defects, it also has some disadvantages. Our aim of this paper is to review the current status and prospect of photo-crosslinkable hydrogel-based 3D bioprinting for repairing osteochondral defects.

Nanoparticle‐Stabilized Emulsion Bioink for Digital Light Processing Based 3D Bioprinting of Porous Tissue Constructs (Adv. Healthcare Mater. 12/2022)

Emulsion Bioinks In article number 2102810 by Rui Liu and co-workers, a nanoparticle-stabilized emulsion bioink via integration of two immiscible aqueous phases is developed to directly bioprint macroporous hydrogel constructs. The bioprinted macroporous hydrogel constructs do excellent service to the proliferation, spreading, and maturation of encapsulated cells in vitro and in vivo.

Suitability of Marine- and Porcine-Derived Collagen Type I Hydrogels for Bioprinting and Tissue Engineering Scaffolds.

Collagens from a wide array of animals have been explored for use in tissue engineering in an effort to replicate the native extracellular environment of the body. Marine-derived biomaterials offer promise over their conventional mammalian counterparts due to lower risk of disease transfer as well as being compatible with more religious and ethical groups within society. Here, collagen type I derived from a marine source (Macruronus novaezelandiae, Blue Grenadier) is compared with the more established porcine collagen type I and its potential in tissue engineering examined. Both collagens were methacrylated, to allow for UV crosslinking during extrusion 3D printing. The materials were shown to be highly cytocompatible with L929 fibroblasts. The mechanical properties of the marine-derived collagen were generally lower than those of the porcine-derived collagen; however, the Young’s modulus for both collagens was shown to be tunable over a wide range. The marine-derived collagen was seen to be a potential biomaterial in tissue engineering; however, this may be limited due to its lower thermal stability at which point it degrades to gelatin.

Development and characterization of pluronic F127 and methylcellulose based hydrogels for 3D bioprinting

Development and characterization of pluronic F127 and methylcellulose based hydrogels for 3D bioprinting

Upgrading a Consumer Stereolithographic 3D Printer to Produce a Physiologically Relevant Model with Human Liver Cancer Organoids

AbstractA widespread application of 3D bioprinting in basic and translational research requires accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open‐source device based on a consumer‐grade 3D stereolithography apparatus (SLA) printer is developed. This SLA bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a parallelized tissue production. To showcase the capability of the bioprinter, constructs consisting of patient‐derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel are produced. A thorough characterization of different GelMA/PEGDA ratios reveals that the mechanical properties of the bioprinted tumor model can be accurately fine‐tuned to mimic a specific tumor micro‐environment. Immunofluorescence and gene expression analyses of tumor markers confirm that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal‐derived reconstituted extracellular matrix.

CyMAD bioreactor: A cyclic magnetic actuation device for magnetically mediated mechanical stimulation of 3D bioprinted hydrogel scaffolds

Mechanical stimulation of bioprinted constructs can enhance the differentiation of cells within these scaffolds, such as driving chondrocytes towards cartilage tissue substitutes. In this study, a holistic approach is presented for designing and engineering a material-specific device based on a magnetic field setup using the Maxwell configuration for a touchless cyclic magnetic stimulation of (bioprinted) hydrogel scaffolds containing magnetic microparticles. We describe the entire development process, from the design of the magnetic field to the construction of the bioreactor and provide an evaluation of the calculation. Finally, an analysis of the distribution and orientation of the particles within the hydrogels and a cytocompatibility test after applying the intended stimulation regime were conducted. As a proof-of-principle, a model system in the shape of a cylindrical bending beam consisting of the established magnetisable bioink based on alginate, methylcellulose and magnetite microparticles (algMC + mag), was used instead of 3D printed, macroporous scaffolds of this material. Requirements for the dimensioning of the force, such as the Young's modulus, were determined experimentally. The magnetic field was calculated using the software Finite Element Method Magnetics (FEMM). The cyclic stimulation of the samples was performed over 14 days with a duration of 3 h per day. The aim was to achieve an elongation of up to 10%. The homogeneous particle distribution in stimulated and non-stimulated samples was proven via μCT and digital image processing (DIP). Even after applying a static magnetic field over 30 min, no structure formation such as chains or agglomeration of the magnetic particles were observed. The deformation behaviour defined as a shifted position of the neutral fibre (centre line of an object) during stimulation was measured via μCT and analysed using DIP. From these data, an elongation of approx. 9% was calculated for day 14. This elongation was achieved for both the stimulated samples and the control group without stimulation, which corresponds to the theoretically calculated value. The cytocompatibility of the bioink, scaffold environment and stimulation approach was demonstrated for bioprinted scaffolds with embedded human mesenchymal stem cells and chondrocytes. These findings proved the suitability and versatility of the bioreactor and the presented approach for stimulation experiments.

A Comprehensive Assessment on the Pivotal Role of Hydrogels in Scaffold-Based Bioprinting.

The past a few decades have seen exponential growth in the field of regenerative medicine. What began as extirpative (complete tissue or organ removal), with little regard to the effects of tissue loss and/or disfigurement, has evolved towards fabricating engineered tissues using personalized living cells (e.g., stem cells), and customizing a matrix or structural organization to support and guide tissue development. Biofabrication, largely accomplished through three-dimensional (3D) printing technology, provides precise, controlled, and layered assemblies of cells and biomaterials, emulating the heterogenous microenvironment of the in vivo tissue architecture. This review provides a concise framework for the bio-manufacturing process and addresses the contributions of hydrogels to biological modeling. The versatility of hydrogels in bioprinting is detailed along with an extensive elaboration of their physical, mechanical, and biological properties, as well as their assets and limitations in bioprinting. The scope of various hydrogels in tissue formation has been discussed through the case studies of biofabricated 3D constructs in order to provide the readers with a glimpse into the barrier-breaking accomplishments of biomedical sciences. In the end, the restraints of bioprinting itself are discussed, accompanied with the identification of available engineering strategies to overcome them.

Bone Healing Materials in the Treatment of Recalcitrant Nonunions and Bone Defects.

The usual treatment for bone defects and recalcitrant nonunions is an autogenous bone graft. However, due to the limitations in obtaining autogenous bone grafts and the morbidity associated with their procurement, various bone healing materials have been developed in recent years. The three main treatment strategies for bone defects and recalcitrant nonunions are synthetic bone graft substitutes (BGS), BGS combined with bioactive molecules, and BGS and stem cells (cell-based constructs). Regarding BGS, numerous biomaterials have been developed to prepare bone tissue engineering scaffolds, including biometals (titanium, iron, magnesium, zinc), bioceramics (hydroxyapatite (HA)), tricalcium phosphate (TCP), biopolymers (collagen, polylactic acid (PLA), polycaprolactone (PCL)), and biocomposites (HA/MONs@miR-34a composite coating, Bioglass (BG)-based ABVF-BG (antibiotic-releasing bone void filling) putty). Bone tissue engineering scaffolds are temporary implants that promote tissue ingrowth and new bone regeneration. They have been developed to improve bone healing through appropriate designs in terms of geometric, mechanical, and biological performance. Concerning BGS combined with bioactive molecules, one of the most potent osteoinductive growth factors is bone morphogenetic proteins (BMPs). In recent years, several natural (collagen, fibrin, chitosan, hyaluronic acid, gelatin, and alginate) and synthetic polymers (polylactic acid, polyglycolic acid, polylactic-coglycolide, poly(e-caprolactone) (PCL), poly-p-dioxanone, and copolymers consisting of glycolide/trimethylene carbonate) have been investigated as potential support materials for bone tissue engineering. Regarding BGS and stem cells (cell-based constructs), the main strategies are bone marrow stromal cells, adipose-derived mesenchymal cells, periosteum-derived stem cells, and 3D bioprinting of hydrogels and cells or bioactive molecules. Currently, significant research is being performed on the biological treatment of recalcitrant nonunions and bone defects, although its use is still far from being generalized. Further research is needed to investigate the efficacy of biological treatments to solve recalcitrant nonunions and bone defects.

Microporosity engineered printable silk/graphene hydrogels and their cytocompatibility evaluations

In this work, regenerated silk fibroin (RSF)/reduced graphene oxide (rGO) hybrid ink system was fabricated in aqueous phase, extrusion printed and photocrosslinked to hydrogels with tunable physicochemical properties and cell-gel interactions. Aqueous isopropyl alcohol was used to disperse the rGO into RSF matrix, where the RSF molecules showed relatively increased radius of gyration with IPA resulting in increased pore size and water uptake capacity of the photocrosslinked hydrogels. Incorporation of IPA along with increase in rGO content systematically decreased the viscosity, contact angle and printing accuracy of RSF/rGO hybrid inks, where the ink with highest rGO content exhibited relatively loose network structure at printing shear rate. However, no secondary structural change and fibrillogenesis was observed under printing shear stress, making it a suitable system for bioprinting silk hydrogel scaffolds. In addition, increase in rGO content systematically increased the crosslink density, β-sheet content, and mechanical properties of RSF/rGO hybrid hydrogels, whereas decreased the micropore size and water uptake capacity. Moreover, with increase in rGO content, the hybrid and bioprinted hydrogels showed good biocompatibility with marginal difference in cell viability on hybrid and bioprinted gels. The developed hydrogel systems could be potentially applied for tissue engineering and other functional applications.

3D Bioprinting of Heterogeneous Constructs Providing Tissue‐Specific Microenvironment Based on Host–Guest Modulated Dynamic Hydrogel Bioink for Osteochondral Regeneration

Abstract3D bioprinting is a promising strategy to develop heterogeneous constructs that mimic osteochondral tissue. However, conventional bioprinted hydrogels suffer from intrinsically weak mechanical strength, limited cell adaptability, and no sustained release of biochemical drugs, restraining their use as bioinks to emulate native osteochondral extracellular matrix. Herein, a novel host–guest modulated dynamic hydrogel is developed for 3D bioprinting heterogeneous cell‐laden constructs for osteochondral regeneration. Apart from gelatin methacryloyl (GelMA), this bioink consists of dopamine‐functionalized GelMA and acrylate β‐cyclodextrin and is crosslinked by host–guest interaction to develop the dynamic network for obtaining promoted cell adaptability, enhanced cell adhesion, reinforced mechanical strength, and tunable modulus. Moreover, based on the sustained drug release provided by the cavity of β‐cyclodextrin, a heterogeneous construct is constructed by employing kartogenin (a chondrogenic factor) into the upper zone with lower Young's modulus and melatonin (an osteogenic factor) into the bottom zone with higher modulus to mimic the osteochondral microenvironment. With the favorable regeneration results in vitro and in vivo, a broad application of this bioink in 3D bioprinting for tissues engineering is expected.

Towards Bioinspired Meniscus-Regenerative Scaffolds: Engineering a Novel 3D Bioprinted Patient-Specific Construct Reinforced by Biomimetically Aligned Nanofibers.

IntroductionThree of the main requirements that remain major challenges in tissue engineering of the knee meniscus are to engineer scaffolds with compatible anatomical shape, good mechanical properties, and microstructure able to mimic the architecture of the extracellular matrix (ECM). In this context, we presented a new biofabrication strategy to develop a three-dimensional (3D) meniscus-regenerative scaffold with custom-made macroscopic size and microarchitecture bioinspired by the organization of structural fibers of native tissue ECM.MethodsThe concept was based on the combination of bioprinted cell-laden hydrogel (type 1 collagen) reinforced by multilayers of biomimetically aligned electrospun nanofibrous mats (polycaprolactone/carbon nanotubes, PCL/CNT), using a patient-specific 3D digital meniscus model reconstructed from MRI data by free and open-source software.ResultsThe results showed that the incorporation of aligned nanofibers sheets between the hydrogel layers enhanced the scaffold’s structural integrity and shape fidelity compared to the nanofiber-free collagen hydrogel. Furthermore, mechanical compression tests demonstrated that the presence of nanofiber layers significantly improved the mechanical properties of the bioprinted construct. Importantly, the introduction of PCL/CNT nanofibrous mats between the layers of the bioprinted collagen hydrogel did not negatively affect cell viability, in which mesenchymal stem cells remained viable even after 7 days of culture within the scaffold.ConclusionOverall, these findings evidence that this bioengineering approach offers a promising strategy for fabricating biomimetic meniscus scaffolds for tissue engineering.

Neural stem cell-laden 3D bioprinting of polyphenol-doped electroconductive hydrogel scaffolds for enhanced neuronal differentiation

The development of three-dimensional (3D) bioprinting technology opens a door for constructing bionic nerve tissue scaffolds on demand for nerve injury repair. However, the electrical insulation of the current nerve tissue scaffolds fabricated by 3D bioprinting hinders the bioelectrical signal transmission between cells, limiting the therapeutic effect in nerve tissue repair. To address this issue, we have developed a neural stem cell (NSC)-laden 3D bioprinted electroconductive hydrogel (ECH) scaffold, composed of modified poly(3,4-ethylenedioxythiophene) (PEDOT), gelatin methacrylate/polyethylene glycol diacrylate hydrogel matrix, and NSCs, to promote the electron propagation in the scaffold for enhanced nerve regeneration. In our strategy, PEDOT is modified by doping with chondroitin sulfate and tannic acid (TA) to improve its water-solubility and electric property. With moderate mechanical strength and good electroconductivity, the 3D ECH scaffold provides a benign conductive microenvironment for the adhesion, growth, and proliferation of the encapsulated NSCs. Clearly, in the 3D ECH scaffolds, the NSCs not only maintain high cell viability (>90%) after bioprinting, but also tend to differentiate into neurons with extended neurites. This study testifies for the first time the effect of polyphenol structure belonging to TA on neuronal differentiation of NSCs, and offers a new insight into designing electroconductive biomaterials to induce neuronal regeneration for nerve injury repair and neurodegenerative disease therapy.

Tunable Microgel-Templated Porogel (MTP) Bioink for 3D Bioprinting Applications.

Micropores are essential for tissue engineering to ensure adequate mass transportation for embedded cells. Despite the considerable progress made by advanced 3D bioprinting technologies, it remains challenging to engineer micropores of 100µm or smaller in cell-laden constructs. Here, a microgel-templated porogel (MTP) bioink platform is reported to introduce controlled microporosity in 3D bioprinted hydrogels in the presence of living cells. Templated gelatin microgels are fabricated with varied sizes (≈10, ≈45, and ≈100µm) and mixed with photo-crosslinkable formulations to make composite MTP bioinks. The addition of microgels significantly enhances the shear-thinning and self-healing viscoelastic properties and thus the printability of bioinks with cell densities up to 1 × 108 mL-1 in matrix. Consistent printability is achieved for a series of MTP bioinks based on different component ratios and matrix materials. After photo-crosslinking the matrix phase, the templated microgels dissociated and diffused under physiological conditions, resulting in corresponding micropores in situ. When embedding osteoblast-like cells in the matrix phase, the MTP bioinks support higher metabolic activity and more uniform mineral formation than bulk gel controls. The approach provides a facile strategy to engineer precise micropores in 3D printed structures to compensate for the limited resolution of current bioprinting approaches.

A thermogelling organic-inorganic hybrid hydrogel with excellent printability, shape fidelity and cytocompatibility for 3D bioprinting

Alginates are the most commonly used bioink in biofabrication, but their rheological profiles make it very challenging to perform real 3D printing. In this study, an advanced hybrid hydrogel ink was developed, a mixture of thermogelling diblock copolymer, alginate and clay i.e. Laponite XLG. The reversible thermogelling and shear thinning properties of the diblock copolymer in the ink system improves handling and 3D printability significantly. Various three-dimensional constructs, including suspended filaments, were printed successfully with high shape fidelity and excellent stackability. Subsequent ionic crosslinking of alginate fixates the printed scaffolds, while the diblock copolymer is washed out of the structure, acting as a fugitive material/porogen on the (macro)molecular level. Finally, cell-laden printing and culture over 21 d demonstrated good cytocompatibility and feasibility of the novel hybrid hydrogels for 3D bioprinting. We believe that the developed approach could be interesting for a wide range of bioprinting applications including tissue engineering and drug screening, potentially enabling also other biological bioinks such as collagen, hyaluronic acid, decellularized extracellular matrices or cellulose based bioinks.

Light-based 3D bioprinting of bone tissue scaffolds with tunable mechanical properties and architecture from photocurable silk fibroin

Three-dimensional (3D) bioprinting based on digital light processing (DLP) offers unique opportunities to prepare scaffolds that mimic the architecture and biomechanical properties of human tissues. Limited availability of biocompatible and biodegradable bioinks amenable for DLP-bioprinting is an impediment in this field. This study presents a bioink prepared from silk fibroin (SF) tailored for DLP bioprinting. Photocurable methacrylated-SF (SF-MA) was synthesized with 67.3% of methacrylation. Physical characterization of rheological and mechanical properties revealed that the 3D printed hydrogels of SF-MA (spanning from 10 to 25 wt%) exhibit bone tissue-like viscoelastic behavior and compressive modulus ranging from ≈12 kPa to ≈96 kPa. The gels exhibited favorable degradation (≈48 to 91% in 21 days). This SF-MA bioink afforded the printing of complex structures, with high precision. Pre-osteoblasts were successfully encapsulated in 3D bioprinted SF-MA hydrogels with high viability. 15% SF-MA DLP bioprinted hydrogels efficiently supported cell proliferation with favorable cell morphology and cytoskeletal organization. A progressive increase in cell-mediated calcium deposition up to 14 days confirmed the ability of the gels to drive osteogenesis, which was further augmented by soluble induction factors. This work demonstrates the potential of silk fibroin-derived bioinks for DLP-based 3D bioprinting of scaffolds for tissue engineering.

Click Chemistry Hydrogels for Extrusion Bioprinting: Progress, Challenges, and Opportunities.

The emergence of 3D bioprinting has allowed a variety of hydrogel-based "bioinks" to be printed in the presence of cells to create precisely defined cell-loaded 3D scaffolds in a single step for advancing tissue engineering and/or regenerative medicine. While existing bioinks based primarily on ionic cross-linking, photo-cross-linking, or thermogelation have significantly advanced the field, they offer technical limitations in terms of the mechanics, degradation rates, and the cell viabilities achievable with the printed scaffolds, particularly in terms of aiming to match the wide range of mechanics and cellular microenvironments. Click chemistry offers an appealing solution to this challenge given that proper selection of the chemistry can enable precise tuning of both the gelation rate and the degradation rate, both key to successful tissue regeneration; simultaneously, the often bio-orthogonal nature of click chemistry is beneficial to maintain high cell viabilities within the scaffolds. However, to date, relatively few examples of 3D-printed click chemistry hydrogels have been reported, mostly due to the technical challenges of controlling mixing during the printing process to generate high-fidelity prints without clogging the printer. This review aims to showcase existing cross-linking modalities, characterize the advantages and disadvantages of different click chemistries reported, highlight current examples of click chemistry hydrogel bioinks, and discuss the design of mixing strategies to enable effective 3D extrusion bioprinting of click hydrogels.

3D Bioprinting of Gelatin-Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells.

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.

Quantification of Cell Contractile Behavior Based on Non-Destructive Macroscopic Measurement of Tension Forces on Bioprinted Hydrogel

Quantification of Cell Contractile Behavior Based on Non-Destructive Macroscopic Measurement of Tension Forces on Bioprinted Hydrogel

Designer DNA biomolecules as a defined biomaterial for 3D bioprinting applications.

DNA has excellent features such as the presence of functional and targeted molecular recognition motifs, tailorability, multifunctionality, high-precision molecular self-assembly, hydrophilicity, and outstanding biocompatibility. Due to these remarkable features, DNA has emerged as a leading next-generation biomaterial of choice to make hydrogels by self-assembly. In recent times, novel routes for the chemical synthesis of DNA, advances in tailorable designs, and affordable production ways have made DNA as a building block material for various applications. These advanced features have made researchers continuously explore the interesting properties of pure and hybrid DNA for 3D bioprinting and other biomedical applications. This review article highlights the topical advancements in the use of DNA as an ideal bioink for the bioprinting of cell-laden three-dimensional tissue constructs for regenerative medicine applications. Various bioprinting techniques and emerging design approaches such as self-assembly, nucleotide sequence, enzymes, and production cost to use DNA as a bioink for bioprinting applications are described. In addition, various types and properties of DNA hydrogels such as stimuli responsiveness and mechanical properties are discussed. Further, recent progress in the applications of DNA in 3D bioprinting are emphasized. Finally, the current challenges and future perspectives of DNA hydrogels in 3D bioprinting and other biomedical applications are discussed.