Fabrication of GelMA - Agarose Based 3D Bioprinted Photocurable Hydrogel with In Vitro Cytocompatibility and Cells Mirroring Natural Keratocytes for Corneal Stromal Regeneration.

The complex anatomy of the cornea and the subsequent keratocyte-fibroblast transition have always made corneal stromal regeneration difficult. Recently, 3D printing has received considerable attention in terms of fabrication of scaffolds with precise dimension and pattern. In the current work, 3D printable polymer hydrogels made of GelMA/agarose are formulated and its rheological properties are evaluated. Despite the variation in agarose content, both the hydrogels exhibited G'>G'' modulus. A prototype for 3D stromal model is created using Solid Works software, mimicking the anatomy of an adult cornea. The fabrication of 3D-printed hydrogels is performed using pneumatic extrusion. The FTIR analysis speculated that the hydrogel is well crosslinked and established strong hydrogen bonding with each other, thus contributing to improved thermal and structural stability. The MTT analysis revealed a higher rate of cell proliferation on the hydrogels. The optical analysis carried out on the 14th day of incubation revealed that the hydrogels exhibit transparency matching with natural corneal stromal tissue. Specific protein marker expression confirmed the keratocyte phenotype and showed that the cells do not undergo terminal differentiation into stromal fibroblasts. The findings of this work point to the potential of GelMA/A hydrogels as a novel biomaterial for corneal stromal tissue engineering.

Matrix design for optimal pancreatic ß cells transplantation.

New therapeutic approaches to treat type 1 diabetes mellitus relies on pancreatic islet transplantation. Here, developing immuno-isolation strategies is essential to eliminate the need for systemic immunosuppression after pancreatic islet grafts. A solution is the macro-encapsulation of grafts in semipermeable matrixes with a double function: separating islets from host immune cells and facilitating the diffusion of insulin, glucose, and other metabolites. This study aims to synthesize and characterize different types of gelatin-collagen matrixes to prepare a macro-encapsulation device for pancreatic islets that fulfill these functions. While natural polymers exhibit superior biocompatibility compared to synthetic ones, their mechanical properties are challenging to reproduce. To address this issue, we conducted a comparative analysis between photo-crosslinked gelatin matrixes and chemically crosslinked collagen matrixes. We show that the different crosslinkers and polymerization methods influence the survival and glucose-stimulated insulin production of pancreatic ß cells (INS1) in vitro, as well as the in vitro and in vivo stability of the matrix and the immuno-isolation in vivo. Among the matrixes, the stiff multilayer GelMA matrixes (8.5 kPa), fabricated by digital light processing, were the best suited for pancreatic ß cells macro-encapsulation regarding these parameters. Within the alveoli of this matrix, pancreatic ß cells spontaneously formed aggregates.

Engineering Microgel Packing to Tailor the Physical and Biological Properties of Gelatin Methacryloyl Granular Hydrogel Scaffolds.

Granular hydrogel scaffolds (GHS) are fabricated via placing hydrogel microparticles (HMP) in close contact (packing), followed by physical and/or chemical interparticle bond formation. Gelatin methacryloyl (GelMA) GHS have recently emerged as a promising platform for biomedical applications; however, little is known about how the packing of building blocks, physically crosslinked soft GelMA HMP, affects the physical (pore microarchitecture and mechanical/rheological properties) and biological (in vitro and in vivo) attributes of GHS. Here, the GHS pore microarchitecture is engineered via the external (centrifugal) force-induced packing and deformation of GelMA HMP to regulate GHS mechanical and rheological properties, as well as biological responses in vitro and in vivo. Increasing the magnitude and duration of centrifugal force increases the HMP deformation/packing, decreases GHS void fraction and median pore diameter, and increases GHS compressive and storage moduli. MDA-MB-231 human triple negative breast adenocarcinoma cells spread and flatten on the GelMA HMP surface in loosely packed GHS, whereas they adopt an elongated morphology in highly packed GHS as a result of spatial confinement. Via culturing untreated or blebbistatin-treated cells in GHS, the effect of non-muscle myosin II-driven contractility on cell morphology is shown. In vivo subcutaneous implantation in mice confirms a significantly higher endothelial, fibroblast, and macrophage cell infiltration within the GHS with a lower packing density, which is in accordance with the in vitro cell migration outcome. These results indicate that the packing state of GelMA GHS may enable the engineering of cell response in vitro and tissue response in vivo. This research is a fundamental step forward in standardizing and engineering GelMA GHS microarchitecture for tissue engineering and regeneration.

Cost-effective and natural-inspired lotus root/GelMA scaffolds enhanced wound healing via ROS scavenging, angiogenesis and reepithelialization.

Skin wounds, prevalent and fraught with complications, significantly impact individuals and society. Wound healing encounters numerous obstacles, such as excessive reactive oxygen species (ROS) production and impaired angiogenesis, thus promoting the development of chronic wound. Traditional clinical interventions like hemostasis, debridement, and surgery face considerable challenges, including the risk of secondary infections. While therapies designed to scavenge excess ROS and enhance proangiogenic properties have shown effectiveness in wound healing, their clinical adoption is hindered by high costs, complex manufacturing processes, and the potential for allergic reactions. Lotus root, distinguished by its natural micro and macro porous architecture, exhibits significant promise as a tissue engineering scaffold. This study introduced a novel scaffold based on hybridization of lotus root-inspired and Gelatin Methacryloyl (GelMA), verified with satisfactory physicochemical properties, biocompatibility, antioxidative capabilities and proangiogenic abilities. In vivo tests employing a full-thickness wound model revealed that these scaffolds notably enhanced micro vessel formation and collagen remodeling within the wound bed, thus accelerating the healing process. Given the straightforward accessibility of lotus roots and the cost-effective production of the scaffolds, the novel scaffolds with ROS scavenging, pro-angiogenesis and re-epithelialization abilities are anticipated to have clinical applicability for various chronic wounds.

l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

Hydrogel encapsulation facilitates a low-concentration cryoprotectant for cryopreservation of mouse testicular tissue.

Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction.

Photo-Cross-linked Gelatin Methacryloyl Hydrogels Enable the Growth of Primary Human Endometrial Stromal Cells and Epithelial Gland Organoids.

In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.

Measurement of covalent bond formation in light-curing hydrogels predicts physical stability under flow.

Photocrosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring, and because stability is dependent on size of the patterned features. To solve both problems, we obtained 1H NMR spectra of cured hydrogels which were enzymatically degraded. This allowed us to take advantage of the high-resolution that solution NMR provides. This unique approach enabled the measurement of degree of crosslinking (DoC) and prediction of material stability under physiological fluid flow. We showed that NMR spectra of enzyme-digested gels successfully reported on DoC as a function of light exposure and wavelength within two classes of photocrosslinkable hydrogels: methacryloyl-modified gelatin and a composite of thiol-modified gelatin and norbornene-terminated polyethylene glycol. This approach revealed that a threshold DoC was required for patterned features in each material to become stable, and that smaller features required a higher DoC for stability. Finally, we demonstrated that DoC was predictive of the stability of architecturally complex features when photopatterning, underscoring the value of monitoring DoC when using light-reactive gels. We anticipate that the ability to quantify chemical crosslinks will accelerate the design of advanced hydrogel materials for structurally demanding applications such as photopatterning and bioprinting.

Dynamic composite hydrogels of gelatin methacryloyl (GelMA) with supramolecular fibers for tissue engineering applications.

In the field of tissue engineering, there is a growing need for biomaterials with structural properties that replicate the native characteristics of the extracellular matrix (ECM). It is important to include fibrous structures into ECM mimics, especially when constructing scar models. Additionally, including a dynamic aspect to cell-laden biomaterials is particularly interesting, since native ECM is constantly reshaped by cells. Composite hydrogels are developed to bring different combinations of structures and properties to a scaffold by using different types and sources of materials. In this work, we aimed to combine gelatin methacryloyl (GelMA) with biocompatible supramolecular fibers made of a small self-assembling sugar-derived molecule (N-heptyl-D-galactonamide, GalC7). The GalC7 fibers were directly grown in the GelMA through a thermal process, and it was shown that the presence of the fibrous network increased the Young's modulus of GelMA. Due to the non-covalent interactions that govern the self-assembly, these fibers were observed to dissolve over time, leading to a dynamic softening of the composite gels. Cardiac fibroblast cells were successfully encapsulated into composite gels for 7 days, showing excellent biocompatibility and fibroblasts extending in an elongated morphology, most likely in the channels left by the fibers after their degradation. These novel composite hydrogels present unique properties and could be used as tools to study biological processes such as fibrosis, vascularization and invasion.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

GelMA hydrogel dual photo-crosslinking to dynamically modulate ECM stiffness.

The dynamic nature of the extracellular matrix (ECM), particularly its stiffness, plays a pivotal role in cellular behavior, especially after myocardial infarction (MI), where cardiac fibroblasts (cFbs) are key in ECM remodeling. This study explores the effects of dynamic stiffness changes on cFb activation and ECM production, addressing a gap in understanding the dynamics of ECM stiffness and their impact on cellular behavior. Utilizing gelatin methacrylate (GelMA) hydrogels, we developed a model to dynamically alter the stiffness of cFb environment through a two-step photocrosslinking process. By inducing a quiescent state in cFbs with a TGF-ß inhibitor, we ensured the direct observation of cFbs-responses to the engineered mechanical environment. Our findings demonstrate that the mechanical history of substrates significantly influences cFb activation and ECM-related gene expression. Cells that were initially cultured for 24 h on the soft substrate remained more quiescent when the hydrogel was stiffened compared to cells cultured directly to a stiff static substrate. This underscores the importance of past mechanical history in cellular behavior. The present study offers new insights into the role of ECM stiffness changes in regulating cellular behavior, with significant implications for understanding tissue remodeling processes, such as in post-MI scenarios.

Injective hydrogel loaded with liposomes-encapsulated MY-1 promotes wound healing and increases tensile strength by accelerating fibroblast migration via the PI3K/AKT-Rac1 signaling pathway.

Failed skin wound healing, through delayed wound healing or wound dehiscence, is a global public health issue that imposes significant burdens on individuals and society. Although the application of growth factor is an effective method to improve the pace and quality of wound healing, the clinically approved factors are limited. Parathyroid hormone (PTH) demonstrates promising results in wound healing by promoting collagen deposition and cell migration, but its application is limited by potentially inhibitory effects when administered continuously and locally. Through partially replacing and repeating the amino acid domains of PTH(1-34), we previously designed a novel PTH analog, PTH(3-34)(29-34) or MY-1, and found that it avoided the inhibitory effects of PTH while retaining its positive functions. To evaluate its role in wound healing, MY-1 was encapsulated in liposomes and incorporated into the methacryloyl gelatin (GelMA) hydrogel, through which an injectable nanocomposite hydrogel (GelMA-MY@Lipo, or GML) was developed. In vitro studies revealed that the GML had similar properties in terms of the appearance, microstructure, functional groups, swelling, and degradation capacities as the GelMA hydrogel. In vitro drug release testing showed a relatively more sustainable release of MY-1, which was still detectable in vivo 9 days post-application. When the GML was topically applied to the wound areas of rat models, wound closure as well as tensile strength were improved. Further studies showed that the effects of GML on wound repair and tensile strength were closely related to the promotion of fibroblast migration to the wound area through the controlled release of MY-1. Mechanically, MY-1 enhanced fibroblast migration by activating PI3K/AKT signaling and its downstream molecule, Rac1, by which it increased fibroblast aggregation in the early stage and resulting in denser collagen deposition at a later time. Overall, these findings demonstrated that the nanocomposite hydrogel system promoted skin wound healing and increased tensile strength, thus offering new potential in the treatment of wound healing.

Photo-crosslinkable hydrogel incorporated with bone matrix particles for advancements in dentin tissue engineering.

The objective of this study was to create injectable photo-crosslinkable biomaterials, using gelatin methacryloyl (GelMA) hydrogel, combined with a decellularized bone matrix (BMdc) and a deproteinized (BMdp) bovine bone matrix. These were intended to serve as bioactive scaffolds for dentin regeneration. The parameters for GelMA hydrogel fabrication were initially selected, followed by the incorporation of BMdc and BMdp at a 1% (w/v) ratio. Nano-hydroxyapatite (nHA) was also included as a control. A physicochemical characterization was conducted, with FTIR analysis indicating that the mineral phase was complexed with GelMA, and BMdc was chemically bonded to the amide groups of gelatin. The porous structure was preserved post-BMdc incorporation, with bone particles incorporated alongside the pores. Conversely, the mineral phase was situated inside the pore opening, affecting the degree of porosity. The mineral phase did not modify the degradability of GelMA, even under conditions of type I collagenase-mediated enzymatic challenge, allowing hydrogel injection and increased mechanical strength. Subsequently, human dental pulp cells (HDPCs) were seeded onto the hydrogels. The cells remained viable and proliferative, irrespective of the GelMA composition. All mineral phases resulted in a significant increase in alkaline phosphatase activity and mineralized matrix deposition. However, GelMA-BMdc exhibited higher cell expression values, significantly surpassing those of all other formulations. In conclusion, our results showed that GelMA-BMdc produced a porous and stable hydrogel, capable of enhancing odontoblastic differentiation and mineral deposition when in contact with HDPCs, thereby showing potential for dentin regeneration.

Curcumin-shellac nanoparticle-loaded GelMA/SilMA hydrogel for colorectal cancer therapy.

In this study, a novel approach was employed to develop a therapeutic system for colorectal cancer treatment. Specifically, a GelMA/SilMA hydrogel loaded with curcumin-shellac nanoparticles (Cur@Lac NPs) was created. A microfluidic swirl mixer was utilized to formulate stable Cur@Lac NPs, ensuring their consistent and effective encapsulation. The pH-specific release of curcumin from the NPs demonstrated their potential for colon cancer treatment. By carefully regulating the ratio of GelMA (gelatin methacrylate) and SilMA (silk fibroin methacrylate), a GelMA/SilMA dual network hydrogel was generated, offering controlled release and degradation capabilities. The incorporation of SilMA notably enhanced the mechanical properties of the dual network matrix, improving compression resistance and mitigating deformation. This mechanical improvement is crucial for maintaining the structural integrity of the hydrogel during in vivo applications. In comparison to the direct incubation of curcumin, the strategy of encapsulating curcumin into NPs and embedding them within the GelMA/SilMA hydrogel resulted in more controlled release mechanisms. This controlled release was achieved through the disintegration of the NPs and the swelling and degradation of the hydrogel matrix. The encapsulating strategy also demonstrated enhanced cellular uptake of curcumin, leveraging the advantages of both NPs and in-situ hydrogel injection. This combination ensures a more efficient and sustained delivery of the therapeutic agent directly to the tumor site. Overall, this approach holds significant promise as a smart drug delivery system, potentially improving the efficacy of colorectal cancer treatments by providing targeted, controlled, and sustained drug release with enhanced mechanical stability and biocompatibility.

Gelatin Methacryloyl (GelMA) - 45S5 Bioactive Glass (BG) Composites for Bone Tissue Engineering: 3D Extrusion Printability and Cytocompatibility Assessment Using Human Osteoblasts.

3D extrusion printing has been widely investigated for low-volume production of complex-shaped scaffolds for tissue regeneration. Gelatin methacryloyl (GelMA) is used as a baseline material for the synthesis of biomaterial inks, often with organic/inorganic fillers, to obtain a balance between good printability and biophysical properties. The present study demonstrates how 45S5 bioactive glass (BG) addition and GelMA concentrations can be tailored to develop GelMA composite scaffolds with good printability and buildability. The experimental results suggest that 45S5 BG addition consistently decreases the compression stiffness, irrespective of GelMA concentration, albeit within 20% of the baseline scaffold (without 45S5 BG). The optimal addition of 2 wt % 45S5 BG in 7.5 wt % GelMA was demonstrated to provide the best combination of printability and buildability in the 3D extrusion printing route. The degradation decreases and the swelling kinetics increases with 45S5 BG addition, irrespective of GelMA concentration. Importantly, the dissolution in simulated body fluid over 3 weeks clearly promoted the nucleation and growth of crystalline calcium phosphate particles, indicating the potential of GelMA-45S5 BG to promote biomineralization. The cytocompatibility assessment using human osteoblasts could demonstrate uncompromised cell proliferation or osteogenic marker expression over 21 days in culture for 3D printable 7.5 wt % GelMA -2 wt % 45S5 BG scaffolds when compared to 7.5 wt % GelMA. The results thus encourage further investigations of the GelMA/45S5 BG composite system for bone tissue engineering applications.

Gelatin methacryloyl microneedle loaded with 3D-MSC-Exosomes for the protection of ischemia-reperfusion.

Exosomes (Exo) generated from mesenchymal stem cells (MSCs) have great therapeutic potential in ischemia-reperfusion treatment. For best therapeutic effect, high quality Exo product and effective delivery system are indispensable. In this study, we developed a new strategy for ischemia-reperfusion recovery by combining MSCs 3D (3D-MSC) culturing technology to generate Exo (3D-MSC-Exo) and microneedle for topical delivery. Firstly, primary MSCs from neonatal mice were isolated and 3D cultured with gelatin methacryloyl (GelMA) hydrogel to prepare 3D-MSC-Exo. The 3D-MSC showed better viability and 3D-MSC-Exo exhibited more effective effects of reducing neuroinflammation, inhibiting glial scarring, and promoting angiogenesis. Subsequently, the biocompatible GelMA was used to construct microneedles for 3D-Exo delivery (GelMA-MN@3D-Exo). The results demonstrated GelMA microneedles had excellent 3D-Exo loading capacity and enabled continuous 3D-Exo release to maintain effective therapeutic concentrations. Furthermore, the rat middle cerebral artery occlusion (MCAO) model was established to evaluate the therapeutic effect of GelMA-MN@3D-Exo in ischemia-reperfusion in vivo. Animal experiments showed that the GelMA-MN@3D-Exo system could effectively reduce the local neuroinflammatory reaction, promote angiogenesis and minimize glial scar proliferation in ischemia-reperfusion. The underlying reasons for the stronger neuroprotective effect of 3D-Exo was further studied using mass spectrometry and transcriptome assays, verifying their effects on immune regulation and cell proliferation. Taken together, our findings demonstrated that GelMA-MN@3D-Exo microneedle can effectively attenuate ischemia-reperfusion cell damage in the MCAO model, which provides a promising therapeutic strategy for ischemia-reperfusion recovery.

Electrofluidic control for textile-based cell culture: Identification of appropriate conditions required to integrate cell culture with electrofluidics.

Electric field-driven microfluidics, known as electrofluidics, is a novel attractive analytical tool when it is integrated with low-cost textile substrate. Textile-based electrofluidics, primarily explored on yarn substrates, is in its early stages, with few studies on 3D structures. Further, textile structures have rarely been used in cellular analysis as a low-cost alternative. Herein, we investigated novel 3D textile structures and develop optimal electrophoretic designs and conditions that are favourable for direct 3D cell culture integration, developing an integrated cell culture textile-based electrofluidic platform that was optimised to balance electrokinetic performance and cell viability requirements. Significantly, there were contrasting electrolyte compositional conditions that were required to satisfy cell viability and electrophoretic mobility requiring the development of and electrolyte that satisfied the minimum requirements of both these components within the one platform. Human dermal fibroblast cell cultures were successfully integrated with gelatine methacryloyl (GelMA) hydrogel-coated electrofluidic platform and studied under different electric fields using 5 mM TRIS/HEPES/300 mM glucose. Higher analyte mobility was observed on 2.5% GelMA-coated textile which also facilitated excellent cell attachment, viability and proliferation. Cell viability also increased by decreasing the magnitude and time duration of applied electric field with good cell viability at field of up to 20 V cm-1.

Lithium Promotes Osteogenesis via Rab11a-Facilitated Exosomal Wnt10a Secretion and ß-Catenin Signaling Activation.

Both bone mesenchymal stem cells (BMSCs) and their exosomes suggest promising therapeutic tools for bone regeneration. Lithium has been reported to regulate BMSC function and engineer exosomes to improve bone regeneration in patients with glucocorticoid-induced osteonecrosis of the femoral head. However, the mechanisms by which lithium promotes osteogenesis have not been elucidated. Here, we demonstrated that lithium promotes the osteogenesis of BMSCs via lithium-induced increases in the secretion of exosomal Wnt10a to activate Wnt/ß-catenin signaling, whose secretion is correlated with enhanced MARK2 activation to increase the trafficking of the Rab11a and Rab11FIP1 complexes together with exosomal Wnt10a to the plasma membrane. Then, we compared the proosteogenic effects of exosomes derived from lithium-treated or untreated BMSCs (Li-Exo or Con-Exo) both in vitro and in vivo. We found that, compared with Con-Exo, Li-Exo had superior abilities to promote the uptake and osteogenic differentiation of BMSCs. To optimize the in vivo application of these hydrogels, we fabricated Li-Exo-functionalized gelatin methacrylate (GelMA) hydrogels, which are more effective at promoting osteogenesis and bone repair than Con-Exo. Collectively, these findings demonstrate the mechanism by which lithium promotes osteogenesis and the great promise of lithium for engineering BMSCs and their exosomes for bone regeneration, warranting further exploration in clinical practice.

Screening of MMP-13 Inhibitors Using a GelMA-Alginate Interpenetrating Network Hydrogel-Based Model Mimicking Cytokine-Induced Key Features of Osteoarthritis In Vitro.

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. Current clinical treatment options lack effective pharmaceutical interventions targeting the disease's root causes. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, very few drugs within this class are in preclinical or clinical trial phases. Hydrogel-based 3D in vitro models have shown promise as preclinical testing platforms due to their resemblance to native extracellular matrix (ECM), abundant availability, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α). The results demonstrate a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. However, inconsistencies in human cartilage explant samples led to inconclusive results. Nonetheless, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening.

Integrating Graphene Oxide-Hydrogels and Electrical Stimulation for Controlled Neurotrophic Factor Encapsulation: A Promising Approach for Efficient Nerve Tissue Regeneration.

Nerve growth factor (NGF) plays a crucial role in cellular growth and neurodifferentiation. To achieve significant neuronal regeneration and repair using in vitro NGF delivery, spatiotemporal control that follows the natural neuronal processes must be developed. Notably, a challenge hindering this is the uncontrolled burst release from the growth factor delivery systems. The rapid depletion of NGF reduces treatment efficacy, leading to poor cellular response. To address this, we developed a highly controllable system using graphene oxygen (GO) and GelMA hydrogels modulated by electrical stimulation. Our system showed superior control over the release kinetics, reducing the burst up 30-fold. We demonstrate that the system is also able to sequester and retain NGF up to 10-times more efficiently than GelMA hydrogels alone. Our controlled release system enabled neurodifferentiation, as revealed by gene expression and immunostaining analysis. The increased retention and reduced burst release from our system show a promising pathway for nerve tissue engineering research toward effective regeneration.