Designing Gelatin Methacryloyl (GelMA)‐Based Bioinks for Visible Light Stereolithographic 3D Biofabrication

Bioinks play a key role in determining the capability of the biofabricatoin processes and the resolution of the printed constructs. Excellent biocompatibility, tunable physical properties, and ease of chemical or biological modifications of gelatin methacryloyl (GelMA) have made it an attractive choice as bioinks for biomanufacturing of various tissues or organs. However, the current preparation methods for GelMA‐based bioinks lack the ability to tailor their physical properties for desired bioprinting methods. Inherently, GelMA prepolymer solution exhibits a fast sol–gel transition at room temperature, which is a hurdle for its use in stereolithography (SLA) bioprinting. Here, synthesis parameters are optimized such as solvents, pH, and reaction time to develop GelMA bioinks which have a slow sol–gel transition at room temperature and visible light crosslinkable functions. A total of eight GelMA combinations are identified as suitable for digital light processing (DLP)‐based SLA (DLP‐SLA) bioprinting through systematic characterizations of their physical and rheological properties. Out of various types of GelMA, those synthesized in reverse osmosis (RO) purified water (referred to as RO‐GelMA) are regarded as most suitable to achieve high DLP‐SLA printing resolution. RO‐GelMA‐based bioinks are also found to be biocompatible showing high survival rates of encapsulated cells in the photocrosslinked gels. Additionally, the astrocytes and fibroblasts are observed to grow and integrate well within the bioprinted constructs. The bioink's superior physical and photocrosslinking properties offer pathways of tuning the scaffold microenvironment and highlight the applicability of developed GelMA bioinks in various tissue engineering and regenerative medicine applications.     

A General Strategy for Extrusion Bioprinting of Bio‐Macromolecular Bioinks through Alginate‐Templated Dual‐Stage Crosslinking

The recently developed 3D bioprinting technology has greatly improved the ability to generate biomimetic tissues that are structurally and functionally relevant to their human counterparts. The selection of proper biomaterials as the bioinks is a key step toward successful bioprinting. For example, viscosity of a bioink is an important rheological parameter to determine the flexibility in deposition of free‐standing structures and the maintenance of architectural integrity following bioprinting. This requirement, however, has greatly limited the selection of bioinks, especially for those naturally derived due to their commonly low mechanical properties. Here the generalization of a mechanism for extrusion bioprinting of bio‐macromolecular components, mainly focusing on collagen and its derivatives including gelatin and gelatin methacryloyl, is reported. Specifically, a templating strategy is adopted using a composite bioink containing both the desired bio‐macromolecular component and a polysaccharide alginate. The physically crosslinkable alginate component serves as the temporal structural support to stabilize the shape of the construct during bioprinting; upon subsequent chemical or physical crosslinking of the bio‐macromolecular component, alginate can be selectively removed to leave only the desired bio‐macromolecule. It is anticipated that this strategy is general, and can be readily expanded for use of a wide variety of other bio‐macromolecular bioinks.     

Sonochemical Degradation of Gelatin Methacryloyl to Control Viscoelasticity for Inkjet Bioprinting

Inkjet printing enables the mimicry of the microenvironment of natural complex tissues by patterning cells and hydrogels at a high resolution. However, the polymer content of an inkjet-printable bioink is limited as it leads to strong viscoelasticity in the inkjet nozzle. Here it is demonstrated that sonochemical treatment controls the viscoelasticity of a gelatin methacryloyl (GelMA) based bioink by shortening the length of polymer chains without causing chemical destruction of the methacryloyl groups. The rheological properties of treated GelMA inks are evaluated by a piezo-axial vibrator over a wide range of frequencies between 10 and 10 000 Hz. This approach enables to effectively increase the maximum printable polymer concentration from 3% to 10%. Then it is studied how the sonochemical treatment effectively controls the microstructure and mechanical properties of GelMA hydrogel constructs after crosslinking while maintaining its fluid properties within the printable range. The control of mechanical properties of GelMA hydrogels can lead fibroblasts more spreading on the hydrogels. A 3D cell-laden multilayered hydrogel constructs containing layers with different physical properties is fabrictated by using high-resolution inkjet printing. The sonochemical treatment delivers a new path to inkjet bioprinting to build microarchitectures with various physical properties by expanding the range of applicable bioinks.     

Three-Dimensional Bioprinting of Decellularized Extracellular Matrix-Based Bioinks for Tissue Engineering

Three-dimensional (3D) bioprinting is one of the most promising additive manufacturing technologies for fabricating various biomimetic architectures of tissues and organs. In this context, the bioink, a critical element for biofabrication, is a mixture of biomaterials and living cells used in 3D printing to create cell-laden structures. Recently, decellularized extracellular matrix (dECM)-based bioinks derived from natural tissues have garnered enormous attention from researchers due to their unique and complex biochemical properties. This review initially presents the details of the natural ECM and its role in cell growth and metabolism. Further, we briefly emphasize the commonly used decellularization treatment procedures and subsequent evaluations for the quality control of the dECM. In addition, we summarize some of the common bioink preparation strategies, the 3D bioprinting approaches, and the applicability of 3D-printed dECM bioinks to tissue engineering. Finally, we present some of the challenges in this field and the prospects for future development.     

In vitro formation and characterization of a perfusable three-dimensional tubular capillary network in microfluidic devices

This paper describes the in vitro formation and characterization of perfusable capillary networks made of human umbilical vein endothelial cells (HUVECs) in microfluidic devices (MFDs). Using this platform, an array of three-dimensional (3D) tubular capillaries of various dimensions (50-150 μm in diameter and 100-1600 μm in length) can be formed reproducibly. To generate connected blood vessels, MFDs were completely filled with fibrin gel and subsequently processed to selectively leave behind gel structures inside the bridge channels. Following gel solidification, HUVECs were coated along the gel walls, on opposite ends of the patterned 3D fibrin gel. After 3-4 days, HUVECs migrating into the fibrin gel from opposite ends fused with each other, spontaneously forming a connected vessel that expressed tight junction proteins (e.g., ZO-1), which are characteristic of post-capillary venules. With ready access to a perfusable capillary network, we demonstrated perfusion of the vessels and imaged red blood cells (RBCs) and beads flowing through them. The results were reproducible (～50% successful perfusable capillaries), consistent, and could be performed in a parallel manner (9 devices per well plate). Additionally, compatibility with high resolution live-cell microscopy and the possibility of incorporating other cell types makes this a unique experimental platform for investigating basic and applied aspects of angiogenesis, anastomosis, and vascular biology.     

A universal self‐eroding sacrificial bioink that enables bioprinting at room temperature

Natural polymer‐based hydrogel bioinks are widely used in bioprinting due to their suitability for recapitulation of in vivo cellular activities. However, preservation of the target geometry in a cell‐laden hydrogel is difficult to achieve. The aim of this study was to develop a universal sacrificial bioink that allows high cell viability and a better shape fidelity in the cell‐laden construct. A polysaccharide‐based universal sacrificial bioink was developed for microextrusion‐based bioprinting and was optimized to erode in 48 hours in the cell culture medium without formation of any undesired by‐products. The sacrificial hydrogel was prepared from alginate and agarose via a microwave oven assisted method and bioprinted at room temperature to generate microchannels in the cell‐laden hydrogel or to support a tubular structure and its biocompatibility determined by live/dead assay. Bioprinting time was significantly reduced, down to a few minutes for a large‐scale tissue model (1 minute 52 seconds for a 2 cm tubular structure), by means of a high bioprinting speed up to 25 mm/s. After 48 hours in the cell culture, the sacrificial bioink completely detached from the cell‐laden construct without causing any changes in its printed shape. Cell viability in the cell‐laden construct was observed to be more than 95% at the end of 3‐day culture. This novel sacrificial bioink enables bioprinting at room temperature without affecting oxygen and nutrient penetration into the cell‐laden hydrogel and allows retention of high cell viability and shape fidelity.     

Biofabrication of Cell‐Loaded 3D Spider Silk Constructs

Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue‐like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non‐immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell‐loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell‐binding motif to the spider silk protein further enables fine‐tuned control over cell–material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.     

Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks

In this review, few established cell printing techniques along with their parameters that affect the cell viability during bioprinting are considered. 3D bioprinting is developed on the principle of additive manufacturing using biomaterial inks and bioinks. Different bioprinting methods impose few challenges on cell printing such as shear stress, mechanical impact, heat, laser radiation, etc., which eventually lead to cell death. These factors also cause alteration of cells phenotype, recoverable or irrecoverable damages to the cells. Such challenges are not addressed in detail in the literature and scientific reports. Hence, this review presents a detailed discussion of several cellular bioprinting methods and their process‐related impacts on cell viability, followed by probable mitigation techniques. Most of the printable bioinks encompass cells within hydrogel as scaffold material to avoid the direct exposure of the harsh printing environment on cells. However, the advantages of printing with scaffold‐free cellular aggregates over cell‐laden hydrogels have emerged very recently. Henceforth, optimal and favorable crosslinking mechanisms providing structural rigidity to the cell‐laden printed constructs with ideal cell differentiation and proliferation, are discussed for improved understanding of cell printing methods for the future of organ printing and transplantation.     

Injectable Supramolecular Hydrogel/Microgel Composites for Therapeutic Delivery

Shear‐thinning hydrogels are useful for biomedical applications, from 3D bioprinting to injectable biomaterials. Although they have the appropriate properties for injection, it may be advantageous to decouple injectability from the controlled release of encapsulated therapeutics. Toward this, composites of hydrogels and encapsulated microgels are introduced with microgels that are fabricated via microfluidics. The microgel cross‐linker controls degradation and entrapped molecule release, and the concentration of microgels alters composite hydrogel rheological properties. For the treatment of myocardial infarction (MI), interleukin‐10 (IL‐10) is encapsulated in microgels and released from composites. In a rat model of MI, composites with IL‐10 reduce macrophage density after 1 week and improve scar thickness, ejection fraction, cardiac output, and the size of vascular structures after 4 weeks when compared to saline injection. Improvements are also observed with the composite without IL‐10 over saline, emphasizing the role of injectable hydrogels alone on tissue repair.     

Simple,Rapid, and Large-Scale Fabrication of Multi-Branched Hydrogels Based on Viscous Fingering for Cell Culture Applications

Hydrogels are widely used in cell culture applications. For fabricating tissues and organs, it is essential to produce hydrogels with specific structures. For instance, multiple-branched hydrogels are desirable for the development of network architectures that resemble the biological vascular network. However, existing techniques are inefficient and time-consuming for this application. To address this issue, a simple, rapid, and large-scale fabrication method based on viscous fingering is proposed. This approach utilizes only two plates. To produce a thin solution, a high-viscosity solution is introduced into the space between the plates, and one of the plates is peeled off. During this procedure, the solution's high viscosity results in the formation of multi-branched structures. Using this strategy, 180 mm × 200 mm multi-branched Pluronic F-127 hydrogels are successfully fabricated within 1 min. These structures are used as sacrificial layers for the fabrication of polydimethylsiloxane channels for culturing human umbilical vein endothelial cells (HUVECs). Similarly, multi-branched Matrigel and calcium (Ca)-alginate hydrogel structures are fabricated, and HUVECs are successfully cultured inside the hydrogels. Also, the hydrogels are collected from the plate, while maintaining their structures. The proposed fabrication technique will contribute to the development of network architectures such as vascular structures in tissue engineering.     

Tripeptide Leu-Ser-Trp Regulates the Vascular Endothelial Cells Phenotype Switching by Mediating the Vascular Smooth Muscle Cells-Derived Small Extracellular Vesicles Packaging of miR-145

Tripeptide LSW, initially identified as a potent ACE inhibitory peptide from soybean protein, was recently reported to exert a protective effect against angiotensin II-induced endothelial dysfunction via extracellular vesicles (EVs). However, the molecular mechanisms, especially in lipid accumulation-induced atherosclerosis, still remain unclear. The study aimed to investigate whether the protective effects of LSW against endothelial dysfunction on vascular endothelial cells (VECs) was via vascular smooth muscle cells (VSMCs)-derived miRNA-145 packaged in EVs. The miRNA-145 was concentrated in EVs from LSW-treated VSMCs (LEVs), internalized into the HVUECs, and targeted the programmed cell death protein 4 (PDCD4) expression of HUVECs. Oxidized low-density lipoprotein (oxLDL) was applied to induce endothelial dysfunction in HUVECs; oxLDL-induced endothelial dysfunction in HUVECs was attenuated by PDCD4 knockout or LEVs incubation. The results of this study suggested a novel function of LSW as a regulator on the functional EVs from vascular cells in the oxLDL-induced atherosclerotic model.     

Cell-Encapsulating Hydrogel Puzzle: Polyrotaxane-Based Self-Healing Hydrogels

Slide-ring hydrogels using polyrotaxanes have been developed as highly tough soft materials. However, they have never been used as biomaterials because of the lack of biocompatibility. Meanwhile, self-healing hydrogels are expected to improve fatigue resistance and extend the period of use. However, owing to the lack of high mechanical strength, they are limited in their use as biomaterials. Here we first developed a biocompatible self-healing/slide-ring hydrogel using glycol chitosan and a water-soluble polyrotaxane. We obtained excellent mechanical toughness and biocompatibility to promote the proliferation of human umbilical vein endothelial cells (HUVECs) encapsulated in the hydrogel. Owing to the rapid self-healing property, the cell-encapsulating gels adjusted arbitrarily, maintaining good cell proliferation function. Therefore, slide-ring hydrogels enable the use of biomaterials for soft-tissue engineering.     

Enhanced Maturation of 3D Bioprinted Skeletal Muscle Tissue Constructs Encapsulating Soluble Factor-Releasing Microparticles

Several microfabrication technologies have been used to engineer native-like skeletal muscle tissues. However, the successful development of muscle remains a significant challenge in the tissue engineering field. Muscle tissue engineering aims to combine muscle precursor cells aligned within a highly organized 3D structure and biological factors crucial to support cell differentiation and maturation into functional myotubes and myofibers. In this study, the use of 3D bioprinting is proposed for the fabrication of muscle tissues using gelatin methacryloyl (GelMA) incorporating sustained insulin-like growth factor-1 (IGF-1)-releasing microparticles and myoblast cells. This study hypothesizes that functional and mature myotubes will be obtained more efficiently using a bioink that can release IGF-1 sustainably for in vitro muscle engineering. Synthesized microfluidic-assisted polymeric microparticles demonstrate successful adsorption of IGF-1 and sustained release of IGF-1 at physiological pH for at least 21 days. Incorporating the IGF-1-releasing microparticles in the GelMA bioink assisted in promoting the alignment of myoblasts and differentiation into myotubes. Furthermore, the myotubes show spontaneous contraction in the muscle constructs bioprinted with IGF-1-releasing bioink. The proposed bioprinting strategy aims to improve the development of new therapies applied to the regeneration and maturation of muscle tissues.     

Recent advances on bioprinting of hydrogels containing carbon materials

The intrinsic properties and versatility of carbon materials (CMs) have recently raised a growing interest in their combination with hydrogels towards the development of advanced materials for biomedical applications. The increasing demand for biomimetic constructs that closely mimic the intricate composition and structure of native tissues has boosted the interest in using three-dimensional (3D) bioprinting technologies for the processing of CMs-containing hydrogels into specialized and more complex constructs capable of steering cell behavior. This review summarizes the progress on the bioprinting of 3D hydrogels containing CMs, focusing on the role of CMs on biomaterial ink design and their impact on both the printing process and the biological function of fabricated constructs. Recent findings demonstrate that CMs are versatile materials that have been mostly used to (1) tune the rheological properties of biomaterial inks, (2) improve the mechanical properties of hydrogels, and/or (3) confer new physical features to hydrogels, such as shape memory, roughness, and thermal and electrical conductivity, which have been shown to modulate the biological response of bioprinted constructs.     

NMR-Based Metabolomic Analysis on the Protective Effects of Apolipoprotein A-I Mimetic Peptide against Contrast Media-Induced Endothelial Dysfunction

Endothelial dysfunction plays key roles in the pathological process of contrast media (CM)-induced acute kidney injury (CI-AKI) in patients undergoing vascular angiography or intervention treatment. Previously, we have demonstrated that an apolipoprotein A-I (apoA-I) mimetic peptide, D-4F, inhibits oxidative stress and improves endothelial dysfunction caused by CM through the AMPK/PKC pathway. However, it is unclear whether CM induce metabolic impairments in endothelial cells and whether D-4F ameliorates these metabolic impairments. In this work, we evaluated vitalities of human umbilical vein endothelial cells (HUVECs) treated with iodixanol and D-4F and performed nuclear magnetic resonance (NMR)-based metabolomic analysis to assess iodixanol-induced metabolic impairments in HUVECs, and to address the metabolic mechanisms underlying the protective effects of D-4F for ameliorating these metabolic impairments. Our results showed that iodixanol treatment distinctly impaired the vitality of HUVECs, and greatly disordered the metabolic pathways related to energy production and oxidative stress. Iodixanol activated glucose metabolism and the TCA cycle but inhibited choline metabolism and glutathione metabolism. Significantly, D-4F pretreatment could improve the iodixanol-impaired vitality of HUVECs and ameliorate the iodixanol-induced impairments in several metabolic pathways including glycolysis, TCA cycle and choline metabolism in HUVECs. Moreover, D-4F upregulated the glutathione level and hence enhanced antioxidative capacity and increased the levels of tyrosine and nicotinamide adenine dinucleotide in HUVECs. These results provided the mechanistic understanding of CM-induced endothelial impairments and the protective effects of D-4F for improving endothelial cell dysfunction. This work is beneficial to further exploring D-4F as a potential pharmacological agent for preventing CM-induced endothelial impairment and acute kidney injury.     

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost‐efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin‐methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co‐culture consisting of human adipose tissue‐derived mesenchymal stem cells (hAD‐MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.     

具生物活性的快速光交联生物可降解水凝胶的设计合成

采用开环聚合方法合成了一系列水溶性生物可降解的低聚(丙交酯-co-丙烯酸酯碳酸酯)-b-聚乙二醇-b-低聚(丙交酯-co-丙烯酸酯碳酸酯)(OLAC-PEG-OLAC)三嵌段共聚物,并通过光交联方法方便制备得到具生物活性的新型生物可降解水凝胶.流变测试表明水凝胶储存模量(170～10000 Pa)和凝胶时间(0.8～8min)均可通过调节丙烯酸酯碳酸酯(AC)单元数、聚合物浓度及光引发剂浓度等得到控制.降解实验表明水凝胶的降解速率可通过改变AC和丙交酯(LA)单元数进行调控.含巯基的生物活性分子如RGDC短肽可通过迈克尔加成反应直接链接到OLAC-PEG-OLAC上,由此可方便制备可注射性的具生物活性的生物可降解水凝胶.MG63成骨细胞实验表明RGDC短肽功能化的OLAC-PEG-OLAC水凝胶可很好地促进细胞黏附和生长.该快速光交联生物可降解水凝胶以其优异的凝胶、降解和生物功能化等性能可望为生物组织工程提供理想的三维活性多孔支架.     

Bioactive polypeptide hydrogels modified with RGD and N-cadherin mimetic peptide promote chondrogenic differentiation of bone marrow mesenchymal stem cells

Cell-material and cell-cell interactions represent two crucial aspects of the regulation of cell behavior. In the present study, poly(L-glutamic acid)(PLG) hydrogels were prepared by catalyst-free click crosslinking via a strain-promoted azide-alkyne cycloaddition(SPAAC) reaction between azido-grafted PLG(PLG-N_3) and azadibenzocyclooctyne-grafted PLG(PLG-ADIBO).The bioactive peptides c(RGDfK) and N-cadherin mimetic peptide(N-Cad) were both conjugated to the PLG hydrogel(denoted PLG+RGD/N-Cad) in order to regulate cell-material and cell-cell interactions. Gelation time and storage modulus of the hydrogels were tunable through variations in the concentration of polypeptide precursors. The hydrogels degraded gradually in the presence of proteinases. The viability of bone marrow mesenchymal stem cells(BMSCs) was maintained when cultured with extracts of the hydrogels or encapsulated within the hydrogels. Degradation was observed within 10 weeks following the subcutaneous injection of hydrogel solution in rats, displaying excellent histocompatibility in vivo. The introduction of RGD into the PLG hydrogel promoted the adhesion of BMSCs onto the hydrogels. Moreover, when encapsulated within the PLG+RGD/NCad hydrogel, BMSCs secreted cartilage-specific matrix, in addition to chondrogenic gene and protein expression being significantly enhanced in comparison with BMSCs encapsulated in hydrogels without N-Cad modification. These findings suggest that these biodegradable, bioactive polypeptide hydrogels have great potential for use in 3D cell culture and in cartilage tissue engineering.     

Different complex surfaces of polyethyleneglycol (PEG) and REDV ligand to enhance the endothelial cells selectivity over smooth muscle cells

Arg-Glu-Asp-Val (REDV) peptide with endothelial cells (ECs) selectivity was immobilized onto PEG based polymeric coating via the active p-nitrophenyloxycarbonyl group. The adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) onto surface modified either by REDV end-tethered polyethylene glycol (PEG) or by the complex of free PEG and REDV were investigated to understand the synergic action of nonspecific resistance of PEG and specific recognitions of REDV. Cell culture results indicated that the surfaces end tethered by REDV peptide via PEG "spacer" (n=1, 6, 10) exhibited slight EC selectivity and showed small difference between different lengths of PEG chain. Both separate-culture and co-culture of HUVECs and HASMCs indicated that the introducing of free PEG into REDV tethered surface inhibited HASMCs adhesion significantly and remained a high level of HUVECs growth. Furthermore, the surface with short free PEG chain (n=6) was much more effective to enhance ECs selectivity than long EG chain (n=23). The combination of nonspecific resistance of short free PEG and the ECs selectivity of REDV peptide presents much better ability to enhance the competitive adhesion of HUVECs over HASMCs.     

In situ forming physical hydrogels for three-dimensional tissue morphogenesis

In situ forming physical hydrogels are formed through a biologically benign reaction between two multi-arm macromers, one containing terminal thiol and the other containing terminal vinyl sulfone groups. One macromer is self-assembled through a coiled-coil domain; and the physical junction of this macromer confers the physical nature to the whole network. Unlike covalently cross-linked hydrogels in which material degradation is a prerequisite for three-dimensional cell movement, these physical hydrogels have junctions that undergo reversible dissociation and re-association, constitutively opening paths for cell movement. Epithelial cells encapsulated in these hydrogels can form hollow spherical cysts without the need for material degradation.