bims-ecemfi 2024-08-18 papers

bims-ecemfi

Biomed News

on ECM and fibroblasts

Issue of 2024–08–18
thirty-two papers selected by
Badri Narayanan Narasimhan, University of California, San Diego

Degradability tunes ECM stress relaxation and cellular mechanics.
Matrix stiffness increases energy efficiency of endothelial cells.
Exploration of Curvature and Stiffness Dual-Regulated Breast Cancer Cell Motility by a Motor-Clutch Model and Cell Traction Force Characterization.
An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells.
Protocol for in vitro evaluation of effects of stiffness on patient-derived glioblastoma.
Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing.
Collagen-rich liver-derived extracellular matrix hydrogels augment survival and function of primary rat liver sinusoidal endothelial cells and hepatocytes.
Local photo-crosslinking of native tissue matrix regulates cell function.
Spatial regulation of substrate adhesion directs fibroblast morphotype and phenotype.
Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system.
3D culture of ovarian follicles in granular and nanofibrillar hydrogels.
Interplay of chromatin organization and mechanics of the cell nucleus.
Bone-derived extracellular matrix hydrogel from thrombospondin-2 knock-out mice for bone repair.
Insights gained from computational modeling of YAP/TAZ signaling for cellular mechanotransduction.
Acrylate-Based PEG Hydrogels with Ultrafast Biodegradability for 3D Cell Culture.
Macroporous PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates Prepared via Radical-Free Cross-Linking for Cartilage Tissue Engineering.
Material Properties Of The Embryonic Small Intestine During Buckling Morphogenesis.
YAP1 Inhibition Induces Phenotype Switching of Cancer-Associated Fibroblasts to Tumor Suppressive in Prostate Cancer.
Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient.
CHD4 R975H mutant activates tumorigenic pathways and promotes stemness and M2-like macrophage polarization in endometrial cancer.
Nuclear α-actinin-4 regulates breast cancer invasiveness and EMT.
A nuclear jamming transition in vertebrate organogenesis.
Time and Space Modulation of Substrate Curvature to Regulate Cell Mechanical Identity.
Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor.
Two-dimensional vascularized liver organoid on extracellular matrix with defined stiffness for modeling fibrotic and normal tissues.
Methods to achieve tissue-mimetic physicochemical properties in hydrogels for regenerative medicine and tissue engineering.
Engineering synthetic agonists for targeted activation of Notch signaling.
Oscillatory hypoxia induced gene expression predicts low survival in human breast cancer patients.
Integrative single-cell analysis of human colorectal cancer reveals patient stratification with distinct immune evasion mechanisms.
Augmentation of Tendon and Ligament Repair with Fiber-Reinforced Hydrogel Composites.
Magneto-Enzymatic Microgels for Precise Hydrogel Sculpturing.
Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

bioRxiv. 2024 Jul 29. pii: 2024.07.28.605514. [Epub ahead of print]

Degradability tunes ECM stress relaxation and cellular mechanics.

Badri Narayanan Narasimhan, Stephanie I Fraley.

In native extracellular matrices (ECM), cells can use matrix metalloproteinases (MMPs) to degrade and remodel their surroundings. Likewise, synthetic matrices have been engineered to facilitate MMP-mediated cleavage that enables cell spreading, migration, and interactions. However, the intersection of matrix degradability and mechanical properties has not been fully considered. We hypothesized that immediate mechanical changes result from the action of MMPs on the ECM and that these changes are sensed by cells. Using atomic force microscopy (AFM) to measure cell-scale mechanical properties, we find that both fibrillar collagen and synthetic degradable matrices exhibit enhanced stress relaxation after MMP exposure. Cells respond to these relaxation differences by altering their spreading and focal adhesions. We demonstrate that stress relaxation can be tuned through the rational design of matrix degradability. These findings establish a fundamental link between matrix degradability and stress relaxation, which may impact a range of biological applications.

DOI: https://doi.org/10.1101/2024.07.28.605514
Matrix Biol. 2024 Aug 13. pii: S0945-053X(24)00106-9. [Epub ahead of print]

Matrix stiffness increases energy efficiency of endothelial cells.

Curtis T Schunk, Wenjun Wang, Lindsey N Sabo, Paul V Taufalele, Cynthia A Reinhart-King.

  To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis.

Keywords:  ATP; cell contractility; energy efficiency; extracellular matrix stiffness; traction stress

DOI:  https://doi.org/10.1016/j.matbio.2024.08.004
ACS Appl Mater Interfaces. 2024 Aug 14.

Exploration of Curvature and Stiffness Dual-Regulated Breast Cancer Cell Motility by a Motor-Clutch Model and Cell Traction Force Characterization.

Cong Wang, Zhao Xu, Xingquan Ma, Yuting Yin, Bo Cheng, Yuqing Dong.

  The migration of breast cancer cells is the main cause of death and significantly regulated by physical factors of the extracellular matrix (ECM). To be specific, the curvature and stiffness of the ECM were discovered to effectively guide cell migration in velocity and direction. However, it is not clear what the extent of effect is when these dual-physical factors regulate cell migration. Moreover, the mechanobiology mechanism of breast cancer cell migration in the molecular level and analysis of cell traction force (CTF) are also important, but there is a lack of systematic investigation. Therefore, we employed a microfluidic platform to construct hydrogel microspheres with an independently adjustable curvature and stiffness as a three-dimensional substrate for breast cancer cell migration. We found that the cell migration velocity was negatively correlated to curvature and positively correlated to stiffness. In addition, curvature was investigated to influence the focal adhesion expression as well as the assignment of F-actin at the molecular level. Further, with the help of a motor-clutch mathematical model and hydrogel microsphere stress sensors, it was concluded that cells perceived physical factors (curvature and stiffness) to cause changes in CTF, which ultimately regulated cell motility. In summary, we employed a theoretical model (motor-clutch) and experimental strategy (stress sensors) to understand the mechanism of curvature and stiffness regulating breast cancer cell motility. These results provide evidence of force driven cancer cell migration by ECM physical factors and explain the mechanism from the perspective of mechanobiology.

Keywords:  breast cancer metastasis; cell migration; cell traction force; curvature and stiffness; mechanobiology mechanism

DOI:  https://doi.org/10.1021/acsami.4c09615
Curr Biol. 2024 Aug 06. pii: S0960-9822(24)00944-8. [Epub ahead of print]

An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells.

Bailey N Warder, Kara A Nelson, Justin Sui, Lauren Anllo, Stephen DiNardo.

  Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

Keywords:  Drosophila; actomyosin contractility; feedback; morphogenesis; niche; stem cell; testis

DOI:  https://doi.org/10.1016/j.cub.2024.07.041
STAR Protoc. 2024 Aug 14. pii: S2666-1667(24)00431-3. [Epub ahead of print]5(3): 103266

Protocol for in vitro evaluation of effects of stiffness on patient-derived glioblastoma.

Alireza Sohrabi, Stephanie K Seidlits.

  Glioblastoma (GBM) is the most common and lethal type of primary brain tumor. Physiologically, GBM cells experience a heterogeneous mechanical landscape. Here, we present an in vitro method to study the effects of tissue stiffness on patient-derived GBM that utilizes hyaluronic acid (HA)-based, mechanically tunable scaffolds for three-dimensional (3D) culture of patient-derived GBM spheroids. We describe steps to fabricate and characterize HA-based scaffolds, culture GBM spheroids within 3D hydrogel scaffolds, and prepare cultured cells for a variety of experimental assessments. For complete details on the use and execution of this protocol, please refer to Sohrabi et al.1.

Keywords:  Cancer; Material sciences; Tissue Engineering

DOI:  https://doi.org/10.1016/j.xpro.2024.103266
Nat Commun. 2024 Aug 09. 15(1): 6820

Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing.

Sergei Butenko, Raji R Nagalla, Christian F Guerrero-Juarez, Francesco Palomba, Li-Mor David, Ronald Q Nguyen, Denise Gay, Axel A Almet, Michelle A Digman, Qing Nie, Philip O Scumpia, Maksim V Plikus, Wendy F Liu.

Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.

DOI: https://doi.org/10.1038/s41467-024-50072-y
Int J Biol Macromol. 2024 Aug 12. pii: S0141-8130(24)05522-3. [Epub ahead of print]278(Pt 2): 134717

Collagen-rich liver-derived extracellular matrix hydrogels augment survival and function of primary rat liver sinusoidal endothelial cells and hepatocytes.

Junyu Wang, Fenghua Zhao, Linda A Brouwer, Manon Buist-Homan, Justina C Wolters, Han Moshage, Martin C Harmsen.

  Liver sinusoidal endothelial cells (LSECs) are key targets for addressing metabolic dysfunction-associated steatotic liver disease (MASLD). However, isolating and culturing primary LSECs is challenging due to rapid dedifferentiation, resulting in loss of function. The extracellular matrix (ECM) likely plays a crucial role in maintaining the fate and function of LSECs. In this study, we explored the influence of liver-ECM (L-ECM) on liver cells and developed culture conditions that maintain the differentiated function of liver cells in vitro for prolonged periods. Porcine liver-derived L-ECM, containing 34.9 % protein, 0.045 % glycosaminoglycans, and negligible residual DNA (41.2 ng/mg), was utilized to culture primary rat liver cells in generated hydrogels. Proteomic analyses and molecular weight distribution of proteins of solubilized L-ECM revealed the typical diverse ECM core matrisome, with abundant collagens. L-ECM hydrogels showed suitable stiffness and stress relaxation properties. Furthermore, we demonstrated that collagen-rich L-ECM hydrogels enhanced LSECs' and hepatocytes' viability, and reduced the dedifferentiation rate of LSECs. In addition, hepatocyte function was maintained longer by culture on L-ECM hydrogels compared to traditional culturing. These beneficial effects are likely attributed to the bioactive macromolecules including collagens, and mechanical and microarchitectural properties of the L-ECM hydrogels.

Keywords:  Biological/mechanical/microarchitectural properties; Extracellular matrix; Liver sinusoidal endothelial cells; Metabolic dysfunction-associated steatotic liver disease

DOI:  https://doi.org/10.1016/j.ijbiomac.2024.134717
bioRxiv. 2024 Aug 10. pii: 2024.08.10.607417. [Epub ahead of print]

Local photo-crosslinking of native tissue matrix regulates cell function.

Donia W Ahmed, Matthew L Tan, Jackson Gabbard, Yuchen Liu, Michael M Hu, Miriam Stevens, Firaol S Midekssa, Lin Han, Rachel L Zemans, Brendon M Baker, Claudia Loebel.

Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.

DOI: https://doi.org/10.1101/2024.08.10.607417
PNAS Nexus. 2024 Aug;3(8): pgae289

Spatial regulation of substrate adhesion directs fibroblast morphotype and phenotype.

Mirko D'Urso, Ignasi Jorba, Atze van der Pol, Carlijn V C Bouten, Nicholas A Kurniawan.

  The switching of the fibroblast phenotype to myofibroblast is a hallmark of a wide variety of tissue pathologies. This phenotypical switch is known to be influenced not only by humoral factors such as TGF-β, but also by mechanical and physical cues in the cellular environment, and is accompanied by distinctive changes in cell morphology. However, the causative link between these cues, the concomitant morphological changes, and the resulting phenotypic switch remain elusive. Here, we use protein micropatterning to spatially control dermal fibroblast adhesion without invoking exogenous mechanical changes and demonstrate that varying the spatial configuration of focal adhesions (FAs) is sufficient to direct fibroblast phenotype. We further developed an automated morphometry analysis pipeline, which revealed FA eccentricity as the primary determinant of cell-state positioning along the spectrum of fibroblast phenotype. Moreover, linear fibronectin patterns that constrain the FAs were found to promote a further phenotype transition, characterized by dispersed expression of alpha-smooth muscle actin, pointing to an interesting possibility of controlling fibroblast phenotype beyond the canonical fibroblast-myofibroblast axis. Together, our study reveals that the spatial configuration of adhesion to the cellular microenvironment is a key factor governing fibroblast morphotype and phenotype, shedding new light on fibroblast phenotype regulation.

Keywords:  fibroblast; focal adhesions; morphometry; myofibroblast; phenotype transition

DOI:  https://doi.org/10.1093/pnasnexus/pgae289
Sci Rep. 2024 08 14. 14(1): 18851

Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system.

M Mohamadian Namaqi, F Moll, S Wiedemeier, A Grodrian, K Lemke.

  The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.

Keywords:  Cancer cell migration; Colon cancer; Dynamic 3D cell culture

DOI:  https://doi.org/10.1038/s41598-024-69261-2
Biomater Adv. 2024 Aug 03. pii: S2772-9508(24)00230-9. [Epub ahead of print]164 213987

3D culture of ovarian follicles in granular and nanofibrillar hydrogels.

Marko Mihajlovic, Dóra Katalin Pásztor-Jánoska, Jesús Cadenas, Cristina Subiran Adrados, Claus Yding Andersen, Stine Gry Kristensen, Johan Ulrik Lind.

  3D culture of ovarian follicles in hydrogel matrices is an important emerging tool for basic scientific studies as well as clinical applications such as fertility preservation. For optimizing and scaling 3D culture of preantral follicles, there is a need for identifying biomaterial matrices that simplifies and improves the current culture procedures. At present, microencapsulation of follicles in alginate beads is the most commonly used approach. However, this technique involves notable manual handling and is best suited for encapsulation of single or several follicles. As a potential alternative, we here explore the suitability of different particle-based hydrogel matrices, where follicles can easily be introduced in tunable 3D environments, in large numbers. Specifically, we study the growth of secondary murine follicles in microgranular alginate and nanofibrillar cellulose matrices, with and without cell-binding cues, and map follicle growth against the viscoelastic properties of the matrices. We cultured follicles within the particle-based hydrogels for 10 days and continuously monitored their size, survival, and tendency to extrude oocytes. Interestingly, we observed that the diameter of the growing follicles increased significantly in the particle-based matrices, as compared to state-of-the-art alginate micro-encapsulation. On the other hand, the follicles displayed an increased tendency for early oocyte extrusion in the granular matrices, leading to a notable reduction in the number of intact follicles. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. Still, our findings suggest that viscoelastic, granular hydrogels represent promising matrices for 3D culture of early-stage ovarian follicles. In particular, these materials may easily be implemented in advanced culturing devices such as micro-perfusion systems.

Keywords:  3D cell culture; Biomaterials; Matrices; Microgranular hydrogels; Nanofibers; Ovarian follicles; Reproductive biology; Scaffolds

DOI:  https://doi.org/10.1016/j.bioadv.2024.213987
Biophys J. 2024 Aug 08. pii: S0006-3495(24)00525-3. [Epub ahead of print]

Interplay of chromatin organization and mechanics of the cell nucleus.

Marco De Corato, Maria Jose Gomez-Benito.

The nucleus of eukaryotic cells is constantly subjected to different kinds of mechanical stimuli, which can impact the organization of chromatin and, subsequently, the expression of genetic information. Experiments from different groups showed that nuclear deformation can lead to transient or permanent condensation or decondensation of chromatin and to the mechanical activation of genes, thus altering the transcription of proteins. Changes in chromatin organization, in turn, change the mechanical properties of the nucleus, possibly leading to an auxetic behavior. Here, we model the mechanics of the nucleus as a chemically-active polymer gel in which the chromatin can exist in two states: a self-attractive state representing the heterochromatin and a repulsive state representing euchromatin. The model predicts reversible or irreversible changes in chromatin condensation levels upon external deformations of the nucleus. We find an auxetic response for a broad range of parameters under small and large deformations. These results agree with experimental observations and highlight the key role of chromatin organization in the mechanical response of the nucleus.

DOI: https://doi.org/10.1016/j.bpj.2024.08.003
Acta Biomater. 2024 Aug 10. pii: S1742-7061(24)00455-0. [Epub ahead of print]

Bone-derived extracellular matrix hydrogel from thrombospondin-2 knock-out mice for bone repair.

Zhuoyue Chen, Junqi Zhang, Francis Y Lee, Themis R Kyriakides.

  Bone extracellular matrix (ECM) has been shown to mimic aspects of the tissue's complex microenvironment, suggesting its potential role in promoting bone repair. However, current ECM-based therapies suffer from limitations such as inefficient scale-up, lack of mechanical integrity, and sub-optimal efficacy. Here, we fabricated hydrogels from decellularized ECM (dECM) from wild type (WT) and thrombospondin-2 knockout (TSP2KO) mouse bones. TSP2KO bone ECM hydrogel was found to have distinct mechanical properties and collagen fibril assembly from WT. Furthermore, TSP2KO hydrogel promoted mesenchymal stem cell (MSC) attachment, spreading, and invasion in vitro. Similarly, it promoted formation of tube-like structures by human umbilical vein endothelial cells (HUVECs). When applied to a murine calvarial defect model, TSP2KO hydrogel enhanced repair, in part, due to increased angiogenesis. Our study suggests the pro-angiogenic therapeutic potential of TSP2KO bone ECM hydrogel in bone repair. STATEMENT OF SIGNIFICANCE: The study describes the first successful preparation of a novel hydrogel made from decellularized mouse bones. Bones from wild-type mice and mice lacking thrombospondin 2 (TSP2) were used to fabricate the gels. Hydrogels from TSP2KO bones have unique characteristics in structure and biomechanics. These gels interacted well with cells in vitro and helped repair damaged bone in a mouse model. Therefore, TSP2KO bone-derived hydrogel has translational potential for accelerating repair of bone defects that are otherwise difficult to heal. This study not only creates a new material with promise for healing, but also validates tunability of native biomaterials by genetic engineering.

Keywords:  Bone repair; angiogenesis; extracellular matrix; hydrogel; thrombospondin

DOI:  https://doi.org/10.1016/j.actbio.2024.08.011
NPJ Syst Biol Appl. 2024 Aug 15. 10(1): 90

Insights gained from computational modeling of YAP/TAZ signaling for cellular mechanotransduction.

Hamidreza Jafarinia, Ali Khalilimeybodi, Jorge Barrasa-Fano, Stephanie I Fraley, Padmini Rangamani, Aurélie Carlier.

YAP/TAZ signaling pathway is regulated by a multiplicity of feedback loops, crosstalk with other pathways, and both mechanical and biochemical stimuli. Computational modeling serves as a powerful tool to unravel how these different factors can regulate YAP/TAZ, emphasizing biophysical modeling as an indispensable tool for deciphering mechanotransduction and its regulation of cell fate. We provide a critical review of the current state-of-the-art of computational models focused on YAP/TAZ signaling.

DOI: https://doi.org/10.1038/s41540-024-00414-9
Biomacromolecules. 2024 Aug 13.

Acrylate-Based PEG Hydrogels with Ultrafast Biodegradability for 3D Cell Culture.

Zhili Li, Dezhong Zhou.

Poly(ethylene glycol) (PEG)-based hydrogels are particularly challenging to degrade, which hinders efficient cell harvesting within the gel matrix. Here, highly branched copolymers of PEG methyl ether acrylate (PEGMA) and disulfide diacrylate (DSDA) (PEG-DS) with short primary chains and multiple pendent vinyl groups were synthesized by a "vinyl oligomer combination" approach. PEG-DS readily cross-links with thiolated gelatin (Gel-SH) to form hydrogels. Results demonstrate that shortening the primary chains of PEG-DS significantly enhances the viability of bone marrow mesenchymal stem cells (BMSCs) by up to 193.2%. Importantly, DS junctions can be easily cleaved into short primary chains using dithiothreitol (DTT), triggering ultrafast degradation of PEG-DS/Gel-SH hydrogels within 2 min under mild conditions and release of the encapsulated BMSCs. This study establishes a novel strategy to enhance the degradation of acrylate-based PEG hydrogels for three-dimensional (3D) cell culture and harvesting. These findings expand the potential applications of such hydrogels in various biomedical fields.

DOI: https://doi.org/10.1021/acs.biomac.4c01051
ACS Appl Bio Mater. 2024 Aug 13.

Macroporous PEG-Alginate Hybrid Double-Network Cryogels with Tunable Degradation Rates Prepared via Radical-Free Cross-Linking for Cartilage Tissue Engineering.

Kaixiang Zhang, Zining Yang, Michael Patrick Seitz, Era Jain.

  Trauma or repeated damage to joints can result in focal cartilage defects, significantly elevating the risk of osteoarthritis. Damaged cartilage has an inherently limited self-healing capacity and remains an urgent unmet clinical need. Consequently, there is growing interest in biodegradable hydrogels as potential scaffolds for the repair or reconstruction of cartilage defects. Here, we developed a biodegradable and macroporous hybrid double-network (DN) cryogel by combining two independently cross-linked networks of multiarm polyethylene glycol (PEG) acrylate and alginate.Hybrid DN cryogels are formed using highly biocompatible click reactions for the PEG network and ionic bonding for the alginate network. By judicious selection of various structurally similar cross-linkers to form the PEG network, we can generate hybrid DN cryogels with customizable degradation kinetics. The resulting PEG-alginate hybrid DN cryogels have an interconnected macroporous structure, high mechanical strength, and rapid swelling kinetics. The interconnected macropores in the cryogels support efficient mesenchymal stem cell infiltration at a high density. Finally, we demonstrate that PEG-alginate hybrid DN cryogels allow sustained release of chondrogenic growth factors and support chondrogenic differentiation of mouse mesenchymal stem cells. This study provides a novel method to generate macroporous hybrid DN cryogels with customizable degradation rates and a potential scaffold for cartilage tissue engineering.

Keywords:  cartilage tissue engineering; click reaction; cryogels; double network; hydrogels

DOI:  https://doi.org/10.1021/acsabm.4c00091
bioRxiv. 2024 Aug 09. pii: 2024.08.07.606927. [Epub ahead of print]

Material Properties Of The Embryonic Small Intestine During Buckling Morphogenesis.

Jenny Gao, Lucia Martin, Elise A Loffet, John F Durel, Panagiotis Oikonomou, Nandan L Nerurkar.

During embryonic development, tissues undergo dramatic deformations as functional morphologies are stereotypically sculpted from simple rudiments. Formation of healthy, functional organs therefore requires tight control over the material properties of embryonic tissues during development, yet the biological basis of embryonic tissue mechanics is poorly understood. The present study investigates the mechanics of the embryonic small intestine, a tissue that is compactly organized in the body cavity by a mechanical instability during development, wherein differential elongation rates between the intestinal tube and its attached mesentery create compressive forces that buckle the tube into loops with wavelength and curvature that are tightly conserved for a given species. Focusing on the intestinal tube, we combined micromechanical testing with histologic analyses and enzymatic degradation experiments to conclude that elastic fibers closely associated with intestinal smooth muscle layers are responsible for the bending stiffness of the tube, and for establishing its pronounced mechanical anisotropy. These findings provide insights into the developmental role of elastic fibers in controlling tissue stiffness, and raise new questions on the physiologic function of elastic fibers in the intestine during adulthood.
GRAPHICAL ABSTRACT:

DOI: https://doi.org/10.1101/2024.08.07.606927
Cancer Res. 2024 Aug 13.

YAP1 Inhibition Induces Phenotype Switching of Cancer-Associated Fibroblasts to Tumor Suppressive in Prostate Cancer.

Hongtao Song, Tong Lu, Donghui Han, Jiayu Zhang, Lunbiao Gan, Chao Xu, Shaojie Liu, Peng Li, Keying Zhang, Zhihao Hu, Hongji Li, Yu Li, Xiaolong Zhao, Jingliang Zhang, Nianzeng Xing, Changhong Shi, Weihong Wen, Fa Yang, Weijun Qin.

Prostate cancer (PCa) rarely responds to immune-checkpoint blockade (ICB) therapies. Cancer-associated fibroblasts (CAFs) are critical components of the immunologically "cold" tumor microenvironment and are considered a promising target to enhance the immunotherapy response. In this study, we aimed to reveal the mechanisms regulating CAF plasticity to identify potential strategies to switch CAFs from pro-tumorigenic to anti-tumor phenotypes and enhance ICB efficacy in PCa. Integration of four PCa single-cell RNA-sequencing datasets defined pro-tumorigenic and anti-tumor CAFs, and RNA-seq, flow cytometry, and a PCa organoid model demonstrated the functions of two CAF subtypes. Extracellular matrix-associated CAFs (ECM-CAF) promoted collagen deposition and cancer cell progression, and lymphocyte-associated CAFs (Lym-CAF) exhibited an anti-tumor phenotype and induced the infiltration and activation of CD8+ T cells. YAP1 activity regulated the ECM-CAF phenotype, and YAP1 silencing promoted switching to Lym-CAFs. NF-κB p65 was the core transcription factor in the Lym-CAF subset, and YAP1 inhibited nuclear translocation of p65. Selective depletion of YAP1 in ECM-CAFs in vivo promoted CD8+ T-cell infiltration and activation and enhanced the therapeutic effects of anti- PD-1 treatment in PCa. Overall, this study revealed a mechanism regulating CAF identity in PCa and highlighted a therapeutic strategy for altering the CAF subtype to suppress tumor growth and increase sensitivity to ICB.

DOI: https://doi.org/10.1158/0008-5472.CAN-24-0932
Biofabrication. 2024 Aug 09.

Droplet-based microfluidics for engineering shape-controlled hydrogels with stiffness gradient.

Bram Soliman, Ian Chin, Yi Wei Li, Melissa Ishii, Minh Hieu Ho, Vinh Khanh Doan, Thomas Cox, Peng-Yuan Wang, Gabriella C J Lindberg, Yu Shrike Zhang, Tim B F Woodfield, Yu Suk Choi, Khoon S Lim.

  Current biofabrication strategies are limited in their ability to replicate native shape-to-function relationships, that are dependent on adequate biomimicry of shape, size and spatial heterogeneity, within cell-laden hydrogels. In this study, a diffusion-based microfluidics platform is presented that meets these needs in a two-step process. In the first step, a hydrogel-precursor solution is dispersed into a continuous oil phase within the microfluidics tubing. By adjusting the dispersed and oil phase flow rates, the physical architecture of hydrogel-precursor phases can be adjusted to generate spherical and plug-like structures, as well as continuous meter-long hydrogel-precursor phases (up to 1.75 m). The second step involves the controlled introduction a small molecule-containing aqueous phase through a T-shaped tube connector to enable controlled small molecule diffusion across the interface of the aqueous phase and hydrogel-precursor. Application of this system is demonstrated by diffusing co-initiator sodium persulfate (SPS) into hydrogel-precursor solutions, where the controlled SPS diffusion into the hydrogel-precursor and subsequent photo-polymerization allows for the formation of unique radial stiffness patterns across the shape- and size-controlled hydrogels, as well as allowing the formation of hollow hydrogels with controllable internal architectures. Mesenchymal stromal cells are successfully encapsulated within hollow hydrogels and hydrogels containing radial stiffness gradient. The cells are observed to respond to the microscale spatial heterogeneity as evidenced by increased cell elongation in softer core regions of the hydrogel as compared to the peripheral stiffer hydrogel regions, as well as stiffness-dependent nuclear accumulation of the yes-associated protein mechano-regulator. Finally, breast cancer cells are found to phenotypically switch in response to stiffness gradients, causing a shift in their ability to aggregate, which may have implications for metastasis. The diffusion-based microfluidics will mimic native shape-to-function relationship and provides a platform to further study the roles of micro- and macroscale architectural features that exist within native tissues.

Keywords:  Architecture; Diffusion; Gradient; Hydrogels; Microfluidics; bioprinting

DOI:  https://doi.org/10.1088/1758-5090/ad6d8e
Sci Rep. 2024 08 10. 14(1): 18617

CHD4 R975H mutant activates tumorigenic pathways and promotes stemness and M2-like macrophage polarization in endometrial cancer.

Qinglin Zhang, Fengzhi Zhu, Yin Tong, Diwen Shi, Jiangwen Zhang.

Endometrial cancer (EC), one of the most prevalent carcinomas in females, is associated with increasing mortality. We identified the CHD4 R975H mutation as a high-frequency occurrence in EC patients through a comprehensive survey of EC databases. Computational predictions suggest that this mutation profoundly impacts the structural and functional integrity of CHD4. Functional assays revealed that the CHD4 R975H mutation enhances EC cell invasion, proliferation, and colony formation, promoting a cancer stem cell (CSC)-like phenotype. RNA-seq analysis of cells expressing CHD4 R975H mutant revealed a transcriptomic landscape marked by the activation of several cancer-promoting signaling pathways, including TNF-α signaling via NF-κB, KRAS, P53, mTOR, TGF-β, EGFR, Myc and growth factor signaling. Validation assays confirmed the activation of these pathways, further demonstrating that CHD4 R975H mutation induces stemness in EC cells and M2-like polarization of tumor-associated macrophages (TAMs). Our study elucidated the oncogenic role of CHD4 R975H mutation, highlighting its dual impact on facilitating cancer stemness and transforming TAMs into an immunosuppressive subtype. These findings contribute valuable insights into the molecular mechanisms driving EC progression and open avenues for targeted therapeutic interventions.

DOI: https://doi.org/10.1038/s41598-024-69233-6
Cytoskeleton (Hoboken). 2024 Aug 15.

Nuclear α-actinin-4 regulates breast cancer invasiveness and EMT.

Sumon Kumar Saha, Madhurima Sarkar, Mahima Srivastava, Sarbajeet Dutta, Shamik Sen.

  Epithelial-to-mesenchymal transition (EMT) is a key process where cells lose their adhesion properties and augment their invasive properties. α-Actinin4 (ACTN4) is an actin crosslinking protein that responds to mechanical stimuli and is found to be elevated in breast cancer patients. While ACTN4 has been implicated in regulating cancer invasiveness by modulating cytoskeletal organization, its nuclear functions remain much less explored. Here we address this question by first establishing a correlation between nuclear localization and invasiveness in breast cancer cells. Using cancer databases, we then establish a correlation between ACTN4 expression and EMT in breast cancer. Interestingly, TGFβ-induced EMT induction in MCF10A normal mammary epithelial cells leads to increased ACTN4 expression and nuclear enrichment. We then show that ACTN4 knockdown in MDA-MB-231 breast cancer cells, which harbor sizeable fraction of nuclear ACTN4, leads to reduced invasiveness and loss of mesenchymal traits. Similar behavior was observed in knockdown cells expressing K255E ACTN4, which is primarily localized to the cytosol. Together, our findings establish a role for nuclear ACTN4 in regulating invasiveness via modulation of EMT.

Keywords:  ACTN4; EMT; K255E; TGFβ; invasiveness

DOI:  https://doi.org/10.1002/cm.21901
Nat Mater. 2024 Aug 12.

A nuclear jamming transition in vertebrate organogenesis.

Sangwoo Kim, Rana Amini, Shuo-Ting Yen, Petr Pospíšil, Arthur Boutillon, Ilker Ali Deniz, Otger Campàs.

Jamming of cell collectives and associated rigidity transitions have been shown to play a key role in tissue dynamics, structure and morphogenesis. Cellular jamming is controlled by cellular density and the mechanics of cell-cell contacts. However, the contribution of subcellular organelles to the physical state of the emergent tissue is unclear. Here we report a nuclear jamming transition in zebrafish retina and brain tissues, where physical interactions between highly packed nuclei restrict cellular movements and control tissue mechanics and architecture. Computational modelling suggests that the nuclear volume fraction and anisotropy of cells control the emerging tissue physical state. Analysis of tissue architecture, mechanics and nuclear movements during eye development show that retina tissues undergo a nuclear jamming transition as they form, with increasing nuclear packing leading to more ordered cellular arrangements, reminiscent of the crystalline cellular packings in the functional adult eye. Our results reveal an important role of the cell nucleus in tissue mechanics and architecture.

DOI: https://doi.org/10.1038/s41563-024-01972-3
Acta Biomater. 2024 Aug 08. pii: S1742-7061(24)00451-3. [Epub ahead of print]

Time and Space Modulation of Substrate Curvature to Regulate Cell Mechanical Identity.

Stefania Saporito, Valeria Panzetta, Paolo Antonio Netti.

  Recently, a variety of microenvironmental biophysical stimuli have been proved to play a crucial role in regulating cell functions. Among them, morpho-physical cues, like curvature, are emerging as key regulators of cellular behavior. Changes in substrate curvature have been shown to impact the arrangement of Focal Adhesions (FAs), influencing the direction and intensity of cytoskeleton generated forces and resulting in an overall alteration of cell mechanical identity. In their native environment, cells encounter varying degrees of substrate curvature, and in specific organs, they are exposed to dynamic changes of curvature due to periodic tissue deformation. However, the mechanism by which cells perceive substrate curvature remains poorly understood. To this aim, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. Employing a combined experimental and simulative approach, human adipose-derived stem cells were exposed to controlled curvature intensity and frequency. During this exposure, measurements were taken on FAs extension and orientation, cytoskeleton organization and cellular/nuclear alignment. The data clearly indicated a significant influence of the substrate curvature on cell adhesion processes. These findings contribute to a better understanding of the mechanisms through which cells perceive and respond to substrate curvature signals. STATEMENT OF SIGNIFICANCE: This work is our contribution to the comprehension of substrate curvature's function as a crucial regulator of cell adhesion at the scale of focal adhesions and cell mechanical identity. In recent years, a large body of knowledge is continuously growing providing comprehension of the role of various microenvironmental biophysical stimuli in regulating cell functions. Nevertheless, little is known about the role of substrate curvature, in particular, when cells are exposed to this stimulus in a dynamic manner. To address the role of substrate curvature on cellular behavior, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. The experiment data made it abundantly evident that the substrate curvature had a major impact on the mechanisms involved in cell adhesion.

Keywords:  Curvature; Cytoskeleton; Finite element models; Focal Adhesion; Micro-pneumatic Platform; Stem Cells

DOI:  https://doi.org/10.1016/j.actbio.2024.08.006
J Am Chem Soc. 2024 Aug 12.

Measuring Integrin Force Loading Rates Using a Two-Step DNA Tension Sensor.

J Dale Combs, Alexander K Foote, Hiroaki Ogasawara, Arventh Velusamy, Sk Aysha Rashid, Joseph Nicholas Mancuso, Khalid Salaita.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (piconewton) integrin-ECM forces have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrin receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop an LR probe composed of an engineered DNA structure that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN (hairpin unfolding) and a high force threshold at 47 pN (duplex shearing). These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live cells. Automated analysis of tens of thousands of events from eight cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 s. A subset of these events mature in magnitude to >47 pN with a median loading rate of 1.1 pN s-1 and primarily localize at the periphery of the cell-substrate junction. The LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of molecular mechanotransduction in living systems.

DOI: https://doi.org/10.1021/jacs.4c03629
J Tissue Eng. 2024 Jan-Dec;15:15 20417314241268344

Two-dimensional vascularized liver organoid on extracellular matrix with defined stiffness for modeling fibrotic and normal tissues.

Lei Ma, Lin Yin, Hai Zhu, Jing Li, Dong Wang.

  Antifibrotic drug screening requires evaluating the inhibitory effects of drug candidates on fibrotic cells while minimizing any adverse effects on normal cells. It is challenging to create organ-specific vascularized organoids that accurately model fibrotic and normal tissues for drug screening. Our previous studies have established methods for culturing primary microvessels and epithelial cells from adult tissues. In this proof-of-concept study, we used rats as a model organism to create a two-dimensional vascularized liver organoid model that comprised primary microvessels, epithelia, and stellate cells from adult livers. To provide appropriate substrates for cell culture, we engineered ECMs with defined stiffness to mimic the different stages of fibrotic tissues and normal tissues. We examined the effects of two TGFβ signaling inhibitors, A83-01 and pirfenidone, on the vascularized liver organoids on the stiff and soft ECMs. We found that A83-01 inhibited fibrotic markers while promoting epithelial genes of hepatocytes and cholangiocytes. However, it inhibited microvascular genes on soft ECM, indicating a detrimental effect on normal tissues. Furthermore, A83-01 significantly promoted the expression of markers of stem cells and cancers, increasing the potential risk of it being a carcinogen. In contrast, pirfenidone, an FDA-approved compound for antifibrotic treatments, did not significantly affect all the genes examined on soft ECM. Although pirfenidone had minor effects on most genes, it did reduce the expression of collagens, the major components of fibrotic tissues. These results explain why pirfenidone can slow fibrosis progression with minor side effects in clinical trials. In conclusion, our study presents a method for creating vascularized liver organoids that can accurately mimic fibrotic and normal tissues for drug screening. Our findings provide valuable insights into the potential risks and benefits of using A83-01 and pirfenidone as antifibrotic drugs. This method can be applied to other organs to create organ-specific vascularized organoids for drug development.

Keywords:  Liver organoid; extracellular matrix; fibrosis; stiffness; vascularization

DOI:  https://doi.org/10.1177/20417314241268344
J Mater Chem B. 2024 Aug 16.

Methods to achieve tissue-mimetic physicochemical properties in hydrogels for regenerative medicine and tissue engineering.

Rabia Fatima, Bethany Almeida.

Hydrogels are water-swollen polymeric matrices with properties that are remarkably similar in function to the extracellular matrix. For example, the polymer matrix provides structural support and adhesion sites for cells in much of the same way as the fibers of the extracellular matrix. In addition, depending on the polymer used, bioactive sites on the polymer may provide signals to initiate certain cell behavior. However, despite their potential as biomaterials for tissue engineering and regenerative medicine applications, fabricating hydrogels that truly mimic the physicochemical properties of the extracellular matrix to physiologically-relevant values is a challenge. Recent efforts in the field have sought to improve the physicochemical properties of hydrogels using advanced materials science and engineering methods. In this review, we highlight some of the most promising methods, including crosslinking strategies and manufacturing approaches such as 3D bioprinting and granular hydrogels. We also provide a brief perspective on the future outlook of this field and how these methods may lead to the clinical translation of hydrogel biomaterials for tissue engineering and regenerative medicine applications.

DOI: https://doi.org/10.1039/d4tb00716f
bioRxiv. 2024 Aug 08. pii: 2024.08.06.606897. [Epub ahead of print]

Engineering synthetic agonists for targeted activation of Notch signaling.

David H Perez, Daniel Antfolk, Elliot Medina, David Gonzalez-Perez, Vincent C Luca.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although several Notch inhibitors are under development as cancer therapies, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins that mimic the tension-generating mechanism of endogenous ligands. SNAGs were designed by fusing a high-affinity variant of the Notch ligand Delta-like 4 (DLL4) to antibody fragments that induce target internalization. This bispecific format enables the SNAG-bound biomarkers to "pull" on Notch receptors, triggering Notch activation in mixed populations of biomarker-expressing and non-expressing cells. SNAGs targeting the immune checkpoint PDL1 potently activated Notch in co-cultures of Notch1-and PDL1-expressing cells, but not in monocultures of Notch1-expressing cells alone. Additional SNAGs targeting the tumor antigens CD19 and HER2 also activated Notch in mixed cell populations, indicating that the SNAG design concept is adaptable to multiple biomarkers. SNAG-mediated Notch activation was blocked by a dynamin inhibitor, and efficacy increased dramatically when SNAGs were dimerized via fusion to antibody Fc domains, suggesting that endocytosis and multimerization are important for optimal SNAG function. These insights will greatly expand our ability to modulate Notch signaling for applications in immunotherapy and regenerative medicine.

DOI: https://doi.org/10.1101/2024.08.06.606897
Mol Carcinog. 2024 Aug 16.

Oscillatory hypoxia induced gene expression predicts low survival in human breast cancer patients.

Yasir Suhail, Yamin Liu, Wenqiang Du, Junaid Afzal, Xihua Qiu, Amina Atiq, Paola Vera-Licona, Eran Agmon, Kshitiz.

  Hypoxia is one of the key factors in the tumor microenvironment regulating nearly all steps in the metastatic cascade in many cancers, including in breast cancer. The hypoxic regions can however be dynamic with the availability of oxygen fluctuating or oscillating. The canonical response to hypoxia is relayed by transcription factor Hypoxia-Inducible Factor 1 (HIF-1), which is stabilized in hypoxia and acts as the master regulator of a large number of downstream genes. However, HIF-1 transcriptional activity can also fluctuate either due to unstable hypoxia, or by lactate mediated noncanonical degradation of HIF-1. Our understanding of how oscillatory hypoxia or HIF-1 activity specifically influences cancer malignancy is very limited. Here, using MDA-MB-231 cells as a model of triple negative breast cancer characterized by severe hypoxia, we measured the gene expression changes induced specifically by oscillatory hypoxia. We found that oscillatory hypoxia can specifically regulate gene expression differently, and at times opposite to stable hypoxia. Using the Cancer Genome Atlas RNAseq data of human cancer samples, we show that the oscillatory specific gene expression signature in MDA-MB-231 is enriched in most human cancers, and prognosticates low survival in breast cancer patients. In particular, we found that oscillatory hypoxia, unlike stable hypoxia, induces unfolded protein folding response in cells resulting in gene expression predicting reduced survival.

Keywords:  HIF‐1 dynamics; cancer hypoxia; hypoxia; oscillatory hypoxia; unfolded protein folding response

DOI:  https://doi.org/10.1002/mc.23810
Nat Cancer. 2024 Aug 15.

Integrative single-cell analysis of human colorectal cancer reveals patient stratification with distinct immune evasion mechanisms.

Xiaojing Chu, Xiangjie Li, Yu Zhang, Guohui Dang, Yuhui Miao, Wenbin Xu, Jinyu Wang, Zemin Zhang, Sijin Cheng.

The tumor microenvironment (TME) considerably influences colorectal cancer (CRC) progression, therapeutic response and clinical outcome, but studies of interindividual heterogeneities of the TME in CRC are lacking. Here, by integrating human colorectal single-cell transcriptomic data from approximately 200 donors, we comprehensively characterized transcriptional remodeling in the TME compared to noncancer tissues and identified a rare tumor-specific subset of endothelial cells with T cell recruitment potential. The large sample size enabled us to stratify patients based on their TME heterogeneity, revealing divergent TME subtypes in which cancer cells exploit different immune evasion mechanisms. Additionally, by associating single-cell transcriptional profiling with risk genes identified by genome-wide association studies, we determined that stromal cells are major effector cell types in CRC genetic susceptibility. In summary, our results provide valuable insights into CRC pathogenesis and might help with the development of personalized immune therapies.

DOI: https://doi.org/10.1038/s43018-024-00807-z
Adv Healthc Mater. 2024 Aug 12. e2400668

Augmentation of Tendon and Ligament Repair with Fiber-Reinforced Hydrogel Composites.

Robert N Kent, Alice H Huang, Brendon M Baker.

  This review highlights the promise of fiber-reinforced hydrogel composites (FRHCs) for augmenting tendon and ligament repair and regeneration. Composed of reinforcing fibers embedded in a hydrogel, these scaffolds provide both mechanical strength and a conducive microenvironment for biological processes required for connective tissue regeneration. Typical properties of FRHCs are discussed, highlighting their ability to simultaneously fulfill essential mechanical and biological design criteria for a regenerative scaffold. Furthermore, features of FRHCs are described that improve specific biological aspects of tendon healing including mesenchymal progenitor cell recruitment, early polarization to a pro-regenerative immune response, tenogenic differentiation of recruited progenitor cells, and subsequent production of a mature, aligned collagenous matrix. Finally, the review offers a perspective on clinical translation of tendon FRHCs and outlines key directions for future work.

Keywords:  biomaterial scaffolds; fiber‐reinforced hydrogel composites; tendon regeneration; tenogenic differentiation; tissue engineering

DOI:  https://doi.org/10.1002/adhm.202400668
Adv Mater. 2024 Aug 13. e2402988

Magneto-Enzymatic Microgels for Precise Hydrogel Sculpturing.

Maria C Mendes, João A Pereira, Ana S Silva, João F Mano.

  The inclusion of hollow channels in tissue-engineered hydrogels is crucial for mimicking the natural physiological conditions and facilitating the delivery of nutrients and oxygen to cells. Although bio-fabrication techniques provide diverse strategies to create these channels, many require sophisticated equipment and time-consuming protocols. Herein, collagenase, a degrading agent for methacrylated gelatin hydrogels, and magnetic nanoparticles (MNPs) are combined and processed into enzymatically active spherical structures using a straightforward oil bath emulsion methodology. The generated microgels are then used to microfabricate channels within biomimetic hydrogels via a novel sculpturing approach that relied on the precise coupling of protein-enzyme pairs (for controlled local degradation) and magnetic actuation (for directional control). Results show that the sculpting velocity can be tailored by adjusting the magnetic field intensity or concentration of MNPs within the microgels. Additionally, varying the magnetic field position or microgel size generated diverse trajectories and channels of different widths. This innovative technology improves the viability of encapsulated cells through enhanced medium transport, outperforming non-sculpted hydrogels and offering new perspectives for hydrogel vascularization and drug/biomolecule administration. Ultimately, this novel concept can help design fully controlled channels in hydrogels or soft materials, even those with complex tortuosity, in a single wireless top-down biocompatible step.

Keywords:  magnetic forces; magneto‐enzymatic microgels; perfusable hydrogels; protein‐enzyme pairing

DOI:  https://doi.org/10.1002/adma.202402988
Elife. 2024 Aug 15. pii: e80622. [Epub ahead of print]13

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

Victor C Wong, Patrick R Houlihan, Hui Liu, Deepika Walpita, Michael C DeSantis, Zhe Liu, Erin K O'Shea.

  AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Keywords:  AMPA receptor; CRISPR/Cas9; actin; cell biology; neuroscience; rat; single-particle tracking; synaptic plasticity; vesicle trafficking

DOI:  https://doi.org/10.7554/eLife.80622