Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel.

Immunoassays, which use antigen-antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 µL. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 µL sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method.

Human umbilical cord mesenchymal stem cell-derived exosomes combined with gelatin methacryloyl hydrogel to promote fractional laser injury wound healing.

To investigate whether human umbilical cord mesenchymal stem cell-derived exosomes combined with gelatin methacryloyl (GelMA) hydrogel are beneficial in promoting healing of laser-injured skin wounds in mice. Supernatants of cultured human umbilical cord mesenchymal stem cells (HUC-MSCs) were collected to obtain human umbilical cord MSC-derived exosomes (HUC-MSCs-Exos), which were combined with GelMA hydrogel complex to treat a mouse fractional laser injury model. The study was divided into PBS group, EX (HUC-MSCs-Exos) group, GEL (GelMA hydrogel) group and EX+GEL (HUC-MSCs-Exos combined with GelMA hydrogel) group. The healing of laser-injured skin in each group was observed by gross view and dermatoscopy, and changes in skin structure, angiogenesis and proliferation-related indexes were observed during the healing process of laser-injured skin in each group. The results of the animal experiments showed that the EX and GEL groups alone and the EL+EX group exhibited less inflammatory response compared to the PBS group. The EX and GEL groups showed marked tissue proliferation and favourable angiogenesis, which promoted the wound healing well. The GEL+EX group had the most significant promotion of wound healing compared to the PBS group. qPCR results showed that the expression levels of proliferation-related factors, including KI67 and VEGF and angiogenesis-related factor CD31, were significantly higher in the GEL+EX group than in the other groups, with a time-dependent effect. The combination of HUC-MSCs-Exos and GelMA hydrogel is beneficial in reducing the early inflammatory response of laser-injured skin in mice and promoting its proliferation and angiogenesis, which in turn promotes wound healing.

Himatanthus bracteatus-Composed In Situ Polymerizable Hydrogel for Wound Healing.

The Himatanthus genus presents anti-inflammatory, antioxidant activities, suggesting potential wound-healing properties. This study aimed to develop and analyze the wound-healing properties of a photopolymerizable gelatin-based hydrogel (GelMA) containing an ethanolic extract of Himatanthus bracteatus in a murine model. The extract was obtained under high pressure conditions, incorporated (2%) into the GelMA (GelMA-HB), and physically characterized. The anti-inflammatory activity of the extract was assessed using a carrageenan-induced pleurisy model and the GelMA-HB scarring properties in a wound-healing assay. The extract reduced IL-1ß and TNF-α levels (48.5 ± 6.7 and 64.1 ± 4.9 pg/mL) compared to the vehicle (94.4 ± 2.3 pg/mL and 106.3 ± 5.7 pg/mL; p < 0.001). GelMA-HB depicted significantly lower swelling and increased resistance to mechanical compression compared to GelMA (p < 0.05). GelMA-HB accelerated wound closure over the time course of the experiment (p < 0.05) and promoted a significantly greater peak of myofibroblast differentiation (36.1 ± 6.6 cells) and microvascular density (23.1 ± 0.7 microvessels) on day 7 in comparison to GelMA (31.9 ± 5.3 cells and 20.2 ± 0.6 microvessels) and the control (25.8 ± 4.6 cells and 17.5 ± 0.5 microvessels) (p < 0.05). In conclusion, GelMA-HB improved wound healing in rodents, probably by modulating the inflammatory response and myofibroblastic and microvascular differentiation.

Carbon Dot-Based Photo-Cross-Linked Gelatin Methacryloyl Hydrogel Enables Dental Pulp Regeneration: A Preliminary Study.

An essential factor in tooth nutritional deficits and aberrant root growth is pulp necrosis. Removing inflammatory or necrotic pulp tissue and replacing it with an inert material are the most widely used therapeutic concepts of endodontic treatment. However, pulp loss can lead to discoloration, increased fracture risk, and the reinfection of the damaged tooth. It is now anticipated that the pulp-dentin complex will regenerate through a variety of application methods based on human dental pulp stem cells (hDPSC). In order to create a photo-cross-linked gelatinized methacrylate hydrogel, GelMA/EUO-CDs-E (ECE), that is biodegradable and injectable for application, we created a novel nanoassembly of ECE based on eucommia carbon dots (EUO-CDs) and epigallocatechin gallate (EGCG). We then loaded it onto gelatin methacryloyl (GelMA) hydrogel. We have evaluated the material and examined its in vivo and in vitro angiogenesis-promoting potential as well as its dentin differentiation-enabling characteristics. The outcomes of the experiment demonstrated that GelMA/ECE was favorable to cell proliferation and enhanced hDPSC's capacity for angiogenesis and dentin differentiation. The regeneration of vascular-rich pulp-like tissues was found to occur in vivo when hDPSC-containing GelMA/ECE was injected into cleaned human root segments (RS) for subcutaneous implantation in nude mice. This suggests that the injectable bioscaffold is appropriate for clinical use in pulp regenerative medicine.

Development of gelatin-methacryloyl composite carriers for bone morphogenetic Protein-2 delivery: A potential strategy for spinal fusion.

To reduce the risk of nonunion after spinal fusion surgery, the in situ transplantation of bone marrow mesenchymal stem cells (BMSCs) induced toward osteogenic differentiation by bone morphogenetic protein-2 (BMP2) has been proven effective. However, the current biological agents used for transplantation have limitations, such as a short half-life and low bioavailability. To address this, our study utilized a safe and effective gelatin-methacryloyl (GelMA) as a carrier for BMP2. In vitro, experiments were conducted to observe the ability of this composite vehicle to induce osteogenic differentiation of BMSCs. The results showed that the GelMA hydrogel, with its critical properties and controlled release performance of BMP2, exhibited a slow release of BMP2 over 30 days. Moreover, the GelMA hydrogel not only enhanced the proliferation activity of BMSCs but also significantly promoted their osteogenic differentiation ability, surpassing the BMP2 effects. To investigate the potential of the GelMA-BMP2 composite vehicle, a rabbit model was employed to explore its ability to induce in situ intervertebral fusion by BMSCs. Transplantation experiments in rabbits demonstrated the effective induction of intervertebral bone fusion by the GelMA-BMP2-BMSC composite vehicle. In conclusion, the GelMA-BMP2-BMSC composite vehicle shows promising prospects in preclinical translational therapy for spinal intervertebral fusion. It addresses the limitations of current biological agents and offers a controlled release of BMP2, enhancing the proliferation and osteogenic differentiation of BMSCs.

UV-polymerizable methacrylated gelatin (GelMA)-based hydrogel containing tannic acids for wound healing.

Gelatin-based photopolymerizable methacrylate hydrogel (GelMA) is a promising biomaterial for in situ drug delivery, while aqueous extract of Punica granatum (AEPG) peel fruit rich in gallic acid and ellagic acid is used to improve wound healing. The aim of this study was to develop and analyze the healing properties of GelMA containing AEPG, gallic acid, or ellagic acid in a rodent model. GelMA hydrogels containing 5% AEPG (GelMA-PG), 1.6% gallic acid (GelMA-GA), or 2.1% ellagic acid (GelMA-EA) were produced and their mechanical properties, enzymatic degradation, and thermogravimetric profile determined. Wound closure rates, healing histological grading, and immunohistochemical counts of myofibroblasts were assessed over time. The swelling of hydrogels varied between 50 and 90%, and GelMA exhibited a higher swelling than the other groups. The GPG samples showed higher compression and Young's moduli than GelMA, GGA, and GAE. All samples degraded around 95% in 48 h. GPG and GGA significantly accelerated wound closure, improved collagenization, increased histological grading, and hastened myofibroblast differentiation in comparison to the control, GelMA, and GEA. GelMA containing AEPG (GPG) improved wound healing, and although gallic acid is the major responsible for such biological activity, a potential synergic effect played by other polyphenols present in the extract is evident.

Gelatin methacryloyl hydrogel scaffold loaded with activated Schwann cells attenuates apoptosis and promotes functional recovery following spinal cord injury.

Spinal cord injury (SCI) is a refractory disease of the central nervous system with a high disability and incidence rate. In recent years, bioactive material combined with cell transplantation has been considered an effective method for the treatment of SCI. The present study encapsulated activated Schwann cells (ASCs) in a 3D gelatin methacryloyl (GelMA) hydrogel in order to investigate its therapeutic effects on SCI. ASCs were isolated from previously ligated rat sciatic nerves. Scanning electron microscopy and live/dead staining were used to evaluate the biocompatibility of hydrogels with the ASCs. The scaffold was transplanted into the spinal cord of rats in the hemisection model. Behavioral tests and hematoxylin and eosin staining were employed to assess the locomotion recovery and lesion areas before and after treatment. Cell apoptosis was evaluated using TUNEL staining and immunochemistry, and apoptosis-related protein expression was detected using western blot analysis. The ASCs exhibited a favorable survival and proliferative ability in the 3D GelMA hydrogel. The scaffold transplantation significantly reduced the cavities and improved functional recovery. Moreover, the GelMA/ASCs implants significantly inhibited cell apoptosis following SCI and this effect may be mediated via the p38 MAPK pathway. Overall, these findings indicated that ASCs combined with the 3D GelMA hydrogel may be a promising therapeutic strategy for SCI.

Preservation of Small Extracellular Vesicle in Gelatin Methacryloyl Hydrogel Through Reduced Particles Aggregation for Therapeutic Applications.

PURPOSE: Small extracellular vesicles (sEV) play an irreplaceable role in cell-cell communication. However, sEV in solution aggregate with each other during preservation, leading to impairment of the structures, contents, and functions of sEV. Therefore, there is a need to develop an optimal preservation method that combines high recovery rate, low cost, convenience, and easy-transportation in one. In this study, a new preservation strategy different from the cryopreservation or lyophilization was developed by reducing sEV particles aggregation. METHODS: The sEV were encapsulated in thermoresponsive gelatin methacryloyl (GelMA) hydrogels at 4°C to reduce particles aggregation during the reversible cross-linking process. The sEV movement was visualized in different mediums and particles' number, size, structure and protein of 28 days preserved sEV were compared to fresh sEV. Human umbilical vein endothelial cells (HUVEC) and rat adipose-derived stromal stem cells (rASC) were isolated and cultured with fresh and preserved sEV to test the cellular response. A mice subcutaneous model was adopted to detect controlled release and angiogenesis ability of preserved sEV. RESULTS: Through particles tracks visualization, GelMA hydrogels significantly decreased the sEV movement. After 28 days preservation in GelMA at 4°C, the particles number, size, structure and protein of sEV were similar to fresh sEV. In vitro, preserved sEV had the same ability to promote cell proliferation, migration and angiogenesis as fresh sEV. In vivo, preserved sEV-GelMA could artificially regulate the absorptivity of GelMA hydrogels and controlled released sEV for therapeutic application, and preserved sEV encapsulated in GelMA significantly promoted angiogenesis in mice. CONCLUSION: Our results demonstrated that sEV encapsulated in GelMA could be a novel strategy for long-term preservation of sEV for therapeutic application.

[Study on the gelatin methacryloyl composite scaffold with exogenous transforming growth factor ß 1 to promote the repair of skull defects].

OBJECTIVE: To prepare a bone tissue engineering scaffold for repairing the skull defect of Sprague Dawley (SD) rats by combining exogenous transforming growth factor ß 1 (TGF-ß 1) with gelatin methacryloyl (GelMA) hydrogel. METHODS: Firstly, GelMA hydrogel composite scaffolds containing exogenous TGF-ß 1 at concentrations of 0, 150, 300, 600, 900, and 1 200 ng/mL (set to groups A, B, C, D, E, and F, respectively) were prepared. Cell counting kit 8 (CCK-8) method was used to detect the effect of composite scaffold on the proliferation of bone marrow mesenchymal stem cells (BMSCs) in SD rats. ALP staining, alizarin red staining, osteocalcin (OCN) immunofluorescence staining, and Western blot were used to explore the effect of scaffolds on osteogenic differentiation of BMSCs, and the optimal concentration of TGF-ß 1/GelMA scaffold was selected. Thirty-six 8-week-old SD rats were taken to prepare a 5 mm diameter skull bone defect model and randomly divided into 3 groups, namely the control group, the GelMA group, and the GelMA+TGF-ß 1 group (using the optimal concentration of TGF-ß 1/GelMA scaffold). The rats were sacrificed at 4 and 8 weeks after operation, and micro-CT, HE staining, and OCN immunohistochemistry staining were performed to observe the repair effect of skull defects. RESULTS: The CCK-8 method showed that the TGF-ß 1/GelMA scaffolds in each group had a promoting effect on the proliferation of BMSCs. Group D had the strongest effect, and the cell activity was significantly higher than that of the other groups ( P<0.05). The results of ALP staining, alizarin red staining, OCN immunofluorescence staining, and Western blot showed that the percentage of ALP positive area, the percentage of alizarin red positive area, and the relative expressions of ALP and OCN proteins in group D were significantly higher than those of the other groups ( P<0.05), the osteogenesis effect in group D was the strongest. Therefore, in vitroexperiments screened out the optimal concentration of TGF-ß 1/GelMA scaffold to be 600 ng/mL. Micro-CT, HE staining, and OCN immunohistochemistry staining of rat skull defect repair experiments showed that the new bone tissue and bone volume/tissue volume ratio in the TGF-ß 1+GelMA group were significantly higher than those in the GelMA group and control group at 4 and 8 weeks after operation ( P<0.05). CONCLUSION: The TGF-ß 1/GelMA scaffold with a concentration of 600 ng/mL can significantly promote the osteogenic differentiation of BMSCs, can significantly promote bone regeneration at the skull defect, and can be used as a bioactive material for bone tissue regeneration.

Gelatin Methacryloyl Hydrogels Control the Localized Delivery of Albumin-Bound Paclitaxel.

Hydrogels are excellent candidates for the sustained local delivery of anticancer drugs, as they possess tunable physicochemical characteristics that enable to control drug release kinetics and potentially tackle the problem of systemic side effects in traditional chemotherapeutic delivery. Yet, current systems often involve complicated manufacturing or covalent bonding processes that are not compatible with regulatory or market reality. Here, we developed a novel gelatin methacryloyl (GelMA)-based drug delivery system (GelMA-DDS) for the sustained local delivery of paclitaxel-based Abraxane®, for the prevention of local breast cancer recurrence following mastectomy. GelMA-DDS readily encapsulated Abraxane® with a maximum of 96% encapsulation efficiency. The mechanical properties of the hydrogel system were not affected by drug loading. Tuning of the physical properties, by varying GelMA concentration, allowed tailoring of GelMA-DDS mesh size, where decreasing the GelMA concentration provided overall more sustained cumulative release (significant differences between 5%, 10%, and 15%) with a maximum of 75% over three months of release, identified to be released by diffusion. Additionally, enzymatic degradation, which more readily mimics the in vivo situation, followed a near zero-order rate, with a total release of the cargo at various rates (2-14 h) depending on GelMA concentration. Finally, the results demonstrated that Abraxane® delivery from the hydrogel system led to a dose-dependent reduction of viability, metabolic activity, and live-cell density of triple-negative breast cancer cells in vitro. The GelMA-DDS provides a novel and simple approach for the sustained local administration of anti-cancer drugs for breast cancer recurrence.

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

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

Visible Light Photoinitiation of Cell-Adhesive Gelatin Methacryloyl Hydrogels for Stereolithography 3D Bioprinting.

We present the first cell-attachable and visible-light-crosslinkable bioinks based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography three-dimensional (3D) bioprinting. To develop a visible-light-crosslinkable hydrogel, we systematically studied five combinations of GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentrations on the characteristics of the hydrogel. Experimental results show that the compressive Young's modulus and pore size are positively affected by the concentration of EY, whereas the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps in improving the compressive Young's modulus and cell attachment. We further employed the developed visible-light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.