Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

Effects of Encapsulated Cells on the Physical-Mechanical Properties and Microstructure of Gelatin Methacrylate Hydrogels.

Gelatin methacrylate (GelMA) has been gaining popularity in recent years as a photo-crosslinkable biomaterial widely used in a variety of bioprinting and tissue engineering applications. Several studies have established the effects of process-based and material-based parameters on the physical-mechanical properties and microstructure of GelMA hydrogels. However, the effect of encapsulated cells on the physical-mechanical properties and microstructure of GelMA hydrogels has not been fully understood. In this study, 3T3 fibroblasts were encapsulated at different cell densities within the GelMA hydrogels and incubated over 96 h. The effects of encapsulated cells were investigated in terms of mechanical properties (tensile modulus and strength), physical properties (swelling and degradation), and microstructure (pore size). Cell viability was also evaluated to confirm that most cells were alive during the incubation. It was found that with an increase in cell density, the mechanical properties decreased, while the degradation and the pore size increased.

Fiber-Based Mini Tissue with Morphology-Controllable GelMA Microfibers.

The use of microscale fibers could facilitate nutrient diffusion in fiber-based tissue engineering and improve cell survival. However, in order to build a functional mini tissue such as muscle fibers, nerve conduits, and blood vessels, hydrogel microfibers should not only mimic the structural features of native tissues but also offer a cell-favorable environment and sufficient strength for tissue functionalization. Therefore, an important goal is to fabricate morphology-controllable microfibers with appropriate hydrogel materials to mimic the structural and functional complexity of native tissues. Here, gelatin methacrylate (GelMA) is used as the fiber material due to its excellent biological performance, and a novel coaxial bioprinting method is developed to fabricate morphology-controllable GelMA microfibers encapsulated in calcium alginate. By adjusting the flow rates, GelMA microfibers with straight, wavy, and helical morphologies could be obtained. By varying the coaxial nozzle design, more complex GelMA microfibers such as Janus, multilayered, and double helix structures could be fabricated. Using these microfibers, mini tissues containing human umbilical cord vein endothelial cells are built, in which cells gradually migrate and connect to form lumen resembling blood vessels. The merits of cytocompatibility, structural diversity, and mechanical tunability of the versatile microfibers may open more avenues for further biomedical research.

Facile One-step Micropatterning Using Photodegradable Methacrylated Gelatin Hydrogels for Improved Cardiomyocyte Organization and Alignment.

Hydrogels are often employed as temporary platforms for cell proliferation and tissue organization in vitro. Researchers have incorporated photodegradable moieties into synthetic polymeric hydrogels as a means of achieving spatiotemporal control over material properties. In this study protein-based photodegradable hydrogels composed of methacrylated gelatin (GelMA) and a crosslinker containing o-nitrobenzyl ester groups have been developed. The hydrogels are able to degrade rapidly and specifically in response to UV light and can be photopatterned to a variety of shapes and dimensions in a one-step process. Micropatterned photodegradable hydrogels are shown to improve cell distribution, alignment and beating regularity of cultured neonatal rat cardiomyocytes. Overall this work introduces a new class of photodegradable hydrogel based on natural and biofunctional polymers as cell culture substrates for improving cellular organization and function.

Decellularized matrix bioink with gelatin methacrylate for simultaneous improvements in printability and biofunctionality.

Gelatin methacrylate (GelMA) bioink has been widely used in bioprinting because it is a printable and biocompatible biomaterial. However, it is difficult to print GelMA bioink without any temperature control because it has a thermally-sensitive rheological property. Therefore, in this study, we developed a temperature-controlled printing system in real time without affecting the viability of the cells encapsulated in the bioink. In addition, a skin-derived decellularized extracellular matrix (SdECM) was printed with GelMA to better mimic the native tissue environment compared with solely using GelMA bioink with the enhancement of structural stability. The temperature setting accuracy was calculated to be 98.58 ± 1.8 % for the module and 99.48 ± 1.33 % for the plate from 5 °C to 37 °C. The group of the temperature of the module at 10 °C and the plate at 20 °C have 93.84 % cell viability with the printable range in the printability window. In particular, the cell viability and proliferation were increased in the encapsulated fibroblasts in the GelMA/SdECM bioink, relative to the GelMA bioink, with a morphology that significantly spread for seven days. The gene expression and growth factors related to skin tissue regeneration were relatively upregulated with SdECM components. In the bioprinting process, the rheological properties of the GelMA/SdECM bioink were successfully adjusted in real time to increase printability, and the native skin tissue mimicked components providing tissue-specific biofunctions to the encapsulated cells. The developed bioprinting strategies and bioinks could support future studies related to the skin tissue reconstruction, regeneration, and other medical applications using the bioprinting process.

Neutrophil Granulopoiesis Optimized Through Ex Vivo Expansion of Hematopoietic Progenitors in Engineered 3D Gelatin Methacrylate Hydrogels.

Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis.

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.

An interpenetrating and patternable conducting polymer hydrogel for electrically stimulated release of glutamate.

Recent advances in drug delivery have made it possible to release bioactive agents from neural implants specifically to local tissues. Conducting polymer coatings have been explored as a delivery platform in bioelectronics, however, their utility is restricted by their limited loading capacity and stability. This study presents the fabrication of a stable conducting polymer hydrogel (CPH), comprising the hydrogel gelatin methacrylate (GelMA), and conducting polymer polypyrrole (PPy) for the electrically controlled delivery of glutamate (Glu). The hybrid GelMA/PPy/Glu can be photolithographically patterned and covalently bonded to an electrode. Fourier-transform infrared (FTIR) analysis confirmed the interpenetrating nature of PPy through the GelMA hydrogels. Electrochemical polymerisation of PPy/Glu through the GelMA hydrogels resulted in a significant increase in the charge storage capacity as determined by cyclic voltammetry (CV). Long-term electrochemical and mechanical stability was demonstrated over 1000 CV cycles and extracts of the materials were cytocompatible with SH-SY5Y neuroblastoma cell lines. Release of Glu from the CPH was responsive to electrical stimulation with almost five times the amount of Glu released upon constant reduction (-0.6 V) compared to when no stimulus was applied. Notably, GelMA/PPy/Glu was able to deliver almost 14 times higher amounts of Glu compared to conventional PPy/Glu films. The described CPH coatings are well suited in implantable drug delivery applications and compared to conducting polymer films can deliver higher quantities of drug in response to mild electrical stimulus. STATEMENT OF SIGNIFICANCE: Conducting polymer hydrogels (CPH) have been explored for the electrically controlled release of bioactives from implantable devices. Typically, the conducting polymer component does not fully penetrate the hydrogel. We report, for the first time, a completely interpenetrating CPH allowing for the full benefits of the composite material to be realised, the hydrogels provide a reservoir for drug delivery, and conducting polymer renders the material responsive to electrical stimulation for drug release. We report a CPH for the electrically controlled delivery of glutamate (excitatory neurotransmitter) where several-fold more glutamate can be delivered compared to conducting polymer films. The described CPH coatings are well suited for use in bioelectronic devices to deliver large quantities of drug in response to mild electrical stimulus.

Improved Osteogenesis by Mineralization Combined With Double-Crosslinked Hydrogel Coating for Proliferation and Differentiation of Mesenchymal Stem Cells.

In consideration of improving the interface problems of poly-L-lactic acid (PLLA) that hindered biomedical use, surface coatings have been explored as an appealing strategy in establishing a multi-functional coating for osteogenesis. Though the layer-by-layer (LBL) coating developed, a few studies have applied double-crosslinked hydrogels in this technique. In this research, we established a bilayer coating with double-crosslinked hydrogels [alginate-gelatin methacrylate (GelMA)] containing bone morphogenic protein (BMP)-2 [alginate-GelMA/hydroxyapatite (HA)/BMP-2], which displayed great biocompatibility and osteogenesis. The characterization of the coating showed improved properties and enhanced wettability of the native PLLA. To evaluate the biosafety and inductive ability of osteogenesis, the behavior (viability, adherence, and proliferation) and morphology of human bone mesenchymal stem cells (hBMSCs) on the bilayer coatings were tested by multiple exams. The satisfactory function of osteogenesis was verified in bilayer coatings. We found the best ratios between GelMA and alginate for biological applications. The Alg70-Gel30 and Alg50-Gel50 groups facilitated the osteogenic transformation of hBMSCs. In brief, alginate-GelMA/HA/BMP-2 could increase the hBMSCs' early transformation of osteoblast lineage and promote the osteogenesis of bone defect, especially the outer hydrogel layer such as Alg70-Gel30 and Alg50-Gel50.

Hybrid Cornea: Cell Laden Hydrogel Incorporated Decellularized Matrix.

The decellularization protocols applied on the corneal stromal constructs in the literature usually fail to provide a corneal matrix with sufficient mechanical and optical properties since they alter the extracellular matrix (ECM) microstructure. In this study, to overcome these limitations, a hybrid cornea stromal construct was engineered by combining gelatin methacrylate (GelMA) and decellularized ECM. Photo-cross-linking of impregnated cell laden GelMA in situ using different UV cross-linking energies (3200, 6210, and 6900 µJ/cm2) and impregnation times (up to 24 h) within a decellularized bovine cornea enhanced light transmission and restored lost mechanical features following the harsh decellularization protocol. The light transmittance value for optimized hybrid constructs (53.6%) was increased nearly 10 fold compared to that of decellularized cornea (5.84%). The compressive modulus was also restored up to 6 fold with calculated values of 5040 and 870 kPa for the hybrid and decellularized samples, respectively. These values were found to be quite close to that of native cornea (48.5%, 9790 kPa). ATR-FTIR analyses were carried out to confirm the final chemical structure. The degradation profiles showed similar decomposition behaviors to that of native cornea. In vitro culture studies showed a high level of cell viability and cell proliferation rate was found remarkable up to the 14th day of the culture period regardless of selected UV energy level. The methodology used in the preparation of the hybrid cornea stromal constructs in this study is a promising approach toward the development of successful corneal transplants.

Tofu-Incorporated Hydrogels for Potential Bone Regeneration.

Food-derived materials possess inherent advantages in tissue engineering applications with appropriate biosafety, availability, and maneuverability. This work takes advantage of gelatin methacrylate (GelMa) to fabricate the tofu-incorporated hydrogels and systematically investigated the potential for bone regeneration. The results affirmed that tofu-incorporated hydrogel possessed porous architecture, satisfactory mechanical performance, and appropriate cytocompatibility. It is worth noting that little inflammation could be caused by the tofu/GelMa hydrogels, and the incorporated tofu powder could also promote the secretion of osteogenesis and immune-related cytokines in the early stage, resulting in improved bone regeneration during the 2-month implantation. All the results suggested that tofu/GelMa hydrogels possessed good potential for bone regeneration with low cost, satisfactory cytocompatibility, and excellent bioactivity.

Uniaxial Stretching of Cell-Laden Microfibers for Promoting C2C12 Myoblasts Alignment and Myofibers Formation.

Fiber-shaped cellular constructs have attracted increasing attention in the regeneration of blood vessels, nerve networks, and skeletal myofibers. Nevertheless, the generation of functional fiber-shaped cellular constructs suffers from limited appropriate microfiber-based fabrication approaches and the maintenance of regenerated tissue functions. Herein, we demonstrate a silicone-tube-based coagulant bath free method to fabricate tens of centimeters long cell-laden microfibers using single UV exposure without pretreatment of nozzles or microchannels. By modulating the exposure time, the gelatin methacrylate microfibers with tissue-like microstructures and mechanical properties are obtained. Then, a culture system integrated with a pillar well-array based stretching device is used to apply uniaxial stretching with various strain ratios in situ to cell-laden microfibers in a 60 mm petri dish. Cells with improved spreading, elongation, and alignment are obtained under uniaxial stretching. Moreover, the promotional effects of uniaxial stretching on the differentiation of C2C12 myoblasts, the formation, and contractility of myofibers become more pronounced with increasing strain ratio and achieve saturation level as strain ratio up to ∼35%.

Molecular interactions and forces of adhesion between single human neural stem cells and gelatin methacrylate hydrogels of varying stiffness.

Single Cell Force Spectroscopy was applied to measure the single cell de-adhesion between human neural stem cells (hNSC) and gelatin methacrylate (GelMA) hydrogel with varying modulus in the range equivalent to brain tissue. The cell de-adhesion force and energy were predominately generated via unbinding of complexes formed between RGD groups of the GelMA and cell surface integrin receptors and the de-adhesion force/energy were found to increase with decreasing modulus of the GelMA hydrogel. For the softer GelMA hydrogels (160 Pa and 450 Pa) it was proposed that a lower degree of cross-linking enables a greater number of polymer chains to bind and freely extend to increase the force and energy of the hNSC-GelMA de-adhesion. In this case, the multiple polymer chains are believed to act together in parallel like 'molecular tensors' to generate tensile forces on the bound receptors until the cell detaches. Counterintuitively for softer substrates, this type of interaction gave rise to higher force loading rates, including the appearance of high and low dynamic force regimes in de-adhesion rupture force versus loading rate analysis. For the stiffer GelMA hydrogel (900 Pa) it was observed that the extension and elastic restoring forces of the polymer chains contributed less to the cell de-adhesion. Due to the apparent lower extent of freely interacting chains on the stiffer GelMA hydrogel the intrinsic RGD groups are presumed to be "more fixed" to the substrate. Hence, the cell de-adhesion is suggested to be mainly governed by the discrete unbinding of integrin-RGD complexes as opposed to elastic restoring forces of polymer chains, leading to smaller piconewton rupture forces and only a single lower dynamic force regime. Intriguingly, when integrin antibodies were introduced for binding integrin α5ß1, ß1- and αv-subunits it was revealed that the cell modifies the de-adhesion force depending on the substrate stiffness. The antibody binding supressed the de-adhesion on the softer GelMA hydrogel while on the stiffer GelMA hydrogel caused an opposing reinforcement in the de-adhesion. STATEMENT OF SIGNIFICANCE: Conceptual models on cell mechanosensing have provided molecular-level insight to rationalize the effects of substrate stiffness. However most experimental studies evaluate the cell adhesion by analysing the bulk material properties. As such there is a discrepancy in the scale between the bulk properties versus the nano- and micro-scale cell interactions. Furthermore there is a paucity of experimental studies on directly measuring the molecular-level forces of cell-material interactions. Here we apply Single Cell Force Spectroscopy to directly measure the adhesion forces between human neural stem cells and gelatin-methacrylate hydrogel. We elucidate the mechanisms by which single cells bind and physically interact with hydrogels of varying stiffness. The study highlights the use of single cell analysis tools to probe molecular-level interactions at the cell-material interface which is of importance in designing material cues for regulating cell function.

Halloysite Nanotube Based Scaffold for Enhanced Bone Regeneration.

Bone regeneration remains a clinical challenge with limited bone substitutes, urging for effective alternative strategies. Nanotubes, especially carbon nanotubes and titanium dioxide nanotubes, have been widely utilized for bone regeneration; however, their further applications were limited by the composition and degradability. As naturally occurring aluminosilicate nanoclay, halloysite nanotubes (HNTs), with good biocompatibility, functionality, and nanotubular structures, may be a promising platform for promoting bone regeneration. Herein, we presented a HNTs incorporated hydrogel and explored the potential bone tissue engineering applications of HNTs. The HNTs encapsulated hydrogel was simply fabricated by using the photopolymerization method with gelatin methacrylate (GelMA) and HNTs. The incorporation of HNTs led to an enhanced mechanical performance while maintaining a good cytocompatibility in vitro. The osteogenic activities of the HNTs incorporated platform have also been studied in vitro and in vivo. Remarkably, the addition of HNTs obviously upregulated the expression of osteogenic differentiation-related genes and concomitant protein of human dental pulp stem cells (hDPSCs) and therefore facilitated subsequent bone regeneration in calvarial defects of rats. Overall, the results obtained in this study highlight the bone regeneration capacity of HNTs, which may enhance current understanding of HNTs, and present a promising alternative strategy for bone regeneration.

Bioprinting of Cell-Laden Microfiber: Can It Become a Standard Product?

Hydrogel microfibers have many fascinating applications as microcarriers for drugs, factors, and cells, such as 3D cell culture, building micro-organoids, and transplantation therapy due to their simple structures. It is unknown whether cell-laden fiber can become a standard-use product like woundplast. Here, from the technical and practical view, the elements required for user-oriented microfibers are first discussed: i) the materials used should promote cell functionalization and be easily processed; ii) follow a manufacturing method for mass fabrication; iii) have the ability to be stored long-term and be available for immediate use. Here, it is demonstrated that bioactive microfibers can be simply fabricated with coaxial bioprinting using gelatin methacrylate due to its tunable biological and mechanical properties. Additionally, programmed microfibers and 3D constructs with controllable composition can also be fabricated. These microfibers can be used to directly build organoids and complex co-culture tissue models. In the present study, vascular organoid, angiogenic sprouts, and tumor angiogenesis are demonstrated. It is also demonstrated, for the first time, that the cell-laden microfibers can be stored long-term via cryopreservation. These results show that cell-laden structures can be developed as a novel type of organoid product, which will open more avenues for tissue engineering and clinical organ repair.

Gelatin-based micro-hydrogel carrying genetically engineered human endothelial cells for neovascularization.

Cell delivery systems based on micro-hydrogels may facilitate the long-term survival of cells upon transplantation. Micro-hydrogels may effectively support cell proliferation, attachment, and migration in ischemic environments. In this study, we report the fabrication of a gelatin methacrylate (GelMA)-based micro-hydrogel for efficient in vivo delivery of genetically engineered endothelial cells. Micro-hydrogels were initially processed via electrospraying of GelMA and alginate (ALG) mixtures (at different ratios) on to calcium chloride (CaCl2) solution. Electrospraying of the GelMA/ALG mixture resulted in the formation of a micro-hydrogel, owing to ALG crosslinking. Secondary crosslinking of GelMA with UV light and ALG hydrogel chelation using sodium citrate solution resulted in GelMA-based micro-hydrogel formation. We observed the angiogenic response of human umbilical vein endothelial cells (HUVECs) in GelMA concentration-dependent manner. The seeding of HUVECs engineered to express human vascular endothelial growth factor on to the GelMA micro-hydrogel and the subsequent transplantation of the micro-hydrogel into a hindlimb ischemia model effectively attenuated the ischemia condition. This facile and simple micro-hydrogel fabrication strategy may serve as a robust method to fabricate efficient cell carriers for various ischemic diseases. STATEMENT OF SIGNIFICANCE: For the therapeutic angiogenesis, it is important to provide the therapeutic cells with a carrier that could stabilize therapeutic cells and facilitate long-term survival of cells. Furthermore, it is also important to administer as many therapeutic cells as possible in a fixed volume. From these cues, we fabricated ECM-based micro-hydrogel produced by the high through-put system. And we intended to facilitate activation of therapeutic cells by coating the therapeutic cells onto the micro-hydrogel. In this manuscript, we fabricated methacrylate gelatin (GelMA) based micro-hydrogels using the electro-spraying method and coated HUVECs engineered to express hVEGF onto the micro-hydrogels. Then, we identified that GelMA concentration-dependent angiogenic response of HUVECs. Furthermore, we demonstrated that the VEGF secreting HUVEC-GelMA micro-hydrogels induced the restoration of blood flow and neovascularization in a hind-limb ischemia mouse model. These findings demonstrate that the high-throughput fabrication of ECM micro-hydrogels could be a novel platform to apply in neovascularization and tissue engineering.

On Low-Concentration Inks Formulated by Nanocellulose Assisted with Gelatin Methacrylate (GelMA) for 3D Printing toward Wound Healing Application.

Cellulose nanofibrils (CNFs) in the form of hydrogels stand out as a platform biomaterial in bioink formulation for 3D printing because of their low cytotoxicity and structural similarity to extracellular matrices. In the present study, 3D scaffolds were successfully printed with low-concentration inks formulated by 1 w/v % 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNF with less than 1 w/v % gelatin methacrylate (GelMA). Quartz crystal microbalance with dissipation monitoring (QCM-D) measurements showed strong interaction between the two biopolymers. The UV cross-linking ability of GelMA (≤1 w/v %) was enhanced in the presence of TEMPO-oxidized CNFs. Multiple factors including strong physical interaction between CNF and GelMA, in situ cross-linking of CNF by Ca2+, and UV cross-linking of GelMA enabled successful 3D printing of low-concentration inks of CNF/GelMA into scaffolds possessing good structural stability. The mechanical strength of the scaffolds was tuned in the range of 2.5 to 5 kPa. The cell culture with 3T3 fibroblasts revealed noncytotoxic and biocompatible features for the formulated inks and printed scaffolds. More importantly, the incorporated GelMA in the CNF hydrogel promoted the proliferation of fibroblasts. The developed low-concentration CNF/GelMA formulations with a facile yet effective approach to fabricate scaffolds showed great potential in 3D printing for wound healing application.

A three dimensional micropatterned tumor model for breast cancer cell migration studies.

Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli, making it difficult to isolate and assess the effects of biochemical or biophysical cues (i.e. tumor architecture, matrix stiffness) on disease progression. In this regard, physiologically relevant tumor models are becoming instrumental to perform studies of cancer cell invasion within well-controlled conditions. Herein, we explored the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique to microengineer a 3D breast tumor model. The microfabrication process enabled precise localization of cell-encapsulated circular constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded separately within the tumor model, all of which maintained high viability throughout the experiments. MDA-MB-231 cells exhibited extensive migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that stayed confined within the circular tumor regions. Additionally, real-time cell tracking indicated that the speed and persistence of MDA-MB-231 cells were substantially higher within the surrounding matrix compared to the circular constructs. Z-stack imaging of F-actin/α-tubulin cytoskeletal organization revealed unique 3D protrusions in MDA-MB-231 cells and an abundance of 3D clusters formed by MCF7 and MCF10A cells. Our results indicate that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in the development of 3D tumor models with well-defined architecture and tunable stiffness.