Photo-crosslinkable Gelatin Methacrylate Research Articles

Enhancement of properties of a cell-laden GelMA hydrogel-based bioink via calcium phosphate phase transition

To improve the properties of the hydrogel-based bioinks, a calcium phosphate phase transition was applied, and the products were examined. We successfully enhanced the mechanical properties of the hydrogels by adding small amounts (< 0.5 wt%) of alpha-tricalcium phosphate (α-TCP) to photo-crosslinkable gelatin methacrylate (GelMA). As a result of the hydrolyzing calcium phosphate phase transition involving α-TCP, which proceeded for 36 h in the cell culture medium, calcium-deficient hydroxyapatite was produced. Approximately 18 times the compressive modulus was achieved for GelMA with 0.5 wt% α-TCP (20.96 kPa) compared with pure GelMA (1.18 kPa). Although cell proliferation decreased during the early stages of cultivation, both osteogenic differentiation and mineralization activities increased dramatically when the calcium phosphate phase transition was performed with 0.25 wt% α-TCP. The addition of α-TCP improved the printability and fidelity of GelMA, as well as the structural stability and compressive modulus (approximately six times higher) after three weeks of culturing. Therefore, we anticipate that the application of calcium phosphate phase transition to hydrogels may have the potential for hard tissue regeneration.

Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and theestablishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery.

Multifunctional and biodegradable methacrylated gelatin/Aloe vera nanofibers for endodontic disinfection and immunomodulation

Currently employed approaches and materials used for vital pulp therapies (VPTs) and regenerative endodontic procedures (REPs) lack the efficacy to predictably achieve successful outcomes due to their inability to achieve adequate disinfection and/or lack of desired immune modulatory effects. Natural polymers and medicinal herbs are biocompatible, biodegradable, and present several therapeutic benefits and immune-modulatory properties; thus, standing out as a clinically viable approach capable of establishing a conducive environment devoid of bacteria and inflammation to support continued root development, dentinal bridge formation, and dental pulp tissue regeneration. However, the low stability and poor mechanical properties of the natural compounds have limited their application as potential biomaterials for endodontic procedures. In this study, Aloe vera (AV), as a natural antimicrobial and anti-inflammatory agent, was incorporated into photocrosslinkable Gelatin methacrylate (GelMA) nanofibers with the purpose of developing a highly biocompatible biomaterial capable of eradicating endodontic infection and modulating inflammation. Stable GelMA/AV nanofibers with optimal properties were obtained at the ratio of (70:30) by electrospinning. In addition to the pronounced antibacterial effect against Enterococcus faecalis, the GelMA/AV (70:30) nanofibers also exhibited a sustained antibacterial activity over 14 days and significant biofilm reduction with minimal cytotoxicity, as well as anti-inflammatory properties and immunomodulatory effects favoring healing. Our results indicate that the novel GelMA/AV (70:30) nanofibers hold great potential as a biomaterial strategy for endodontic infection eradication and enhanced healing.

Bioengineering for vascularization: Trends and directions of photocrosslinkable gelatin methacrylate hydrogels.

Gelatin methacrylate (GelMA) hydrogels have been widely used in various biomedical applications, especially in tissue engineering and regenerative medicine, for their excellent biocompatibility and biodegradability. GelMA crosslinks to form a hydrogel when exposed to light irradiation in the presence of photoinitiators. The mechanical characteristics of GelMA hydrogels are highly tunable by changing the crosslinking conditions, including the GelMA polymer concentration, degree of methacrylation, light wavelength and intensity, and light exposure time et al. In this regard, GelMA hydrogels can be adjusted to closely resemble the native extracellular matrix (ECM) properties for the specific functions of target tissues. Therefore, this review focuses on the applications of GelMA hydrogels for bioengineering human vascular networks in vitro and in vivo. Since most tissues require vasculature to provide nutrients and oxygen to individual cells, timely vascularization is critical to the success of tissue- and cell-based therapies. Recent research has demonstrated the robust formation of human vascular networks by embedding human vascular endothelial cells and perivascular mesenchymal cells in GelMA hydrogels. Vascular cell-laden GelMA hydrogels can be microfabricated using different methodologies and integrated with microfluidic devices to generate a vasculature-on-a-chip system for disease modeling or drug screening. Bioengineered vascular networks can also serve as build-in vasculature to ensure the adequate oxygenation of thick tissue-engineered constructs. Meanwhile, several reports used GelMA hydrogels as implantable materials to deliver therapeutic cells aiming to rebuild the vasculature in ischemic wounds for repairing tissue injuries. Here, we intend to reveal present work trends and provide new insights into the development of clinically relevant applications based on vascularized GelMA hydrogels.

Tissue-Specific Hydrogels for Three-Dimensional Printing and Potential Application in Peripheral Nerve Regeneration.

Decellularized extracellular matrix hydrogel (dECM-G) has demonstrated its significant tissue-specificity, high biocompatibility, and versatile utilities in tissue engineering. However, the low mechanical stability and fast degradation are major drawbacks for its application in three-dimensional (3D) printing. Herein, we report a hybrid hydrogel system consisting of dECM-Gs and photocrosslinkable gelatin methacrylate (GelMA), which resulted in significantly improved printability and structural fidelity. These premixed hydrogels retained high bioactivity and tissue-specificity due to their containing dECM-Gs. More specifically, it was realized that the hydrogel containing dECM-G derived from porcine peripheral nerves (GelMA/pDNM-G) effectively facilitated neurite growth and Schwann cell migration from two-dimensional cultured dorsal root ganglion explants. The nerve cells were also encapsulated in the GelMA/pDNM-G hydrogel for 3D culture or underwent cell-laden bioprinting with high cell viability. The preparation of such GelMA/dECM-G hydrogels enabled the recapitulation of functional tissues through extrusion-based bioprinting, which holds great potential for applications in regenerative medicine. Impact statement Tissue-derived decellularized matrices have drawn broad interests for their versatile applications in tissue engineering and regenerative medicine, especially the decellularized peripheral nerve matrix, which can effectively facilitate axonal extension, remyelination, and neural functional restoration after peripheral nerve injury. However, neither decellularized porcine nerve matrix (pDNM) nor pDNM hydrogel (pDNM-G) can be directly used in three-dimensional printing for personalized nerve constructs or cell transplantation. This work developed a hybrid hydrogel consisting of decellularized extracellular matrix hydrogel (dECM-G) and photocrosslinkable gelatin methacrylate (GelMA), which resulted in significantly improved printability and structural fidelity. The GelMA/pDNM-G hydrogel retained high bioactivity and tissue-specificity due to its dECM-G content. Such hybrid hydrogel systems built up a springboard in advanced biomaterials for neural tissue engineering, as well as a promising strategy for dECM containing bioprinting.

Development of a Dual Hydrogel Model System for Vascularization

Numerous hydrogel-based culture systems are used to create in vitro model for prevascularization. Hydrogels used to induce a microenvironment conducive to microvessel formation are typically soft and fast degradable, but often suffer from maintaining a lasting perfusable channel in vitro. Here, a dual hydrogel system that consists of photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol dimethacrylate (PEGDMA) is reported. GelMA hydrogels present soft and rapidly degradable properties and show microporous structures while PEGDMA is relatively stiff, almost nondegradable in vitro, and less porous. The dual hydrogel system is sequentially photo-crosslinked to construct an endothelial cell (EC)-lined perfusable PEGDMA channel and surrounding GelMA for endothelial vascular networks. Such dual hydrogel system exhibits seamless integration of the stiff PEGDMA channel and the surrounding soft GelMA, and facilitates rapid EC sprouting and extensive microvessel formation from a stable endothelium on the PEGDMA channel into the GelMA. Furthermore, diffusivity of biomolecules in the perfusable dual hydrogel system is affected by both the structural and physicochemical properties of the hydrogel system and the microvascular networks formed in the system. The establishment of the dual hydrogel system for vascularization holds great promise as an in vitro angiogenesis model and prevascularization strategy of large tissue constructs.

Carbon nanotube, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) and Ag nanoparticle doped gelatin based electro-active hydrogel systems

In this study, gelatin-based conductive hydrogels doped with carbon nanotube (CNT), poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and silver (Ag) nanoparticle were prepared for use in biopotential measurements. Gelatin was modified by various concentrations of methacrylic anhydride (MA) to obtain photo-crosslinkable gelatin methacrylate (GelMA) polymer with low (21%), medium (44%) and high (78%) methacrylation degree. Chemical modification of gelatin was characterized by 1H-NMR spectroscopy. It was observed that mechanical properties of the GelMA hydrogels were highly dependent on methacrylation degree. Ultimate compressive stress of low, medium and high methacrylated gelatin hydrogel was measured to be 9.97 ± 0.14 kPa, 17.83 ± 1.73 kPa and 38.23 ± 2.96 kPa, respectively. Surface morphology of hydrogels was visualized by scanning electron microscope (SEM). The pore size of methacrylated gelatin hydrogel significantly reduced with the addition of CNT, PEDOT:PSS and Ag nanoparticle into the hydrogel matrix. The results of thermal gravimetric analysis showed that conductive hydrogels were of higher thermal stability than nonconductive GelMA hydrogel. The suitability of the prepared hydrogels for biopotential measurements was determined by equivalent impedance measurements. Impedance values of hydrogel samples were obtained in the frequency range of 20 Hz–500 Hz. The hydrogel materials prepared in this study exhibited high impedance at low frequencies and low impedance at high frequencies. The results of this study showed that the hydrogels produced with Ag nanoparticle are more suitable electrode materials for physiological measurement systems such as electrocardiography (ECG) and electroencephalography (EEG).

3D printing of complex GelMA-based scaffolds with nanoclay

Photo-crosslinkable gelatin methacrylate (GelMA) has become an attractive ink in 3D printing due to its excellent biological performance. However, limited by low viscosity and long cross-linking time, it is still a challenge to directly print GelMA by extrusion-based 3D printing. Here, to balance the printability and biocompatibility, biomaterial ink composed of GelMA and nanoclay was specially designed. Using this ink, complex scaffolds with high shape fidelity can be easily printed based on the thixotropic property of nanoclay. In this study, we tried to answer some basic printing-required questions of this ink, including the printability window, general properties (porosity, mechanical strength, et al), and biocompatibility. We found that the GelMA/Nanoclay ink enabled printing complex 3D scaffolds, such as a bionic ear and a branched vessel. Furthermore, the addition of nanoclay improved the porosity, increased the mechanical strength, reduced the degradation ratio, and maintained a good biocompatibility of the printed scaffolds. Therefore, this method offers an easy way to print complex scaffolds with good shape fidelity and biological performance, and it might open up new potential applications for the customized therapy of tissue defects.

Temperature-sensitive biocompatible IPN hydrogels based on poly(NIPA-PEGdma) and photocrosslinkable gelatin methacrylate

ABSTRACTThe future of tissue engineering and regenerative medicine relies on the development of biomimetic extracellular matrices (ECM). Hydrogels based on biological and synthetic macromolecules could mimic native ECM which is an interpenetrating network (IPN) of proteins and other biomacromolecules. In this study, gelatin and N-isopropylacrylamide-based IPN hydrogels, named as NPxG, were developed to fabricate 3D matrix systems. A series of IPN hydrogels with different contents in poly(NIPA-PEGdma) (NPx) and gelatin methacrylate (GelMA) were synthesized. NPx and GelMA were characterized by Habeeb’s assay, FTIR, and 1H-NMR. IPN hydrogels were fully characterized by ATR-FTIR, swelling measurements, optical microscopy, SEM, DSC, and rheology. Optical microscopy images and rheology demonstrated that hydrogel properties change around 37°C. Above LCST, the translucent colorless hydrogel shifted to an opaque white hydrogel and the storage modulus increased. A wide range of IPN gels with different swelling properties and potential applications in different fields can be produced by controlling experimental parameters.

Uniform-sized neurosphere-mediated motoneuron differentiation in microwell arrays.

We developed the photocrosslinkable hydrogel microwell arrays for uniform-sized neurosphere-mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform-sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform-sized neurospheres harvested from microwell arrays were replated into laminin-coated substrate. In parallel, uniform-sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three-dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150μm in diameter) on motoneuron differentiation, showing that the largest uniform-sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform-sized neurosphere-mediated motoneuron differentiation.

Electro-mechano responsive properties of gelatin methacrylate (GelMA) hydrogel on conducting polymer electrodes quantified using atomic force microscopy.

Electrical stimulation of hydrogels has been performed to enable micro-actuation or controlled movement of ions and biomolecules such as in drug release applications. Hydrogels are also increasingly used as low modulus, biocompatible coatings on electrode devices and thus are exposed to the effects of electrical stimulation. As such, there is growing interest in the latter, especially on the dynamic and nanoscale physical properties of hydrogels. Here, we report on the electro-mechano properties of photocrosslinkable gelatin methacrylate (GelMA) hydrogel applied as coatings on conducting polymer polypyrrole-dodecylbenze sulfonate (PPy-DBSA) electrodes. In particular, Electrochemical-Atomic Force Microscopy (EC-AFM) was used to quantify the nanoscale actuation and dynamic changes in Young's modulus as the GelMA coating was electrically stimulated via the underlying PPy-DBSA electrode. Pulsed electrical stimulation was shown to induce dynamic expansion and contraction, or nanoscale actuation, of the GelMA hydrogel due to the reversible ingress of electrolyte ions and associated changes in osmotic pressure during oxidation and reduction of the PPy-DBSA film. In addition, dynamic changes in the Young's modulus of up to 50% were observed in the hydrogel and correlated with the actuation process and ion diffusion during oxidation and reduction of the underlying PPy-DBSA film. These dynamic properties were investigated for hydrogels with varying degrees of cross-linking, porosity and modulus, the latter ranging from ≈0.2-1 kPa. The study demonstrates an AFM-based approach to quantify the dynamic physical properties of hydrogels, which are shown to be modulated via electrical stimulation. This can enable a better understanding of the electro-mechano mechanisms that are important for the controlled release of drugs or controlling cell interactions at the hydrogel-cell interface.

Enhancing vascularization of a gelatin-based micro-cavitary hydrogel by increasing the density of the micro-cavities

The transport of nutrients and oxygen by vascular networks into engineered tissue constructs is critical to their successful integration into host tissues. Hydrogel has achieved some promising results as scaffolds for vascularization. However, the vascularization of hydrogel is still constrained by its inherent submicron- or nano-sized pores. In this study, two gelatin-based micro-cavitary gel (Gel-MCG) constructs with varying densities of micro-cavities were developed with a photocrosslinkable gelatin methacrylate (Gel-MA) precursor and porogenic gelatin microspheres (MS), and their functions in supporting vascularization within hydrogels were evaluated with endothelial progenitor outgrowth cells (EPOCs). The increase of cavitary density could enhance the vascularization of Gel-MCG constructs. After 14 d of culture in vitro, the vascularization of Gel-MCG constructs with higher cavitary density was significantly superior to that of gelatin spongy control and the fusion of vascularized cavities in the constructs could be observed. Further subcutaneous implantation of the Gel-MCG constructs with higher cavitary density into nude mice also showed obvious vascular invasion from host tissues. Taken together, these results indicate that the increase in cavitary density can efficiently facilitate the vascularization of Gel-MCG constructs both in vitro and in vivo and that such highly-porous Gel-MCG constructs have great potential to be a promising scaffold for the development of vascularized tissue constructs.

Hydrogel-encapsulated 3D microwell array for neuronal differentiation

We developed a photo-crosslinkable hydrogel-encapsulated three-dimensional (3D) microwell array for studying embryonic stem (ES) cell-derived neuronal differentiation. ES cells were cultured for 5 d in microwells and were subsequently encapsulated by photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol (PEG) hydrogels for an additional 7 d. We observed that ES cells cultured in PEG microwells became uniform-sized embryoid bodies (EBs) compared to those in GelMA microwells. Although ES cells were encapsulated by photo-crosslinkable GelMA and PEG hydrogels, they were highly viable. We demonstrated that uniform-sized EBs encapsulated by GelMA hydrogels in PEG microwells are largely differentiated into neuronal cells. It was revealed that neurites at the periphery of EBs in PEG microwells largely extended into the interface between GelMA hydrogels and PEG microwells for generating neuronal networks. Therefore, this photo-crosslinkable GelMA hydrogel-encapsulated PEG microwell array could be a potentially powerful tool for neurodegenerative disease applications.

Photo-crosslinkable hydrogel-based 3D microfluidic culture device.

We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.

Cell-induced flow-focusing instability in gelatin methacrylate microdroplet generation.

Photo-crosslinkable gelatin methacrylate (GelMa) microspheres are applicable to deliver cells or drugs in biological or biomedical applications. To fabricate GelMa microdroplets, a flow focusing technique with advantages of size control and rapid production was used in a T-junction microfluidic device. Instability played an important role in promoting microdroplet uniformity. 5 wt. % GelMa prepolymer solution mixed with cells affected cell-induced instability. At low flow rate ratio of GelMa to mineral oil below 0.200, stability was maintained regardless of GelMa concentration (5 and 8 wt. %) and cell presence, which led to uniform microdroplet generation. In contrast, instability at high flow rate ratio above 0.200 was worsened by cell presence and unstable jetting length, resulting in the generation of non-uniform cell-laden microdroplets. Therefore, the effect of cell-induced instability on microdroplet generation was minimized at a low flow rate ratio.

Release of Magnetic Nanoparticles from Cell-Encapsulating Biodegradable Nanobiomaterials

The future of tissue engineering requires development of intelligent biomaterials using nanoparticles. Magnetic nanoparticles (MNPs) have several applications in biology and medicine; one example is Food and Drug Administration (FDA)-approved contrast agents in magnetic resonance imaging. Recently, MNPs have been encapsulated within cell-encapsulating hydrogels to create novel nanobiomaterials (i.e., M-gels), which can be manipulated and assembled in magnetic fields. The M-gels can be used as building blocks for bottom-up tissue engineering to create 3D tissue constructs. For tissue engineering applications of M-gels, it is essential to study the release of encapsulated MNPs from the hydrogel polymer network and the effect of MNPs on hydrogel properties, including mechanical characteristics, porosity, swelling behavior, and cellular response (e.g., viability, growth). Therefore, we evaluated the release of MNPs from photocrosslinkable gelatin methacrylate hydrogels as the polymer network undergoes biodegradation using inductively coupled plasma atomic emission spectroscopy. MNP release correlated linearly with hydrogel biodegradation rate with correlation factors (Pearson product moment correlation coefficient) of 0.96 ± 0.03 and 0.99 ± 0.01 for MNP concentrations of 1% and 5%, respectively. We also evaluated the effect of MNPs on hydrogel mechanical properties, porosity, and swelling behavior, as well as cell viability and growth in MNP-encapsulating hydrogels. Fibroblasts encapsulated with MNPs in hydrogels remained viable (>80% at t = 144 h) and formed microtissue constructs in culture (t = 144 h). These results indicated that MNP-encapsulating hydrogels show promise as intelligent nanobiomaterials, with great potential to impact broad areas of bioengineering, including tissue engineering, regenerative medicine, and pharmaceutical applications.