Cell-laden Gelatin Methacryloyl Research Articles

Integration of Melt Electrowritten Polymeric Scaffolds and Bioprinting for Epithelial Healing via Localized Periostin Delivery.

Management of skin injuries imposes a substantial financial burden on patients and hospitals, leading to diminished quality of life. Periostin (rhOSF), an extracellular matrix component, regulates cell function, including a proliferative healing phase, representing a key protein to promote wound healing. Despite its proven efficacy in vitro, there is a lack of scaffolds that facilitate the in situ delivery of rhOSF. In addition, there is a need for a scaffold to not only support cell growth, but also to resist the mechanical forces involved in wound healing. In this work, we synthesized rhOSF-loaded mesoporous nanoparticles (MSNs) and incorporated them into a cell-laden gelatin methacryloyl (GelMA) ink that was bioprinted into melt electrowritten poly(ε-caprolactone) (PCL) microfibrous (MF-PCL) meshes to develop mechanically competent constructs. Diffraction light scattering (DLS) analysis showed a narrow nanoparticle size distribution with an average size of 82.7 ± 13.2 nm. The rhOSF-loaded hydrogels showed a steady and controlled release of rhOSF over 16 days at a daily dose of ∼40 ng/mL. Compared with blank MSNs, the incorporation of rhOSF markedly augmented cell proliferation, underscoring its contribution to cellular performance. Our findings suggest a promising approach to address challenges such as prolonged healing, offering a potential solution for developing robust, biocompatible, and cell-laden grafts for burn wound healing applications.

Low-Temperature Extrusion of Waterborne Polyurethane-Polycaprolactone Composites for Multi-Material Bioprinting of Engineered Elastic Cartilage.

3D bioprinting of elastic cartilage tissues that are mechanically and structurally comparable to their native counterparts, while exhibiting favorable cellular behavior, is an unmet challenge. A practical solution for this problem is the multi-material bioprinting of thermoplastic polymers and cell-laden hydrogels using multiple nozzles. However, the processing of thermoplastic polymers requires high temperatures, which can damage hydrogel-encapsulated cells. In this study, the authors developed waterborne polyurethane (WPU)-polycaprolactone (PCL) composites to allow multi-material co-printing with cell-laden gelatin methacryloyl (GelMA) hydrogels. These composites can be extruded at low temperatures (50-60 °C) and high speeds, thereby reducing heat/shear damage to the printed hydrogel-capsulated cells. Furthermore, their hydrophilic nature improved the cell behavior in vitro. More importantly, the bioprinted structures exhibited good stiffness and viscoelasticity compared to native elastic cartilage. In summary, this study demonstrated low-temperature multi-material bioprinting of WPU-PCL-based constructs with good mechanical properties, degradation time-frames, and cell viability, showcasing their potential in elastic cartilage bio-fabrication and regeneration to serve broad biomedical applications in the future.

A well plate-based GelMA photo-crosslinking system with tunable hydrogel mechanical properties to regulate the PTH-mediated osteogenic fate

Versatile and efficient regulation of the mechanical properties of the extracellular matrix is crucial not only for understanding the dynamic changes in biological systems, but also for obtaining precise and effective cellular responses in drug testing. In this study, we developed a well plate-based hydrogel photo-crosslinking system to effectively control the mechanical properties of hydrogels and perform high-throughput assays. We improved cell biocompatibility by using gelatin methacryloyl (GelMA) with a visible light photo-crosslinking method. Multiple cell-laden GelMA hydrogels were simultaneously and uniformly created using multi-arrayed 520 nm light-emitting diodes in a well plate format. The elastic modulus of the hydrogels can be widely adjusted (0.5–30 kPa) using a photo-crosslinking system capable of independently controlling the light intensity or exposure time for multiple samples. We demonstrate the feasibility of our system by observing enhanced bone differentiation of human mesenchymal stem cells (hMSCs) cultured on stiffer hydrogels. Additionally, we observed that the osteogenic fate of hMSCs, affected by the different mechanical properties of the gel, was regulated by parathyroid hormone (PTH). Notably, in response to PTH, hMSCs in a high-stiffness microenvironment upregulate osteogenic differentiation while exhibiting increased proliferation in a low-stiffness microenvironment. Overall, the developed system enables the generation of multiple cell-laden three-dimensional cell culture models with diverse mechanical properties and holds significant potential for expansion into drug testing.

Harnessing decellularised extracellular matrix microgels into modular bioinks for extrusion-based bioprinting with good printability and high post-printing cell viability.

The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting. A proper bioink not only provides mechanical support for structural fidelity, but also serves as suitable three-dimensional (3D) microenvironment for cell encapsulation and protection. In this study, a hydrogel-based composite bioink was developed consisting of gelatin methacryloyl (GelMA) as the continuous phase and decellularised extracellular matrix microgels (DMs) as the discrete phase. A flow-focusing microfluidic system was employed for the fabrication of cell-laden DMs in a high-throughput manner. After gentle mixing of the DMs and GelMA, both rheological characterisations and 3D printing tests showed that the resulting DM-GelMA hydrogel preserved the shear-thinning nature, mechanical properties, and good printability from GelMA. The integration of DMs not only provided an extracellular matrix-like microenvironment for cell encapsulation, but also considerable shear-resistance for high post-printing cell viability. The DM sizes and inner diameters of the 3D printer needles were correlated and optimised for nozzle-based extrusion. Furthermore, a proof-of-concept bioink composedg of RSC96 Schwann cells encapsulated DMs and human umbilical vein endothelial cell-laden GelMA was successfully bioprinted into 3D constructs, resulting in a modular co-culture system with distinct cells/materials distribution. Overall, the modular DM-GelMA bioink provides a springboard for future precision biofabrication and will serve in numerous biomedical applications such as tissue engineering and drug screening.

3D Bioprinted Hydrogel Microfluidic Devices for Parallel Drug Screening.

Conventional high-throughput screening (HTS) platforms suffer from the need for large cell volumes, high reagent consumption, significant assembly cost, and handling efforts. The assembly of three-dimensional (3D) bioprinted hydrogel-based microfluidic chips within platforms can address these problems. We present a continuous and seamless manufacturing approach to create a bioprinted microfluidic chips with a circular pattern scalable toward HTS platforms. Digital light processing 3D bioprinting is used to tune the local permeability of our chip, made of polyethylene glycol diacrylate and cell-laden gelatin methacryloyl, for creating predefined gradients of biochemical properties. We measured the flow-induced physical characteristics, the mass transport of drug agents, and the biological features of the proposed chip. We measured reactive oxygen species from the encapsulated cells through an integrated process and showed the capacity of the hydrogel-based chip for creating drug/agent gradients. This work introduces a chip design based on a hydrogel that can be changed and could be used for modern HTS platforms such as in vitro organoids.

Low-intensity pulsed ultrasound promotes cell viability and inhibits apoptosis of H9C2 cardiomyocytes in 3D bioprinting scaffolds via PI3K-Akt and ERK1/2 pathways.

The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using an extrusion-based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double-staining assays. Western blot analysis was performed to determine the protein expression levels. We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5MHz and an exposure time of 10min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. A frequency of 0.5MHz and exposure time of 10min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways.

Bisulfite-initiated crosslinking of gelatin methacryloyl hydrogels for embedded 3D bioprinting

Recent studies on three-dimensional (3D) bioprinting of cell-laden gelatin methacryloyl (GelMA) hydrogels have provided promising outcomes for tissue engineering applications. However, the reliance on the use of photo-induced gelation processes for the bioprinting of GelMA and the lack of an alternative crosslinking process remain major challenges for the fabrication of cell-laden structures. Here, we present a novel crosslinking approach to form cell-laden GelMA hydrogel constructs through 3D embedded bioprinting without using any external irradiation that could drastically affect cell viability and functionality. This approach consists of a one-step type of crosslinking via bisulfite-initiated radical polymerization, which is combined with embedded bioprinting technology to improve the structural complexity of printed structures. By this means, complex-shaped hydrogel bio-structures with cell viability higher than 90% were successfully printed within a support bath including sodium bisulfite. This study offers an important alternative to other photo-induced gelation processes to improve the bio-fabrication of GelMA hydrogel with high cell viability.

Biofabrication of Cell-Laden Gelatin Methacryloyl Hydrogels with Incorporation of Silanized Hydroxyapatite by Visible Light Projection.

Gelatin methacryloyl (GelMA) hydrogel is a photopolymerizable biomaterial widely used for three-dimensional (3D) cell culture due to its high biocompatibility. However, the drawback of GelMA hydrogel is its poor mechanical properties, which may compromise the feasibility of biofabrication techniques. In this study, a cell-laden GelMA composite hydrogel with a combination incorporating silanized hydroxyapatite (Si-HAp) and a simple and harmless visible light crosslinking system for this hydrogel were developed. The incorporation of Si-HAp into the GelMA hydrogel enhanced the mechanical properties of the composite hydrogel. Moreover, the composite hydrogel exhibited low cytotoxicity and promoted the osteogenic gene expression of embedded MG63 cells and Human bone marrow mesenchymal stem cells (hBMSCs). We also established a maskless lithographic method to fabricate a defined 3D structure under visible light by using a digital light processing projector, and the incorporation of Si-HAp increased the resolution of photolithographic hydrogels. The GelMA-Si-HAp composite hydrogel system can serve as an effective biomaterial in bone regeneration.

Additively Manufactured Gradient Porous Ti-6Al-4V Hip Replacement Implants Embedded with Cell-Laden Gelatin Methacryloyl Hydrogels.

Laser additive manufacturing has led to a paradigm shift in the design of next-generation customized porous implants aiming to integrate better with the surrounding bone. However, conflicting design criteria have limited the development of fully functional porous implants; increasing porosity improves body fluid/cell-laden prepolymer permeability at the expense of compromising mechanical stability. Here, functionally gradient porosity implants and scaffolds designed based on interconnected triply periodic minimal surfaces (TPMS) are demonstrated. High local porosity is defined at the implant/tissue interface aiming to improve the biological response. Gradually decreasing porosity from the surface to the center of the porous constructs provides mechanical strength in selective laser melted Ti-6Al-4V implants. The effect of unit cell size is studied to discover the printability limit where the specific surface area is maximized. Furthermore, mechanical studies on the unit cell topology effects suggest that the bending-dominated architectures can provide significantly enhanced strength and deformability, compared to stretching-dominated architectures. A finite element (FE) model developed also showed great predictability (within ∼13%) of the mechanical responses of implants to physical activities. Finally, in vitro biocompatibility studies were conducted for two-dimensional (2D) and three-dimensional (3D) cases. The results of the 2D in conjunction with surface roughness show favored physical cell attachment on the implant surface. Also, the results of the 3D biocompatibility study for the scaffolds incorporated with a cell-laden gelatin methacryloyl (GelMA) hydrogel show excellent viability. The design procedure proposed here provides new insights into the development of porous hip implants with simultaneous high mechanical and biological responses.

Gelatin Methacryloyl-Riboflavin (GelMA-RF) Hydrogels for Bone Regeneration.

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA–RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA–RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA–RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA–RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.

HDPSC-laden GelMA microspheres fabricated using electrostatic microdroplet method for endodontic regeneration

The microsphere system has attracted considerable attention as a stem-cell delivery vehicle in regeneration medicine owing to its injectability, fast substance transfer ability, and mimicry of the three-dimensional native environment. However, suitable biomaterials for preparation of microspheres optimal for endodontic regeneration are still being explored. Owing to its excellent bioactivity and biodegradability, gelatin methacryloyl (GelMA) was used to fabricate hydrogel microspheres by the electrostatic microdroplet method, and the potential of GelMA microspheres applied in endodontic regeneration was studied. The average size of GelMA microspheres encapsulating human dental pulp stem cells (hDPSCs) was ~200 μm, and the Young’s modulus was approximately 582.8 ± 66.0 Pa, which was close to that of the natural human dental pulp. The encapsulated hDPSCs could effectively adhere, spread, proliferate, and secrete extracellular matrix proteins in the microspheres, and tended to occupy the outer layer. Moreover, the cell-laden GelMA microsphere system could withstand cryopreservation, and the thawed cells exhibited normal functions. After subcutaneous implantation in a nude mouse model, more vascularized pulp-like tissues were generated in the cell-laden GelMA microsphere group compared with that in the cell-laden bulk GelMA group, and this was accompanied by a suitable degradation rate. The GelMA microspheres showed remarkable performances and great potential as cell delivery vehicles in endodontic regeneration.

Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities.

Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine–based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Our approach to fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population, previously validated biomaterials, and a smart scaffold design. Specifically, we generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell–laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Our results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Our strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.

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.

Bioprinted Injectable Hierarchically Porous Gelatin Methacryloyl Hydrogel Constructs with Shape-Memory Properties.

Direct injection of cell-laden hydrogels shows high potentials in tissue regeneration for translational therapy. The traditional cell-laden hydrogels are often used as bulk space fillers to tissue defects after injection, likely limiting their structural controllability. On the other hand, patterned cell-laden hydrogel constructs often necessitate invasive surgical procedures. To overcome these problems, herein, we report a unique strategy for encapsulating living human cells in a pore-forming gelatin methacryloyl (GelMA)-based bioink to ultimately produce injectable hierarchically macro-micro-nanoporous cell-laden GelMA hydrogel constructs through three-dimensional (3D) extrusion bioprinting. The hydrogel constructs can be fabricated into various shapes and sizes that are defect-specific. Due to the hierarchically macro-micro-nanoporous structures, the cell-laden hydrogel constructs can readily recover to their original shapes, and sustain high cell viability, proliferation, spreading, and differentiation after compression and injection. Besides, in vivo studies further reveal that the hydrogel constructs can integrate well with the surrounding host tissues. These findings suggest that our unique 3D-bioprinted pore-forming GelMA hydrogel constructs are promising candidates for applications in minimally invasive tissue regeneration and cell therapy.

Fibroblast encapsulation in gelatin methacryloyl (GelMA) versus collagen hydrogel as substrates for oral mucosa tissue engineering

PurposeOver the past decades, a variety of biomaterials have been investigated in terms of their suitability for oral mucosa tissue engineering. The aim of this study was to compare collagen and GelMA hydrogels as connective tissue scaffolds for fibroblasts and as substrates for seeding and culture of oral epithelial keratinocyte cells. MethodsHuman primary oral fibroblast and keratinocyte cells were isolated from gingival biopsies. The mixture of fibroblasts with GelMA or collagen gel were aliquoted within six-well tissue culture plate inserts and cross-linked using visible light or reconstitution buffer/heat, respectively. The viability of fibroblasts in the hydrogels was investigated after one and three days of cultivation using the PrestoBlue assay. Following the addition and culture of oral keratinocytes onto the connective tissue constructs, the tissue-engineered oral mucosa was assessed histologically. ResultsThe tissue viability assay shows that collagen hydrogels encapsulating fibroblasts displayed significantly higher cell viability than cell-laden GelMA constructs after 24 and 72 h (p < 0.05). A stratified and differentiated epithelium has formed on the surface of cell-laden collagen hydrogel but not on the surface of the GelMA-based substrate. ConclusionCollagen-based scaffold offers superior biological properties compared to GelMA hydrogel in terms of oral fibroblast growth, as well as epithelial cell adhesion and differentiation. Therefore, collagen-based hydrogels remain the preferred choice for oral mucosa tissue engineering.

Peptide Chitosan/Dextran Core/Shell Vascularized 3D Constructs for Wound Healing.

Three-dimensional (3D) bioprinting has emerged to create novel cell-based therapies for regenerative medicine applications. Vascularized networks within engineered constructs are required, and toward this end, we report a promising strategy using core-shell (c/s) extrusion 3D-bioprinting technology that employs biomimetic biomaterials to construct regenerative, prevascularized scaffolds for wound care. A custom-designed cell-responsive bioink consisting of a 13% (w/v) cell-laden gelatin methacryloyl (GelMA) shell surrounding a peptide-functionalized, succinylated chitosan (C)/dextran aldehyde (D) cell-laden core was successfully bioprinted resulting in organized microdesigns exhibiting excellent cell viability and subsequent vessel formation. Our templating strategy takes advantage of GelMA's intrinsic thermoreversible properties of low degree of acryloyl functionalization used in combination with a lightly, chemically cross-linked peptide-CD core to serve as temporal structural supports that stabilize during extrusion onto a cooled platform. Mechanical integrity was further strengthened layer-by-layer via GelMA UV photo-cross-linking. We report the first example of GelMA used in combination with a peptide-CD bioink to c/s 3D-bioprint regenerative, prevascularized constructs for wound care. Particular cell adhesion and proteolytic peptide-CD functionalized pair combinations, P15/MMP-2 and P15/cRGD, were found to significantly increase growth of human bone-marrow-derived mesenchymal stems cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs). The constructs delivered two cell types: hBMSCs in the shell bioink and HUVECs within the core bioink. Cord-like, natural microvascularization was shown with endothelial cell marker expression as confirmed by immunofluorescence (IF) staining exhibiting tubelike structures. In addition, in vitro skin wound healing activity of the construct showed a ∼twofold rate of wound closure. Overall, c/s 3D-bioprinted, peptide-CD/GelMA constructs provided the appropriate microenvironment for in vitro stem and endothelial cell viability, delivery, and differentiation. We foresee these custom constructs as representing a fundamental step toward engineering larger scale regenerative, prevascularized tissues.

Permeability and mechanical properties of gradient porous PDMS scaffolds fabricated by 3D-printed sacrificial templates designed with minimal surfaces

In the present study, polydimethylsiloxane (PDMS) porous scaffolds are designed based on minimal surface architectures and fabricated through a low-cost and accessible sacrificial mold printing approach using a fused deposition modeling (FDM) 3D printer. The effects of pore characteristics on compressive properties and fluid permeability are studied. The results suggest that radially gradient pore distribution (as a potential way to enhance mechanically-efficient scaffolds with enhanced cell/scaffold integration) has higher elastic modulus and fluid permeability compared to their uniform porosity counterparts. Also, the scaffolds are fairly strain-reversible under repeated loading of up to 40% strain. Among different triply periodic minimal surface pore architectures, P-surface was observed to be stiffer, less permeable and have lower densification strain compared to the D-surface and G-surface-based pore shapes. The biocompatibility of the created scaffolds is assessed by filling the PDMS scaffolds using mouse embryonic fibroblasts with cell-laden gelatin methacryloyl which was cross-linked in situ by UV light. Cell viability is found to be over 90% after 4 days in 3D culture. This method allows for effectively fabricating biocompatible porous organ-shaped scaffolds with detailed pore features which can potentially tailor tissue regenerative applications. Statement of SignificancePrinting polymers with chemical curing mechanism required for materials such as PDMS is challenging and impossible to create high-resolution uniformly cured structures due to hard control on the base polymer and curing process. An interconnected porous mold with ordered internal architecture with complex geometries were 3D printed using low-cost and accessible FDM technology. The mold acted as a 3D sacrificial material to form internally architected flexible PDMS scaffolds for tissue engineering applications. The scaffolds are mechanically stable under high strain cyclic loads and provide enough pore and space for viably integrating cells within the gradient architecture in a controllable manner.

Visible Light Cross-Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth.

In this study, the cyto-compatibility and cellular functionality of cell-laden gelatin-methacryloyl (Gel-MA) hydrogels fabricated using a set of photo-initiators which absorb in 400-450 nm of the visible light range are investigated. Gel-MA hydrogels cross-linked using ruthenium (Ru) and sodium persulfate (SPS), are characterized to have comparable physico-mechanical properties as Gel-MA gels photo-polymerized using more conventionally adopted photo-initiators, such as 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP). It is demonstrated that the Ru/SPS system has a less adverse effect on the viability and metabolic activity of human articular chondrocytes encapsulated in Gel-MA hydrogels for up to 35 days. Furthermore, cell-laden constructs cross-linked using the Ru/SPS system have significantly higher glycosaminoglycan content and re-differentiation capacity as compared to cells encapsulated using I2959 and LAP. Moreover, the Ru/SPS system offers significantly greater light penetration depth as compared to the I2959 system, allowing thick (10 mm) Gel-MA hydrogels to be fabricated with homogenous cross-linking density throughout the construct. These results demonstrate the considerable advantages of the Ru/SPS system over traditional UV polymerizing systems in terms of clinical relevance and practicability for applications such as cell encapsulation, biofabrication, and in situ cross-linking of injectable hydrogels.

Cell-laden gelatin methacryloyl fibres fabricated using bessel beams for controlled endothelial cord formation

In order to engineer viable three-dimensional (3D) tissue, vascular networks need to be incorporated into the tissue to prevent cell necrosis caused by insufficient access to nutrients. In this project, we propose a method to produce viable vascular endothelial cords in vitro. Human Umbilical Vein Endothelial Cells (HUVECs) were encapsulated within Gelatin Methacryloyl (Gelma) hydrogel fibres by exposing a pre-polymer cell-loaded solution to a UV laser Bessel Beam, which possesses several unique properties, such as self-healing and non-diffractive properties, that enable the polymerization of a free-standing cell-laden Gelma fibre from a single exposure. By manipulating the optical setup and laser power, Gelma fibre diameters fabricated experimentally could be tuned based on mathematical models. After exposure to UV, cell viability was high (>95%) and cells proliferated with time. Under confocal microscopy, encapsulated HUVECs were observed to self-assemble into endothelial cords with circular actin cross-sections. Compared to other techniques, the free-standing nature of the fabricated cords offers several advantages including the ability to be harvested and co-cultured with parenchymal cells. The fabricated cords could potentially find applications in regenerative medicine as well as in vitro models to study vascular physiology.

Microfluidics-Enabled Multimaterial Maskless Stereolithographic Bioprinting.

A stereolithography-based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell-loaded) hydrogel bioinks, to achieve layer-by-layer multimaterial bioprinting. Compared to conventional stereolithography-based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell-laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high-fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.