Hydrogel-based Matrix Research Articles

Matrigel-Encapsulated Articular cartilage derived fibronectin adhesion assay derived Chondroprogenitors for Enhanced Chondrogenic Differentiation: An In Vitro Evaluation

In cartilage research, three-dimensional (3D) culture models are pivotal for assessing chondrogenic differentiation potential. Standard pellet cultures, despite their utility, pose challenges like uneven differentiation and handling difficulties. This study explores the use of Matrigel, an extracellular matrix-based hydrogel, to encapsulate fibronectin adhesion assay-derived chondroprogenitors (FAA-CPs) and evaluate their chondrogenic differentiation potential. FAA-CPs, isolated from human articular cartilage and expanded to passage 2, were either polymerized in Matrigel or cultured as standard pellets. Both groups underwent chondrogenic differentiation for 28 days and osteogenic differentiation for 21 days. Comprehensive analyses included histological staining, gene expression (SOX-9, ACAN, COL2A1 for chondrogenesis; COL1A1, RUNX2, COL10A1 for osteogenesis), and biochemical assays for glycosaminoglycans (GAG) and Collagen type II. The results demonstrated that Matrigel-encapsulated FAA-CPs achieved greater GAG accumulation, as evidenced by enhanced Alcian Blue and Safranin O staining, compared to standard pellets. However, the Collagen type II deposition, both histologically and quantitatively, was reduced in Matrigel constructs. Gene expression analysis showed no significant differences in key chondrogenic and osteogenic markers between the two groups. Despite improved handling and GAG deposition, Matrigel did not enhance uniform chondrogenic differentiation nor offer significant benefits for osteogenic differentiation, showing comparable hypertrophic markers to the standard method. While Matrigel encapsulation offers advantages in handling and enhances GAG accumulation quantitatively, these benefits were not reflected in staining results. Furthermore, Matrigel did not significantly outperform standard pellet cultures in chondrogenic or osteogenic differentiation. These findings suggest a need for further refinement and in vivo validation.

Recapitulating Glioma Stem Cell Niches Using 3D Spheroid Models for Glioblastoma Research.

Glioblastoma multiforme (GBM) is among the most aggressive brain cancers, and it contains glioma stem cells (GSCs) that drive tumor initiation, progression, and recurrence. These cells resist conventional therapies, contributing to high recurrence rates in GBM patients. Developing in vitro models that mimic the tumor microenvironment (TME), particularly the GSC niche, is crucial for understanding GBM growth and therapeutic resistance. Three-dimensional (3D) spheroid models provide a more physiologically relevant approach than traditional two-dimensional (2D) cultures, recapitulating key tumor features like hypoxia, cell heterogeneity, and drug resistance. This review examines scaffold-free and scaffold-based methods for generating 3D GBM spheroids, focusing on their applications in studying the cancer stem cell niche. The discussion encompasses methods such as the hanging drop, low-adhesion plates, and magnetic levitation, alongside advancements in embedding spheroids within extracellular matrix-based hydrogels and employing 3D bioprinting to fabricate more intricate tumor models. These 3D culture systems offer substantial potential for enhancing our understanding of GBM biology and devising more effective targeted therapies.

Dissolvable microneedle-based wound dressing transdermally and continuously delivers anti-inflammatory and pro-angiogenic exosomes for diabetic wound treatment

Due to overactive inflammation and hindered angiogenesis, self-healing of diabetic wounds (DW) remains challenging in the clinic. Platelet-derived exosomes (PLT-Exos), a novel exosome capable of anti-inflammation and pro-angiogenesis, show great potential in DW treatment. However, previous administration of exosomes into skin wounds is topical daub or intradermal injection, which cannot intradermally deliver PLT-Exos into the dermis layer, thus impeding its long-term efficacy in anti-inflammation and pro-angiogenesis. Herein, a dissolvable microneedle-based wound dressing (PLT-Exos@ADMMA-MN) was developed for transdermal and long-term delivery of PLT-Exos. Firstly, a photo-crosslinking methacrylated acellular dermal matrix-based hydrogel (ADMMA-GEL), showing physiochemical tailorability, fast-gelling performance, excellent biocompatibility, and pro-angiogenic capacities, was synthesized as a base material of our dressing. For endowing the dressing with anti-inflammation and pro-angiogenesis, PLT-Exos were encapsulated into ADMMA-GEL with a minimum effective concentration determined by our in-vitro experiments. Then, in-vitro results show that this dressing exhibits excellent properties in anti-inflammation and pro-angiogenesis. Lastly, in-vivo experiments showed that this dressing could continuously and transdermally deliver PLT-Exos into skin wounds to switch local macrophage into M2 phenotype while stimulating neovascularization, thus proving a low-inflammatory and pro-angiogenic microenvironment for DW healing. Collectively, this study provides a novel wound dressing capable of suppressing inflammation and stimulating vascularization for DW treatment.

Omnidirectional elastic constraint-based embedded 3D printing in non-yield stress fluids for engineering complex tissues

Embedded 3D printing, relying on yield-stress fluid-based supporting baths, has significantly expanded our ability to create complex tissues and organs. However, many extracellular matrix-based hydrogels, despite their excellent biological performance, such as gelatin and hyaluronic acid (HA) hydrogels, lack yield-stress properties, limiting their use in embedded 3D printing and hindering functional maturation of complex tissue constructs. To address this challenge, we present a method for embedded printing in non-yield stress fluids using sequential printing in a reversible ink template (SPIRIT) strategy. By leveraging an omnidirectional elastic constraint provided by conventional supporting baths, we can precisely fabricate GelMA and HAMA hydrogels into functional tissue constructs with both intricate external structures and internal vessel systems, which cannot be achieved with extant embedded printing techniques. As a proof of concept, we successfully printed a complex ventricle model with high cell density and internal channels using GelMA hydrogel, which demonstrated high cell viability and synchronous beating performance. The omnidirectional elastic constraint-based SPIRIT technique holds promise for advancing organ printing with remarkable structural complexities and biomaterials affinity akin to natural tissue constructs by decoupling from yield-stress fluids.

Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture.Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings. Statement of significanceThe quest to improve treatments for kidney disease is crucial, given the challenges faced by patients on dialysis or waiting for transplants. Exciting new therapies combining biomaterials with cells can revolutionize kidney repair. In this study, researchers created a hydrogel from pig kidney. This gel could be used to deliver cells and other substances that help in kidney regeneration. Despite coming from pigs, it's safe for use in humans, with no harmful substances and reduced risk of immune reactions. Importantly, it promotes a balanced healing response in the body. This research not only advances our knowledge of kidney repair but also offers hope for more effective treatments for kidney diseases.

Cartilage Decellularized Extracellular Matrix-Based Hydrogel with Enhanced Tissue Adhesion and Promoted Chondrogenesis for Cartilage Tissue Engineering

Cartilage Decellularized Extracellular Matrix-Based Hydrogel with Enhanced Tissue Adhesion and Promoted Chondrogenesis for Cartilage Tissue Engineering

Bioactive decellularized extracellular matrix-based hydrogel supports human adipose tissue-derived stem cell maintenance and fibrocartilage phenotype.

Articular cartilage is a highly specialized tissue able to tolerate physical stress. However, its capacity for restoration is restricted, and injuries to the cartilage do not recover spontaneously. Interest in mesenchymal stem cells derived from human adipose tissue (hASCs) is growing due to their potential to improve tissue healing and recovery. Decellularized extracellular matrix (dECM)-based hydrogels combined with hASCs could serve as an interface for studying behavior and differentiation properties in a cartilage microenvironment. In the present study, we described the behavior of hASCs cultured in a commercial dECM MatriXpec™. The structural microtopography of MatriXpec™ was analyzed by scanning electron micrography, and its protein composition was accessed by mass spectrometry. The protein composition of MatriXpec™ is mainly represented by collagen proteins, building its fibrous ultrastructure. hASCs were cultured three-dimensionally (3D) on MatriXpec™ to perform cell viability, growth, and cartilage differentiation analysis. We showed that MatriXpec™ could be loaded with hASCs and that it supports cell maintenance for several days. We observed that the three-dimensional ultrastructure of the biomaterial is composed of nanofibers, and its protein composition reflects the tissue from which it was harvested. Finally, we showed that the molecular cues from the hydrogel are biologically active as these influence cell behavior and differentiation phenotype, increasing the expression of fibrocartilage-related genes such as SOX9, COL1, COL10, and MMP13. MatriXpec™ hydrogel can be used as an interface for 3D hASCs culture studies as it maintains cell viability and supports its differentiation process.

MODL-35. BIOENGINEERED HYDROGEL AND BLOOD BRAIN BARRIER-ON-A-CHIP SYSTEM TO STUDY GLIAL CELL ACTIVATION AND BLOOD BRAIN BARRIER DYSFUNCTION IN GLIOBLASTOMA

Abstract Treatments for glioblastoma (GBM) are limited leading to short patient life expectancies after diagnosis. Current preclinical models of GBM fail to accurately replicate the function of the blood brain barrier (BBB) and the extreme heterogeneity of the GBM tumor hindering the drug development process. Therefore, there is a pressing need for improved model systems to study how different GBM subpopulations interact with glial cells and the BBB. To build such a model, we first focused on designing a 3D extracellular matrix-based hydrogel biomaterial that would support human astrocytes and a functional BBB. The hydrogel chosen for these studies is composed of functionalized hyaluronic acid (HA) and gelatin and has similar mechanical properties to the human brain. Using confocal imaging and qPCR, we have shown that astrocytes encapsulated in this hydrogel have greater branching and express fewer markers of activation compared to astrocytes grown in commonly used 3D biomaterials such as collagen I and Matrigel. Treatment with inflammatory cytokines and GBM cell derived extracellular vesicles (EVs) revealed that astrocytes in this environment display the activation and inflammatory phenotypes observed in vivo in GBM. Next, this hydrogel biomaterial was deployed in a bioengineered BBB-on-a-chip. We have shown that this system supports the formation of 3D microvessel structures with human brain endothelial cells, pericytes, and astrocytes that accurately mimic the function of the human BBB. Studies using this BBB-on-a-chip have shown that invasive glioma stem cells alter BBB permeability influencing the transport of certain therapeutics to the tumor site. We are now beginning to test how various subpopulations of GBM tumor cells behave in this model and how changes to glial cells and the BBB impact drug delivery and efficacy. This study shows that engineered in vitro models can provide critical insight into disease mechanisms and inform treatment design in GBM.

Effect of Matrix Stiffness and Hepatocyte Growth Factor on Small Cell Lung Cancer Cells in Decellularized Extracellular Matrix-Based Hydrogels.

Small cell lung cancer (SCLC) is one of lethal cancers resulting in very low 5-year-survival rate. Although its clinical treatment largely relies on chemotherapy, SCLC cell physiology in three-dimenstional (3D) matrix has been less explored. In this work, the tumor microenvironment is reconstructed with decellularized porcine pulmonary extracellular matrix (dECM) with hyaluronic acid. To modulate matrix stiffness, the methacrylate groups are introduced into both dECM and hyaluronic acid, followed by photocrosslinking with photoinitiator. The stiffness of the resulting dECM-based hydrogel covers the stiffness of normal or cancerous tissue with varying dECM content. The proliferation and cancer stem cell marker expression of encapsulated SCLC cells are promoted in a compliant hydrogel matrix, which has a low shear modulus similar to that of the normal tissue. The hepatocyte growth factor (HGF) that induces SCLC cell invasion and chemoresistance markedly increases invasiveness and gene expression levels of CD44 and Sox2 in the hydrogel matrix. In addition, HGF treatment causes higher resistance against anticancer drugs (cisplatin and paclitaxel) in the 3D microenvironment. These findings indicate that malignant SCLC can be recapitulated in a pulmonary dECM-based matrix.

Biocomposite-based electrochemical chip for ethylene detection

This work introduces the integration of a biocomposite hydrogel with a microfabricated electrochemical chip, enabling a novel approach for detecting ethylene gas. Molybdate ions were impregnated into a chitosan matrix-based hydrogel, resulting in an ethylene-responsive interface that coated a miniaturized electrochemical chip. The electrochemical process involved the oxidation of molybdenum ions from Mo5+ to Mo6+, followed by their reduction back to Mo5+ due to ethylene complexation. This approach provides a sensitive and efficient method for ethylene detection. The composite-functionalized device was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy, demonstrating its sensitivity and selectivity for ethylene detection. This research offers a promising approach to developing efficient sensors for rapid ethylene detection, with potential applications in postharvest, food storage, and ripening, as well as plant growth and development monitoring.

Imageable AuNP-ECM Hydrogel Tissue Implants for Regenerative Medicine.

In myocardial infarction, a blockage in one of the coronary arteries leads to ischemic conditions in the left ventricle of the myocardium and, therefore, to significant death of contractile cardiac cells. This process leads to the formation of scar tissue, which reduces heart functionality. Cardiac tissue engineering is an interdisciplinary technology that treats the injured myocardium and improves its functionality. However, in many cases, mainly when employing injectable hydrogels, the treatment may be partial because it does not fully cover the diseased area and, therefore, may not be effective and even cause conduction disorders. Here, we report a hybrid nanocomposite material composed of gold nanoparticles and an extracellular matrix-based hydrogel. Such a hybrid hydrogel could support cardiac cell growth and promote cardiac tissue assembly. After injection of the hybrid material into the diseased area of the heart, it could be efficiently imaged by magnetic resonance imaging (MRI). Furthermore, as the scar tissue could also be detected by MRI, a distinction between the diseased area and the treatment could be made, providing information about the ability of the hydrogel to cover the scar. We envision that such a nanocomposite hydrogel may improve the accuracy of tissue engineering treatment.

Abstract LB113: Bio-printed 3D cell models and high-content imaging for testing anti-cancer compounds

Abstract The discovery and screening of anti-cancer drugs is a vast field that is constantly evolving. More advanced cell-based models are needed to have an efficient way of studying cellular and subcellular effects of compound treatments, especially in early drug discovery. 3D cellular models that more accurately represent various microenvironments are very important for accurate drug screening and disease modeling. However, the complexity of those models is a limiting factor for the wider adoption of 3D models for research and screening. To address this, we have developed an automated workflow that includes a multitool 3D printing/automation robot enabling printing cells in a hydrogel-based matrix and high-content imaging for the characterization of phenotypic effects of compounds. We demonstrate examples of several bio-printed 3D cell models that can be used for testing anti-cancer drugs. For the development of 3D cellular models, we used VitroGel/VitroInk, xeno-free (animal origin-free) bio-functional hydrogel matrices. Hydrogel matrices with cells were used for printing 3D cellular patterns in 96 well plates, or dispensing cells in hydrogel “domes”. We have utilized a variety of widely used cancer lines HCT-116, HELA, HepG2, and MCF-7, as well as patient-derived triple-negative breast cancer cell line 4IC. Cells mixed with hydrogels/inks were dispensed/printed into a 96-well format using the multi-tool robotic platform, BioAssemblyBot. The platform enabled efficient dispensing/printing of cells into domes, lines, or other patterns. This assay was used for compound testing and evaluation of the anti-cancer effects of various drugs. The integrated system included a high-content confocal imager (IXM-C) and enabled automated seeding, bioprinting, liquid handling, plate transferring, as well as high-content imaging. The 3D bioprinted cells were monitored daily using imaging in transmitted light. In 2-3 days, cells formed spheroids in the matrix. Those were treated with a panel of anti-cancer drugs including doxorubicin, cytarabine, taxol, mitomycin, romidepsin, cisplatin, trametinib, and other compounds with various modes of action. Cells were treated with compounds for 72h, then stained and imaged for the endpoint measurements. 3D models were stained with viability dyes including Calcein AM, EthD, and Hoechst stain to evaluate numbers of live, dead, and total cells, then imaged using the IXM-C confocal imaging system. Image analysis allowed the characterization of cytostatic and cytotoxic effects of various compounds measuring the impact of compounds on cell proliferation, number of spheroids, spheroid size, cell death, and apoptosis. Different measurements, including spheroid size, and numbers of live and dead cells were used to determine EC50s for different compounds and cell types. The results showed the feasibility of 3D cellular models bioprinted with ECM matrices for anti-cancer drug screening workflows. An increase in throughput and ease of operation was achieved through automation. Also, imaging and data analysis methods provided valuable information about complex compound effects in 3D printed and cell-tissue-engineered cancer models. Citation Format: Prathyushakrishna Macha, John Huang, Zhisong Tong, James B. Hoying, Oksana Sirenko. Bio-printed 3D cell models and high-content imaging for testing anti-cancer compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB113.

A follow-up study on “A sensitive determination of morphine in plasma using AuNPs@UiO-66/PVA hydrogel as an advanced optical scaffold”

A double solvent-assisted approach was developed for the preparation of AuNPs@UiO-66 based polyvinyl alcohol hydrogel nanocomposite and evaluated its potential as a nanoprobe for the determination of morphine. The characterization and morphology of the synthesized platform were studied and performance comparison for morphine determination was done between the synthesized scaffold and the reported one in our previous work and discussed in detail. Due to the encapsulation of AuNPs inside UiO-66 in a double solvent-assisted approach, no energy transfer was performed with UiO-66 and finally, morphine could not bind with AuNPs. Given these values, such a hydrogel-based matrix prepared with different methodologies with the same thermal stability demonstrates dissimilar potential toward morphine determination in biological samples.

Three-dimensional printing of microfiber- reinforced hydrogel loaded with oxymatrine for treating spinal cord injury.

Spinal cord injury (SCI) causes severe neural tissue damage and motor/sensory dysfunction. Since the injured spinal cord tissue has limited self-regeneration ability, several strategies, including cell therapy, drug delivery, and tissue engineering scaffold implantation, have been employed to treat SCI. However, each of these strategies fails to obtain desirable outcomes due to their respective limitations. In comparison, advanced tissue engineering scaffolds with appropriate topographical features, favorable composition, and sustained drug delivery capability can be employed to recruit endogenous neural stem cells (NSCs), induce neuronal differentiation, and facilitate neuron maturation. This can lead to the regeneration of injured spinal cord tissue and the recovery of motor function. In this study, fiber bundle-reinforced spinal cord extracellular matrix hydrogel scaffolds loaded with oxymatrine (OMT) were produced through nearfield direct write electrospinning. The spinal cord extracellular matrix-based hydrogel was then coated with OMT. The physical/chemical properties and in vitro degradation behavior of the composite scaffolds were investigated. The in vitro cell culture results showed that composite scaffolds loaded with OMT promoted the differentiation of NSCs into neurons and inhibited differentiation into astrocytes. The in vivo results showed that the composite scaffolds loaded with OMT recruited NSCs from the host tissue, promoted neuronal differentiation and axon extension at the lesion site, inhibited glial scar formation at/around the lesion site, and improved the recovery of motor function in rats with SCI. To sum up, 3D-printed microfiber-reinforced spinal cord extracellular matrix hydrogel scaffolds loaded with OMT are promising biomaterials for the treatment of SCI.

Bio-inspired conductive adhesive based on calcium-free alginate hydrogels for bioelectronic interfaces

Electrode impedance is one of the greatest challenges facing neural interfacing medical devices and the use of electrical stimulation-based therapies in the fields of neurology and regenerative medicine. Maximizing contact between electronics and tissue would allow for more accurate recordings of neural activity and to stimulate with less power in implantable devices as electric signals could be more precisely transferred by a stable interfacial area. Neural environments, inherently wet and ion-rich, present a unique challenge for traditional conductive adhesives. As such, we look to marine mussels that use a 3,4-dihydroxyphenyl-L-analine (DOPA)-containing proteinaceous excretion to adhere to a variety of substrates for inspiration. By functionalizing alginate, which is an abundantly available natural polymer, with the catechol residues DOPA contains, we developed a hydrogel-based matrix to which carbon-based nanofiller was added to render it conductive. The synthesized product had adhesive energy within the range of previously reported mussel-based polymers, good electrical properties and was not cytotoxic to brain derived neural precursor cells.

The Rheology and Printability of Cartilage Matrix-Only Biomaterials.

The potential chondroinductivity from cartilage matrix makes it promising for cartilage repair; however, cartilage matrix-based hydrogels developed thus far have failed to match the mechanical performance of native cartilage or be bioprinted without adding polymers for reinforcement. There is a need for cartilage matrix-based hydrogels with robust mechanical performance and paste-like precursor rheology for bioprinting/enhanced surgical placement. In the current study, our goals were to increase hydrogel stiffness and develop the paste-like precursor/printability of our methacryl-modified solubilized and devitalized cartilage (MeSDVC) hydrogels. We compared two methacryloylating reagents, methacrylic anhydride (MA) and glycidyl methacrylate (GM), and varied the molar excess (ME) of MA from 2 to 20. The MA-modified MeSDVCs had greater methacryloylation than GM-modified MeSDVC (20 ME). While GM and most of the MA hydrogel precursors exhibited paste-like rheology, the 2 ME MA and GM MeSDVCs had the best printability (i.e., shape fidelity, filament collapse). After crosslinking, the 2 ME MA MeSDVC had the highest stiffness (1.55 ± 0.23 MPa), approaching the modulus of native cartilage, and supported the viability/adhesion of seeded cells for 15 days. Overall, the MA (2 ME) improved methacryloylation, hydrogel stiffness, and printability, resulting in a stand-alone MeSDVC printable biomaterial. The MeSDVC has potential as a future bioink and has future clinical relevance for cartilage repair.

Antioxidant-biocompatible and stable catalase-based gelatin\u2013alginate hydrogel scaffold with thermal wound healing capability: immobilization and delivery approach

Hydrogel-based matrix prepared using biopolymers is a new frontier of emerging platforms for enzyme immobilization for biomedical applications. Catalase (CAT) delivery can be effective in inhibiting reactive oxygen species (ROS)-mediated prolongation of the wound healing process. In this study, to improve CAT stability for effective application, gelatin(Gel)–alginate (Alg) biocompatible hydrogel (Gel–Alg), as immobilization support, was prepared using calcium chloride as an ionic cross-linker. High entrapment efficiency of 92% was obtained with 2% Gel and 1.5% Alg. Hydrogel immobilized CAT (CAT–Gel–Alg) showed a wide range of pH from 4 to 9 and temperature stability between 20 to 60 °C, compared to free CAT. CAT–Gel–Alg kinetic parameters revealed an increased Km (24.15 mM) and a decreased Vmax (1.39 µmol H2O2/mg protein min) × 104. CAT–Gel–Alg retained 52% of its original activity after 20 consecutive catalytic runs and displayed improved thermal stability with a higher t1/2 value (half-life of 100.43 vs. 46 min). In addition, 85% of the initial activity was maintained after 8 weeks’ storage at 4 °C. At 24 h after thermal injury, a statistically significant difference in lesion sizes between the treated group and the control group was reported. Finally, our findings suggest that the superior CAT–Gel–Alg stability and reusability are resonant features for efficient biomedical applications, and ROS scavenging by CAT in the post-burn phase offers protection for local treatment of burned tissues with encouraging wound healing kinetics.

Ovarian extracellular matrix-based hydrogel for human ovarian follicle survival in vivo: A pilot work.

To successfully assemble a bio-engineered ovary, we need to create a three-dimensional matrix able to accommodate isolated follicles and cells. The goal of this study was to develop an extracellular matrix hydrogel (oECM) derived from decellularized bovine ovaries able to support, in combination with alginate, human ovarian follicle survival and growth in vitro. Two different hydrogels (oECM1, oECM2) were produced and compared in terms of decellularization efficiency (dsDNA), ECM preservation (collagen and glycosaminoglycan levels), ultrastructure, rigidity, and cytotoxicity. oECM2 showed significantly less dsDNA, greater retention of glycosaminoglycans and better rigidity than oECM1. Isolated human ovarian follicles were then encapsulated in four selected hydrogel combinations: (1) 100% oECM2, (2) 90% oECM2 + 10% alginate, (3) 75% oECM2 + 25% alginate, and (4) 100% alginate. After 1 week of in vitro culture, follicle recovery rate, viability, and growth were analyzed. On day 7 of in vitro culture, follicle recovery rates were 0%, 23%, 65%, 82% in groups 1-4, respectively, rising proportionally with increased alginate content. However, there was no difference in follicle viability or growth between groups 2 and 3 and controls (group 4). In conclusion, since pure alginate cannot be used to graft preantral follicles due to its poor revascularization and degradation after grafting, oECM2 hydrogel combined with alginate may provide a new and promising alternative to graft isolated human follicles in a bio-engineered ovary.

An injectable, self-healing, electroconductive extracellular matrix-based hydrogel for enhancing tissue repair after traumatic spinal cord injury

Injectable biomaterial-based treatment is a promising strategy to enhance tissue repair after traumatic spinal cord injury (SCI) by bridging cavity spaces. However, there are limited reports of injectable, electroconductive hydrogels with self-healing properties being employed for the treatment of traumatic SCI. Hence, a natural extracellular matrix (ECM) biopolymer (chondroitin sulphate and gelatin)-based hydrogel containing polypyrrole, which imparted electroconductive properties, is developed for traumatic SCI repair. The resulting hydrogels showed mechanical (~928 Pa) and conductive properties (4.49 mS/cm) similar to natural spinal cord tissues. Moreover, the hydrogels exhibited shear-thinning and self-healing abilities, which allows it to be effectively injected into the injury site and to fill the lesion cavity to accelerate the tissue repair of traumatic SCI. In vitro, electroconductive ECM hydrogels promoted neuronal differentiation, enhanced axon outgrowth, and inhibited astrocyte differentiation. The electroconductive ECM hydrogel activated endogenous neural stem cell neurogenesis in vivo (n = 6), and induced myelinated axon regeneration into the lesion site via activation of the PI3K/AKT and MEK/ERK pathways, thereby achieving significant locomotor function restoration in rats with spinal cord injury (p < 0.001, compared to SCI group). Overall, the injectable self-healing electroconductive ECM-based hydrogels developed in this study are ideal biomaterials for treatment of traumatic SCI.

Incorporating a structural extracellular matrix gradient into a porcine urinary bladder matrix-based hydrogel dermal scaffold.

The increasing prevalence of chronic, nonhealing wounds necessitates the investigation of full-thickness skin substitutes conducive to host integration and wound closure. Extracellular matrix (ECM)-based hydrogel scaffolds mimic the physiological matrix environment of dermal cells, thereby conferring favorable cellular adhesion, infiltration, and proliferation. However, low-concentration ECM hydrogels rapidly lose mechanical strength as they degrade, leaving them susceptible to shrinkage from fibroblast-mediated contraction. Conversely, high-concentration ECM hydrogels are typically too dense to permit nutrient diffusion and cellular migration. This study investigates the design and fabrication of a graded-concentration hydrogel composed of porcine urinary bladder matrix (UBM) as a dermal scaffold for potential use in chronic wound treatment. Our method of UBM isolation and decellularization effectively removed native DNA while preserving matrix proteins. Hydrogels composed of a range of decellularized UBM (dUBM) concentrations were characterized and used to design a three-tiered gradient hydrogel that promoted cellular activity and maintained structural integrity. The gradient dUBM hydrogel showed stability of cross-sectional area during collagenase degradation, despite considerable loss of mass. The gradient dUBM hydrogel also resisted fibroblast-mediated contraction while supporting high surface cell viability, demonstrating the mechanical support provided by denser layers of dUBM. Overall, incorporation of an ECM concentration gradient into a porcine UBM-based hydrogel scaffold capitalizes on the unique advantages of both high and low-concentration ECM hydrogels, and mitigates the structural weaknesses that have limited the efficacy of hydrogel dermal scaffolds for chronic wounds. Our gradient design shows promise for future development of stable, pro-regenerative wound scaffolds with customized architectures using 3D printing.