Properties Of Extracellular Matrix Research Articles

Hyaluronic acid hydrogel to modulate tumor cell clustering and programmed death ligand 1 signaling in cancer stem cells.

e14584 Background: The root of malignancy lies in the extracellular matrix (ECM) surrounding the stromal cells. Thus, current therapeutic targets manipulate abnormal tumor microenvironments (TME), which nourish cancer stem cells (CSC) and induce tumor cell clustering (TCC). Notably, despite the first line of clinical therapy with surgery and chemotherapy, uncontrolled self-renewal of CSCs, conversion of non-CSCs to CSCs, and TCC trigger cancer recurrence. The transmembrane glycoprotein CD44 is thought to be a potential master regulator in cancer recurrence as it interacts with hyaluronic acid (HA) in TME, expresses aberrantly on CSCs, induces TCC by homophilic interactions like Plakoglobin despite less understanding on their crosstalk, and promotes expression of pro-tumorigenic factor programmed death ligand 1 (PD-L1). Notably, CD44 is expressed in multiple isoforms, and splice isoform switching of CD44 dictates the properties of CSC, which still needs to be clarified. Hence, we aimed to characterize CSCs based on CD44 isoform expression, investigate the crosstalk between CD44 and Plakoglobin, and then design a novel injectable HA hydrogel that can disguise TME and suppress PD-L1 to combat cancer recurrence. Methods: To characterize CSCs, we prepared different scaffolds of HA of varied molecular weights and other ECM proteins, cultured cancer cells (MDA-MB-231, HeLa), and determined the expression of CD44 isoforms. We sorted CD44+/CD24- expressing CSC by flow cytometry. We implemented immunoprecipitation to detect the interaction between CD44 and Plakoglobin. We created a novel HA hydrogel composed of low and very high molecular weight HA, thiolated gelatin, and other components crosslinked with PEGDA. Using a torsional rheometer, we measured matrix stiffness. We assessed the cell proliferation efficiency of CSC by a colony-forming assay. Results: The study disclosed that CD44 isoforms expressed differently based on ECM properties. Matrix stiffness dictated CD44 isoform expression. We observed that CD44 interacted with Plakoglobin. CD44 ablation upregulated Plakoglobin significantly. Tumor suppressor p53 played a significant role in CD44-Plakoglobin crosstalk. We observed that the injectable hydrogel modulated CD44-Plakoglobin signaling and inhibited TCC, likely promoting anoikis. The hydrogel affected CD44 splicing and modified the activity of the CD44 intracellular domain, which interacts with PD-L1 and thus suppressed PD-L1 expression significantly in CSC. Conclusions: Currently, antibodies targeting PD-1/PD-L1 signaling have been clinically approved for various cancers. However, this regimen of treatment is expensive. We report a novel injectable hydrogel that can disguise TME, modulate CSCs and PD-L1, and may advance an alternative cost-effective avenue for cancer immunotherapy.

Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3

Extracellular matrix (ECM) stiffening is a typical characteristic of cartilage aging, which is a quintessential feature of knee osteoarthritis (KOA). However, little is known about how ECM stiffening affects chondrocytes and other molecules downstream. This study mimicked the physiological and pathological stiffness of human cartilage using polydimethylsiloxane (PDMS) substrates. It demonstrated that epigenetic Parkin regulation by histone deacetylase 3 (HDAC3) represents a new mechanosensitive mechanism by which the stiffness matrix affected chondrocyte physiology. We found that ECM stiffening accelerated cultured chondrocyte senescence in vitro, while the stiffness ECM downregulated HDAC3, prompting Parkin acetylation to activate excessive mitophagy and accelerating chondrocyte senescence and osteoarthritis (OA) in mice. Contrarily, intra-articular injection with an HDAC3-expressing adeno-associated virus restored the young phenotype of the aged chondrocytes stimulated by ECM stiffening and alleviated OA in mice. The findings indicated that changes in the mechanical ECM properties initiated pathogenic mechanotransduction signals, promoted the Parkin acetylation and hyperactivated mitophagy, and damaged chondrocyte health. These results may provide new insights into chondrocyte regulation by the mechanical properties of ECM, suggesting that the modification of the physical ECM properties may be a potential OA treatment strategy.

Durable lymph-node expansion is associated with the efficacy of therapeutic vaccination.

Following immunization, lymph nodes dynamically expand and contract. The mechanical and cellular changes enabling the early-stage expansion of lymph nodes have been characterized, yet the durability of such responses and their implications for adaptive immunity and vaccine efficacy are unknown. Here, by leveraging high-frequency ultrasound imaging of the lymph nodes of mice, we report more potent and persistent lymph-node expansion for animals immunized with a mesoporous silica vaccine incorporating a model antigen than for animals given bolus immunization or standard vaccine formulations such as alum, and that durable and robust lymph-node expansion was associated with vaccine efficacy and adaptive immunity for 100 days post-vaccination in a mouse model of melanoma. Immunization altered the mechanical and extracellular-matrix properties of the lymph nodes, drove antigen-dependent proliferation of immune and stromal cells, and altered the transcriptional features of dendritic cells and inflammatory monocytes. Strategies that robustly maintain lymph-node expansion may result in enhanced vaccination outcomes.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ Tcells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ Tcells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ Tcell subset by coupled Tcell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ Tcells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ Tcell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ Tcell exhaustion and could be targeted to potentiate cancer immunotherapy.

Schiff base crosslinked hyaluronic acid hydrogels with tunable and cell instructive time-dependent mechanical properties

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.

Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization

The three-dimensional (3D) organization of cells determines tissue function and integrity, and changes markedly in development and disease. Cell-based simulations have long been used to define the underlying mechanical principles. However, high computational costs have so far limited simulations to either simplified cell geometries or small tissue patches. Here, we present SimuCell3D, an efficient open-source program to simulate large tissues in three dimensions with subcellular resolution, growth, proliferation, extracellular matrix, fluid cavities, nuclei and non-uniform mechanical properties, as found in polarized epithelia. Spheroids, vesicles, sheets, tubes and other tissue geometries can readily be imported from microscopy images and simulated to infer biomechanical parameters. Doing so, we show that 3D cell shapes in layered and pseudostratified epithelia are largely governed by a competition between surface tension and intercellular adhesion. SimuCell3D enables the large-scale in silico study of 3D tissue organization in development and disease at a great level of detail.

Active viscoelastic models for cell and tissue mechanics.

Living cells are out of equilibrium active materials. Cell-generated forces are transmitted across the cytoskeleton network and to the extracellular environment. These active force interactions shape cellular mechanical behaviour, trigger mechano-sensing, regulate cell adaptation to the microenvironment and can affect disease outcomes. In recent years, the mechanobiology community has witnessed the emergence of many experimental and theoretical approaches to study cells as mechanically active materials. In this review, we highlight recent advancements in incorporating active characteristics of cellular behaviour at different length scales into classic viscoelastic models by either adding an active tension-generating element or adjusting the resting length of an elastic element in the model. Summarizing the two groups of approaches, we will review the formulation and application of these models to understand cellular adaptation mechanisms in response to various types of mechanical stimuli, such as the effect of extracellular matrix properties and external loadings or deformations.

Application of hydrogel scaffolds as a cell substrate for cartilage tissue regeneration

Cell therapy or tissue engineering is currently one of the potentially effective strategies for repairing cartilage defects. Mesenchymal stromal cells and autologous and allogeneic chondrocytes obtained from cartilage biopsies from patients undergoing arthroscopic surgery are the most commonly used cells for tissue engineering. Matrices (scaffolds) often used during cell implantation can facilitate successful cell implantation. These can be static supporting or plastic hydrogel substances obtained by various methods from natural or synthetic materials, allowing cells to be delivered to the site damaged by injection. Significant advantages of biocompatible hydrogels in cartilage injury therapy are determined based on the properties of the natural extracellular matrix and the high plasticity required for adapting to irregular surfaces of the tissue defect and allowing injectable cell delivery. These characteristics make injectable hydrogels a promising tool for cartilage bioengineering.
 This study highlights the progress in injectable hydrogels as carriers of different cells in the repair of cartilage tissue defects, as well as the current requirements and unresolved issues related to the use of such therapeutic approaches.

Impact of TRP Channels on Extracellular Matrix Remodeling: Focus on TRPV4 and Collagen.

Disturbed remodeling of the extracellular matrix (ECM) is frequently observed in several high-prevalence pathologies that include fibrotic diseases of organs such as the heart, lung, periodontium, liver, and the stiffening of the ECM surrounding invasive cancers. In many of these lesions, matrix remodeling mediated by fibroblasts is dysregulated, in part by alterations to the regulatory and effector systems that synthesize and degrade collagen, and by alterations to the functions of the integrin-based adhesions that normally mediate mechanical remodeling of collagen fibrils. Cell-matrix adhesions containing collagen-binding integrins are enriched with regulatory and effector systems that initiate localized remodeling of pericellular collagen fibrils to maintain ECM homeostasis. A large cadre of regulatory molecules is enriched in cell-matrix adhesions that affect ECM remodeling through synthesis, degradation, and contraction of collagen fibrils. One of these regulatory molecules is Transient Receptor Potential Vanilloid-type 4 (TRPV4), a mechanically sensitive, Ca2+-permeable plasma membrane channel that regulates collagen remodeling. The gating of Ca2+ across the plasma membrane by TRPV4 and the consequent generation of intracellular Ca2+ signals affect several processes that determine the structural and mechanical properties of collagen-rich ECM. These processes include the synthesis of new collagen fibrils, tractional remodeling by contractile forces, and collagenolysis. While the specific mechanisms by which TRPV4 contributes to matrix remodeling are not well-defined, it is known that TRPV4 is activated by mechanical forces transmitted through collagen adhesion receptors. Here, we consider how TRPV4 expression and function contribute to physiological and pathological collagen remodeling and are associated with collagen adhesions. Over the long-term, an improved understanding of how TRPV4 regulates collagen remodeling could pave the way for new approaches to manage fibrotic lesions.

Combined Amniotic Membrane and Self-Powered Electrical Stimulator Bioelectronic Dress Promotes Wound Healing.

Human amniotic membranes (hAMs) are widely used as wound management biomaterials, especially as grafts for corneal reconstruction due to the structure of the extracellular matrix and excellent biological properties. However, their fragile nature and rapid degradation rate hinder widespread clinical use. In this work, we engineered a novel self-powered electronic dress (E-dress), combining the beneficial properties of an amniotic membrane and a flexible electrical electrode to enhance wound healing. The E-dress displayed a sustained discharge capacity, leading to increased epidermal growth factor (EGF) release from amniotic mesenchymal interstitial stem cells. Live/dead staining, CCK-8, and scratch-wound-closure assays were performed in vitro. Compared with amniotic membrane treatment alone, the E-dress promoted cell proliferation and migration of mouse fibroblast cells and lower cytotoxicity. In a mouse full-skin defect model, the E-dress achieved significantly accelerated wound closure. Histological analysis revealed that E-dress treatment promoted epithelialization and neovascularization in mouse skin. The E-dress exhibited a desirable flexibility that aligned with tissue organization and displayed maximum bioactivity within a short period to overcome rapid degradation, implying great potential for clinical applications.

Abstract 4268: Engineered hydrogel elucidates contributions of matrix mechanics to pathobiology of adenocarcinoma and identify matrix-activated therapeutic targets

Abstract Dysregulation of the extracellular matrix (ECM) properties disturbs tissue homeostasis and contributes to pathological conditions, such as cancer. Recent studies have demonstrated that ECM stiffening is associated with metaplasia-dysplasia-adenocarcinoma sequence in the esophagus. This underscores a compelling need to develop a physiologically-relevant 3D culture model to enable the study of the independent contribution of matrix stiffness to the initiation and progression of esophageal adenocarcinoma (EAC). Such a model will then serve as a platform for pre-clinical drug screening in the context of matrix properties. In this context, we have engineered a hyaluronic acid-based 3D hydrogel platform that supports the growth and differentiation of patient-derived Barrett’s esophagus (BE) organoids, a precursor to EAC, as well as patient-derived EAC organoids. The engineered biomaterial 3D platform allows control over matrix stiffness to better recapitulate the mechanically dynamic esophageal cancer microenvironment and help identify therapeutic targets in EAC.Our data demonstrates that BE and EAC organoid density, growth and proliferation can be controlled by matrix stiffness. RNA sequencing data show that increased matrix stiffness promotes changes in the transcriptional profiles of BE and EAC organoids, revealing enrichment of pathways associated with tumorigenesis and disease progression. Furthermore, we demonstrate through molecular and functional assays that increased matrix stiffness endows stem-like properties to the EAC organoids via Yap-Sox9 mechano-activation. Finally, targeted therapy studies in our in vitro and in vivo engineered environments revealed that Yap inhibition regressed the effects of increased matrix stiffness in EAC organoids. In summary, our data suggest that matrix mechanics have a significant role in activation of EAC-associated signaling pathways in patient-derived BE and EAC organoids. We also demonstrate that the engineered hydrogel serves as a platform to identify potential therapeutic targets to disrupt the contribution of pro-tumorigenic matrix mechanics in EAC. Together, these studies establish an engineered patient-derived organoid culture platform that can be used to elucidate underlying matrix-mediated mechanisms of the metaplasia-dysplasia-adenocarcinoma sequence and inform the development of novel therapeutics that target ECM stiffness in EAC. FUNDING SOURCES: NCI P01CA098101, U54 CA163004 and NIDDK K01 DK133620. Citation Format: Ricardo Cruz-Acuna, Secunda W. Kariuki, Kensuke Sugiura, Spyros Karaiskos, Eleanor M. Plaster, Claudia Loebel, Gizem Efe, Tatiana Karakasheva, Joel T. Gabre, Jianhua Hu, Jason A. Burdick, Anil K. Rustgi. Engineered hydrogel elucidates contributions of matrix mechanics to pathobiology of adenocarcinoma and identify matrix-activated therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4268.

Decellularized tissue exhibits large differences of extracellular matrix properties dependent on decellularization method: novel insights from a standardized characterization on skeletal muscle

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

3D micro/nano hydrogel structures fabricated by two-photon polymerization for biomedical applications.

Hydrogels are three-dimensional natural or synthetic cross-linked networks composed of polymer chains formed by hydrophilic monomers. Due to the ability to simulate many properties of natural extracellular matrix, hydrogels have been widely used in the biomedical field. Hydrogels can be obtained through a variety of polymerization strategies such as heating and redox. However, photochemistry is one of the most interesting methods for researchers in this field. Gelatin-methacryloyl (GelMA) inherits the biological activity of gelatin and has become one of the gold standards in the field of biomaterials. GelMA, as a photopolymerizable hydrogel precursor, can be used to fabricate 3D porous structures for biomedical applications through two-photon polymerization. We report a new formulation of GelMA-based photoresist and used it to manufacture a series of two-photon polymerization structures, with a maximum resolution less than 120nm. The influence of process parameters on 3D structures manufacturing is studied by adjusting the scanning speed, laser power, and layer spacing values in two-photon polymerization processing. In vitro biological tests show that the 3D hydrogel produced by two-photon polymerization in this paper is biocompatible and suitable for MC3T3-E1 cell.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics

Background: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. Purpose: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. Study Design: Controlled laboratory study. Methods: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. Results: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). Conclusion: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. Clinical Relevance: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Impact of the Different Chemical-Based Decellularization Protocols on the Properties of the Caprine Pericardium.

This study aims to decellularized caprine pericardium tissue with varied non-ionic surfactant and anionic detergent concentrations. Protocol A consists of 1%, 0.5%, and 0.25% (w/v) sodium dodecyl sulphate (SDS). Protocol B uses 1%, 0.5%, and 0.25% (w/v) Triton X-100. Protocol C comprised 0.5% SDS + 0.5% Triton X-100, 0.5% + 0.25%, and 0.25% SDS + 0.5% Triton X-100. Protocol B left a few countable cells in the pericardium tissue, but treatments A and C removed all cells. DNA quantification also demonstrated that protocol B had the most leftover DNA after decellularization. The pericardium tissue treated with an equal combination of anionic detergent and non-ionic surfactant preserves the matrix. However, changing the anionic detergent-non-ionic surfactant ratio disrupted the microstructure. Protocol A decreased pericardium tissue secant modulus (p < 0.05). Protocol B-treated pericardium tissue matched native tissue secant modulus and ultimate tensile stress. Protocol C strengthened pericardium tissue. The intact extracellular matrix and biomechanical properties like native tissues require optimal chemical doses and combinations.

The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell-nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell-substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell-matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

Hydrogel-Based Skin Regeneration.

The skin is subject to damage from the surrounding environment. The repair of skin wounds can be very challenging due to several factors such as severe injuries, concomitant infections, or comorbidities such as diabetes. Different drugs and wound dressings have been used to treat skin wounds. Tissue engineering, a novel therapeutic approach, revolutionized the treatment and regeneration of challenging tissue damage. This field includes the use of synthetic and natural biomaterials that support the growth of tissues or organs outside the body. Accordingly, the demand for polymer-based therapeutic strategies for skin tissue defects is significantly increasing. Among the various 3D scaffolds used in tissue engineering, hydrogel scaffolds have gained special significance due to their unique properties such as natural mimicry of the extracellular matrix (ECM), moisture retention, porosity, biocompatibility, biodegradability, and biocompatibility properties. First, this article delineates the process of wound healing and conventional methods of treating wounds. It then presents an examination of the structure and manufacturing methods of hydrogels, followed by an analysis of their crucial characteristics in healing skin wounds and the most recent advancements in using hydrogel dressings for this purpose. Finally, it discusses the potential future advancements in hydrogel materials within the realm of wound healing.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.