Gelatin Methacryloyl Bioink Research Articles

3D bioprinting of fish skin-based gelatin methacryloyl (GelMA) bio-ink for use as a potential skin substitute.

Gelatin methacryloyl (GelMA), typically derived from mammalian sources, has recently emerged as an ideal bio-ink for three-dimensional (3D) bioprinting. Herein, we developed a fish skin-based GelMA bio-ink for the fabrication of a 3D GelMA skin substitute with a 3D bioprinter. Several concentrations of methacrylic acid anhydride were used to fabricate GelMA, in which their physical-mechanical properties were assessed. This fish skin-based GelMA bio-ink was loaded with human adipose tissue-derived mesenchymal stromal cells (ASCs) and human platelet lysate (HPL) and then printed to obtain 3D ASCs + HPL-loaded GelMA scaffolds. Cell viability test and a preliminary investigation of its effectiveness in promoting wound closure were evaluated in a critical-sized full thickness skin defect in a rat model. The cell viability results showed that the number of ASCs increased significantly within the 3D GelMA hydrogel scaffold, indicating its biocompatibility property. In vivo results demonstrated that ASCs + HPL-loaded GelMA scaffolds could delay wound contraction, markedly enhanced collagen deposition, and promoted the formation of new blood vessels, especially at the wound edge, compared to the untreated group. Therefore, this newly fish skin-based GelMA bio-ink developed in this study has the potential to be utilized for the printing of 3D GelMA skin substitutes.

Printing GelMA bioinks: a strategy for buildingin vitromodel to study nanoparticle-based minocycline release and cellular protection under oxidative stress.

Owing to its thermoresponsive and photocrosslinking characteristics, gelatin methacryloyl (GelMA)-based biomaterials have gained widespread usage as a novel and promising bioink for three-dimensional bioprinting and diverse biomedical applications. However, the flow behaviors of GelMA during the sol-gel transition, which are dependent on time and temperature, present significant challenges in printing thick scaffolds while maintaining high printability and cell viability. Moreover, the tunable properties and photocrosslinking capabilities of GelMA underscore its potential for localized drug delivery applications. Previous research has demonstrated the successful incorporation of minocycline (MH) into GelMA scaffolds for therapeutic applications. However, achieving a prolonged and sustained release of concentrated MH remains a challenge, primarily due to its small molecular size. The primary aim of this study is to investigate an optimal extrusion printing method for GelMA bioink in extrusion bioprinting, emphasizing its flow behaviors that are influenced by time and temperature. Additionally, this research seeks to explore the potential of GelMA bioink as a carrier for the sustained release of MH, specifically targeting cellular protection against oxidative stress. The material properties of GelMA were assessed and further optimization of the printing process was conducted considering both printability and cell survival. To achieve sustained drug release within GelMA, the study employed a mechanism using metal ion mediation to facilitate the interaction between MH, dextran sulfate (DS), and magnesium, leading to the formation of nanoparticle complexes (MH-DS). Furthermore, a GelMA-basedin vitromodel was developed in order to investigate the cellular protective properties of MH against oxidative stress. The experimental results revealed that the printability and cell viability of GelMA are significantly influenced by the printing duration, nozzle temperature, and GelMA concentrations. Optimal printing conditions were identified based on a thorough assessment of both printability and cell viability. Scaffolds printed under these optimal conditions exhibited exceptional printability and sustained high cell viability. Notably, it was found that lower GelMA concentrations reduced the initial burst release of MH from the MH-dextran sulfate (MH-DS) complexes, thus favoring more controlled, sustained release profiles. Additionally, MH released under these conditions significantly enhanced fibroblast viability in anin vitromodel simulating oxidative stress.

Fabrication of Fish Scale-Based Gelatin Methacryloyl for 3D Bioprinting Application.

Gelatin methacryloyl (GelMA) is an ideal bioink that is commonly used in bioprinting. GelMA is primarily acquired from mammalian sources; however, the required amount makes the market price extremely high. Since garbage overflow is currently a global issue, we hypothesized that fish scales left over from the seafood industry could be used to synthesize GelMA. Clinically, the utilization of fish products is more advantageous than those derived from mammals as they lower the possibility of disease transmission from mammals to humans and are permissible for practitioners of all major religions. In this study, we used gelatin extracted from fish scales and conventional GelMA synthesis methods to synthesize GelMA, then tested it at different concentrations in order to evaluated and compared the mechanical properties and cell responses. The fish scale GelMA had a printing accuracy of 97%, a swelling ratio of 482%, and a compressive strength of about 85 kPa at a 10% w/v GelMA concentration. Keratinocyte cells (HaCaT cells) were bioprinted with the GelMA bioink to assess cell viability and proliferation. After 72 h of culture, the number of cells increased by almost three-fold compared to 24 h, as indicated by many fluorescent cell nuclei. Based on this finding, it is possible to use fish scale GelMA bioink as a scaffold to support and enhance cell viability and proliferation. Therefore, we conclude that fish scale-based GelMA has the potential to be used as an alternative biomaterial for a wide range of biomedical applications.

Sacrificial-Rotating Rod-Based 3D Bioprinting Technique for the Development of an In Vitro Cardiovascular Model.

Several studies have attempted to develop complex cardiovascular models, but the use of multiple cell types and poor cell alignments after fabrication have limited the practical application of these models. Among various bioprinting methods, extrusion-based bioprinting is the most widely used in the bioengineering field. This method not only has the potential to construct complex 3D biological structures but it also enables the alignment of cells in the printing direction owing to the application of shear stress to the cells during the printing process. Therefore, this study developed an in vitro cardiovascular model using an extrusion-based bioprinting method that utilizes a rotating rod as a printing platform. The rotating rod was made of polyvinyl alcohol (PVA) and used as a sacrificial rod. This rotating platform approach enabled the printing of longer tubular-vascular structures of multiple shapes, including disease models, and the water-soluble properties of PVA facilitated the isolation of the printed vascular models. In addition, this method enabled the printing of the endothelial cells in the bloodstream direction and smooth muscle cells in the circumferential direction to better mimic the anatomy of real blood vessels. Consequently, a cardiovascular model was successfully printed using a gelatin methacryloyl bioink with cells. In conclusion, the proposed fabrication method can facilitate the fabrication of various cardiovascular models that mimic the alignment of real blood vessels.

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 21days. 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.

Combined light-cured and sacrificial hydrogels for fabrication of small-diameter bionic vessels by 3D bioprinting.

Bionic grafts can replace autologous tissue through tissue engineering in cases of cardiovascular disease. However, small-diameter vessel grafts remain challenging to precellularize. Bionic small-diameter vessels with endothelial and smooth muscle cells (SMCs) manufactured with a novel approach. A 1-mm-diameter bionic blood vessel was constructed by combining light-cured hydrogel gelatin-methacryloyl (GelMA) with sacrificial hydrogel Pluronic F127. Mechanical properties of GelMA (Young's modulus and tensile stress) were tested. Cell viability and proliferation were detected using Live/dead staining and CCK-8 assays, respectively. The histology and function of the vessels were observed using hematoxylin and eosin and immunofluorescence staining. GelMA and Pluronic were printed together using extrusion. The temporary Pluronic support was removed by cooling during GelMA crosslinking, yielding a hollow tubular construct. A bionic bilayer vascular structure was fabricated by loading SMCs into the GelMA bioink, followed by perfusion with endothelial cells. In the structure, both cell types maintained good cell viability. The vessel showed good histological morphology and function. Using light-cured and sacrificial hydrogels, we formed a small ca bionic vessel with a small caliber containing SMCs and endothelial cells, demonstrating an innovative approach for construction of bionic vascular tissues.

Precise Printing of Microfiber Scaffold with Gelatin Methacryloyl (GelMA)/Polyethylene Oxide (PEO) Bioink.

Gelatin methacryloyl scaffolds with microscale fiber structures own great significance because they can effectively mimic the extracellular matrix environment. Compared with extruding bioprinting, electrospinning technology is more suitable for establishing accurate hydrogel microfibers. However, electrospinning accurate gelatin methacryloyl microfiber remains a big challenge restricted by its bad spinnability. In this paper, polyethylene oxide, which owns promising spinnability, is added into gelatin methacryloyl hydrogel precursor to improve the spinnability of gelatin methacryloyl bioink. A three-dimensional motion platform for electrospinning is designed and built and the spinning process of microfibers under far-electric-field and near-electric-field conditions is systematically studied, respectively. As a result, scaffolds consisted of unordered and ordered microfibers are successfully fabricated under far-electric-field and near-electric field, respectively. In vitro culture experiments of human umbilical vein endothelial cells are carried out using the prepared gelatin methacryloyl microfiber scaffolds. The results show that the cells can easily attach to the microfibers and grow well. Moreover, the gelatin methacryloyl/ polyethylene oxide microfiber scaffold was directly spun on the polycaprolactone mesh scaffold printed by fused modeling printing method. The results showed that the macroscopic ordered and microscopic disordered microfiber scaffold could be successfully established, which could lead to directed cell growth. We believe that this method can effectively solve the problem of hydrogel spinnability and be a powerful tool for various biomedical engineering methods in the future.

Three-dimensional bioprinting of artificial ovaries by an extrusion-based method using gelatin-methacryloyl bioink

Purpose The aim of this study was to design and fabricate a three-dimensional (3D) printed artificial ovary. Methods We first compared the printability of gelatin-methacryloyl (GelMA), alginate and GelMA–alginate bioinks, of which GelMA was selected for further investigation. The swelling properties, degradation kinetics and shape fidelity of GelMA scaffolds were characterized by equilibrium swelling/lyophilization, collagenase processing and micro-computed tomography evaluation. Commercial ovarian tumor cell lines (COV434, KGN, ID8) and primary culture ovarian somatic cells were utilized to perform cell-laden 3D printing, and the results were evaluated by live/dead assays and TUNEL detection. Murine ovarian follicles were seeded in the ovarian scaffold and their diameters were recorded every day. Finally, in vitro maturation was performed, and the ovulated oocytes were collected and observed. Results Our results indicated that GelMA was suitable for 3D printing fabrication. Its scaffolds performed well in terms of hygroscopicity, degradation kinetics and shape fidelity. The viability of ovarian somatic cells was lower than that of commercial cell lines, suggesting that extrusion-based 3D culture fabrication is not suitable for primary ovarian cells. Nevertheless, the GelMA-based 3D printing system provided an appropriate microenvironment for ovarian follicles, which successfully grew and ovulated in the scaffolds. Metaphase II oocytes were also observed after in vitro maturation. Conclusions The GelMA-based 3D printing culture system is a viable alternative option for follicular growth, development and transfer. Accordingly, it shows promise for clinical application in the treatment of female endocrine and reproductive conditions.

Non-invasive in vivo monitoring of transplanted stem cells in 3D-bioprinted constructs using near-infrared fluorescent imaging.

Cell‐based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue‐engineered scaffolds to the process of tissue regeneration remain poorly understood. Near‐infrared (NIR) fluorescence imaging systems have great potential to non‐invasively monitor the transplanted cell‐based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127‐labeled MSCs (NIR‐MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR‐MSCs‐loaded bioink showed excellent printability. In addition, NIR‐MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non‐invasively monitor the NIR‐MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR‐based imaging system for non‐invasive cell monitoring in vivo could play an active role in validating the cell fate in cell‐based tissue engineering applications.

Cell-Laden Gelatin Methacryloyl Bioink for the Fabrication of Z-Stacked Hydrogel Scaffolds for Tissue Engineering

Hydrogel-based scaffolds have been widely used to fabricate artificial tissues capable of replacing tissues and organs. However, several challenges inherent in fabricating tissues of large size and complex morphology using such scaffolds while ensuring cell viability remain. To address this problem, we synthesized gelatin methacryloyl (GelMA) based bioink with cells for fabricating a scaffold with superior characteristics. The bioink was grafted onto a Z-stacking bioprinter that maintained the cells at physiological temperature during the printing process, without exerting any physical pressure on the cells. Various parameters, such as the bioink composition and light exposure time, were optimized. The printing accuracy of the scaffolds was evaluated using photorheological studies. The internal morphology of the scaffolds at different time points was analyzed using electron microscopy. The Z-stacked scaffolds were fabricated using high-speed printing, with the conditions optimized to achieve high model reproducibility. Stable adhesion and high proliferation of cells encapsulated within the scaffold were confirmed. We introduced various strategies to improve the accuracy and reproducibility of Z-stack GelMA bioprinting while ensuring that the scaffolds facilitated cell adhesion, encapsulation, and proliferation. Our results demonstrate the potential of the present method for various applications in tissue engineering.

Role of temperature on bio-printability of gelatin methacryloyl bioink in two-step cross-linking strategy for tissue engineering applications

Additive manufacturing has shown promising results in reconstructing three-dimensional (3D) living tissues for various applications, including tissue engineering, regenerative medicine, drug discovery, and high-throughput drug screening. In extrusion-based bioprinters, stable formation of filaments and high-fidelity deposition of bioinks are the primary challenges in fabrication of physiologically relevant tissue constructs. Among various bioinks, gelatin methacryloyl (GelMA) is known as a photocurable and physicochemically tunable hydrogel with a demonstrated biocompatibility and tunable biodegradation properties. The two-step crosslinking of GelMA (reversible thermal gelation and permanent photo-crosslinking) has attracted researchers to make complex tissue constructs. Despite promising results in filament formation and printability of this hydrogel, the effect of temperature on physicochemical properties, cytocompatibility, and biodegradation of the hydrogel are to be investigated. This work studies the effect of thermoreversible, physical crosslinking on printability of GelMA. The results of 3D printing of GelMA at different temperatures followed by irreversible chemical photo-crosslinking show that the decrease in temperature improves the filament formation and shape fidelity of the deposited hydrogel, particularly at the temperatures around 15 °C. Time dependant mechanical testing of the printed samples revealed that decreasing the extruding temperature increases the elastic properties of the extruded filaments. Furthermore, our novel approach in minimizing the slippage effect during rheological study enabled to measure changes in linear and non-linear viscoelastic properties of the printed samples at different temperatures. A considerable increase in storage modulus of the extruded samples printed at lower temperatures confirms their higher solid behavior. Scanning electron microscopy revealed a remarkable decrease in porosity of the extruded hydrogels by decreasing the temperature. Chemical analysis by Fourier-transform infrared spectroscopy and circular dichroism showed a direct relationship between the coil-helix transition in hydrogel macromers and its physical alterations. Finally, biodegradation and cytocompatibility of the extruded hydrogels decreased at lower extruding temperatures.

3D bioprinting of bicellular liver lobule-mimetic structures via microextrusion of cellulose nanocrystal-incorporated shear-thinning bioink

3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

Effect of sterilization treatment on mechanical properties, biodegradation, bioactivity and printability of GelMA hydrogels

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol–gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

Bioprinting small diameter blood vessel constructs with an endothelial and smooth muscle cell bilayer in a single step

Bioengineered artificial blood vessels have been a major area of interest over the last decade. Of particular interest are small diameter vessels, as surgical options are currently limited. This study aimed to fabricate a small diameter, heterogeneous bilayer blood vessel-like construct in a single step with gelatin methacryloyl (GelMA) bioink using a 3D micro-extrusion bioprinter on a solid platform. GelMA was supplemented with Hyaluronic acid (HA), glycerol and gelatin to form a GelMA bioink with good printability, mechanical strength, and biocompatibility. Two separate concentrations of GelMA bioink with unique pore sizes were selected to fabricate a heterogeneous bilayer. A higher concentration of GelMA bioink (6% w/v GelMA, 2% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load human umbilical vein endothelial cells (HUVECs) and form an inner, endothelial tissue layer. A lower concentration of GelMA bioink (4% w/v GelMA, 4% gelatin, 0.3% w/v HA, 10% v/v glycerol) was used to load smooth muscle cells (SMCs) and form an outer, muscular tissue layer. Bioprinted blood vessel-like grafts were then assessed for mechanical properties with Instron mechanical testing, and suture-ability, and for biological properties including viability, proliferation, and histological analysis. The resulting 20 mm long, 4.0 mm diameter lumen heterogeneous bilayer blood vessel-like construct closely mimics a native blood vessel and maintains high cell viability and proliferation. Our results represent a novel strategy for small diameter blood vessel biofabrication.

3D Bioprinting of Oxygenated Cell-Laden Gelatin Methacryloyl Constructs.

Cell survival during the early stages of transplantation and before new blood vessels formation is a major challenge in translational applications of 3D bioprinted tissues. Supplementing oxygen (O2 ) to transplanted cells via an O2 generating source such as calcium peroxide (CPO) is an attractive approach to ensure cell viability. Calcium peroxide also produces calcium hydroxide that reduces the viscosity of bioinks, which is a limiting factor for bioprinting. Therefore, adapting this solution into 3D bioprinting is of significant importance. In this study, a gelatin methacryloyl (GelMA) bioink that is optimized in terms of pH and viscosity is developed. The improved rheological properties lead to the production of a robust bioink suitable for 3D bioprinting and controlled O2 release. In addition, O2 release, bioprinting conditions, and mechanical performance of hydrogels having different CPO concentrations are characterized. As a proof of concept study, fibroblasts and cardiomyocytes are bioprinted using CPO containing GelMA bioink. Viability and metabolic activity of printed cells are checked after 7 days of culture under hypoxic condition. The results show that the addition of CPO improves the metabolic activity and viability of cells in bioprinted constructs under hypoxic condition.

Low-Temperature Three-Dimensional Printing of Tissue Cartilage Engineered with Gelatin Methacrylamide

Low-concentration gelatin methacryloyl (GelMA) hydrogels have been found to be promising cell-laden bioinks with excellent cell viability. Herein, we report a strategy that accurately deposits cell-containing bioinks at 5% (w/v) GelMA using extrusion three-dimensional (3D) bioprinting technology by utilizing its photo-crosslinkable and thermosensitive properties without the need for any sacrificial materials. During the 3D printing process, regular, smooth microfibers were formed without any discontinuity of extrusion or clogging, and photo-crosslinking was then used to stabilize the printed GelMA structure. After printing, the scaffolds were cultured in a chondrogenic medium to evaluate their significant roles in directing the behaviors of bone mesenchymal stem cells (BMSCs). Evidence of chondrogenic differentiation was demonstrated by Alcian blue staining and immunofluorescence (Col2a1) as well as the expression of chondrogenic genes. Finally, after platelet-rich plasma treatment, the in vivo effects of the BMSCs on cartilage regeneration on the thigh muscles of female nude mice were measured by using immunohistochemical techniques. The results showed that with this strategy, GelMA bioink displays excellent printability and a high cell survival rate. In vitro and in vivo, the cell-laden scaffold successfully regenerated mature cartilage via a cartilage-specific extracellular matrix, which seems to be suitable for cartilage regeneration and repair.

Free-form co-axial bioprinting of a gelatin methacryloyl bio-ink by direct in situ photo-crosslinking during extrusion

3D bioprinting is an emerging technology for arranging cells and biomaterials in 3D, with the goal to develop functional substitutes for damaged tissue. Photo-crosslinkable hydrogels are promising materials for formulating printable bio-inks. However, owing to conflicting constraints around printability and cell survival, achieving good shape fidelity is a challenge. The typical approach to ‘post-crosslink’ a 3D bioprinted structure necessitates highly viscous bio-inks. Meanwhile ‘pre-crosslinking’ can result in the extrusion of overly gelated bio-inks, which increases shear-stresses experienced by encapsulated cells. Neither of these strategies are amenable to creating free-standing structures, or structures with overhangs, without using secondary support materials. Here, for the first time, an on board light exposure strategy is demonstrated which enables rapid (<1s) and direct crosslinking of the bio-ink as it is being extruded from the nozzle, and without the use of transparent tubing to stabilize the filament. The utilization of this technique on a manually operated device is described, allowing for free-form additive sculpting of bio-ink structures in 3D. Finally, a co-axial freeform extrusion system is demonstrated, allowing for encapsulation of a very soft, or entirely liquid, core within the rapidly photocrosslinked shell filament.

Fiber engraving for bioink bioprinting within 3D printed tissue engineering scaffolds

In this work, we describe a new 3D printing methodology for the fabrication of multimaterial scaffolds involving the combination of thermoplastic extrusion and low temperature extrusion of bioinks. A fiber engraving technique was used to create a groove on the surface of a thermoplastic printed fiber using a commercial 3D printer and a low viscosity bioink was deposited into this groove. In contrast to traditional extrusion bioinks that rely on increased viscosity to prevent lateral spreading, this groove creates a defined space for bioink deposition. By physically constraining bioink spreading, a broader range of viscosities can be used. As proof-of-concept, we fabricated and characterized a multimaterial scaffold containing poly(ε-caprolactone) (PCL) as the thermoplastic polymer and a gelatin-based bioink. A 7.5 w/v% gelatin methacryloyl (GelMA) bioink loaded with either 5 w/v% poly(lactic-co-glycolic acid) (PLGA) microparticles containing fluorescent albumin or mouse fibroblasts (1 ​× ​106 ​cell/mL) was printed at 24 ​°C. The structure of the composite scaffolds had no significant decrease in porosity or mechanical properties as compared to the PCL control scaffolds, demonstrating the engraving technique did not significantly compromise the mechanical or structural integrity of the scaffold. The encapsulated PLGA microparticles were homogeneously distributed in the GelMA and remained in the scaffolds after incubation in PBS for 24 ​h ​at 37 ​°C. In addition, the viability of the fibroblasts encapsulated in the GelMA bioink and printed in the grooves of the PCL scaffolds was confirmed after 24 ​h of incubation. Overall, this work provides a new methodology for the preparation of 3D printed scaffolds containing a robust thermoplastic structure in combination with low viscosity bioinks.

Hydrogel Bioink with Multilayered Interfaces Improves Dispersibility of Encapsulated Cells in Extrusion Bioprinting.

One of the challenges for extrusion bioprinting using low-viscosity bioinks is the fast gravity-driven sedimentation of cells. Cells in a hydrogel bioink that features low viscosity tend to settle to the bottom of the bioink reservoir, and as such, their bioprintability is hindered by association with the inhomogeneous cellularized structures that are deposited. This is particularly true in cases where longer periods are required to print complex or larger tissue constructs. Increasing the bioink's viscosity efficiently retards sedimentation but gives rise to cell membranolysis or functional disruption due to increased shear stress on the cells during the extrusion process. Inspired by the rainbow cocktail, we report the development of a multilayered modification strategy for gelatin methacryloyl (GelMA) bioink to manipulate multiple liquid interfaces, providing interfacial retention to retard cell sedimentation in the bioink reservoir. Indeed, the interfacial tension in our layer-by-layer bioink system, characterized by the pendant drop method, was found to be exponentially higher than the sedimental pull (ΔGravity-Buoyancy = ∼10-9 N) of cells, indicating that the interfacial retention is crucial for preventing cell sedimentation across the adjacent layers. It was demonstrated that the encapsulated cells displayed better dispersibility in constructs bioprinted using the multilayered GelMA bioink system than that of pristine GelMA where the index of homogeneity of the cell distribution in the multilayered bioink was 4 times that of the latter.

Reversible physical crosslinking strategy with optimal temperature for 3D bioprinting of human chondrocyte-laden gelatin methacryloyl bioink.

Gelatin methacryloyl is a promising material in tissue engineering and has been widely studied in three-dimensional bioprinting. Although gelatin methacryloyl possesses excellent biocompatibility and tunable mechanical properties, its poor printability/processability has hindered its further applications. In this study, we report a reversible physical crosslinking strategy for precise deposition of human chondrocyte-laden gelatin methacryloyl bioink at low concentration without any sacrificial material by using extrusive three-dimensional bioprinting. The precise printing temperature was determined by the rheological properties of gelatin methacryloyl with temperature. Ten percent (w/v) gelatin methacryloyl was chosen as the printing formula due to highest biocompatibility in three-dimensional cell cultures in gelatin methacryloyl hydrogel disks. Primary human chondrocyte-laden 10% (w/v) gelatin methacryloyl was successfully printed without any construct deformation or collapse and was permanently crosslinked by ultraviolet light. The printed gelatin methacryloyl hydrogel constructs remained stable in long-term culture. Chondrocyte viability and proliferation that were printed under this optimal temperature were better than that of chondrocytes printed under lower temperatures and were similar to that of chondrocytes in the non-printed gelatin methacryloyl hydrogels. The results indicate that with this strategy, 10% (w/v) gelatin methacryloyl bioink presented excellent printability and printing resolution with high cell viability, which appears to be suitable for printing primary human chondrocytes in cartilage biofabrication and can be extensively applied in tissue engineering of other organs or in other biomedical fields.