Extracellular Matrix Architecture Research Articles

Cell-derived Matrix Assays to Assess Extracellular Matrix Architecture and Track Cell Movement.

The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell-ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell-ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes. This protocol was validated in: Sci Adv (2021), DOI: 10.1126/sciadv.abh0363.

3115 – THE COLLAGEN-MODIFYING LYSYL HYDROXYLASE, PLOD2, IS REQUIRED FOR HEMATOPOIETIC STEM CELL SPECIFICATION

Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis. Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis.

Patterned photocrosslinking to establish stiffness anisotropies in fibrous 3D hydrogels

Cells are known to constantly interact with their local extracellular matrix (ECM) and respond to a variety of biochemical and mechanical cues received from the ECM. Nonetheless, comprehensive understanding of cell-ECM interactions has been elusive. Many studies rely on analysis of cell behavior on 2D substrates, which do not reflect a natural cell environment. Further, lack of dynamic control over local stiffness anisotropies and fiber alignment hinders progress in studies in naturally derived fibrous 3D cultures. Here, we present a cell-safe method of patterned photocrosslinking, which can aid in studying biological hypotheses related to mechanotransduction in 3D hydrogels. As previously described by our group, ruthenium-catalyzed photocrosslinking (RCP) of selected ECM regions promotes localized increase in stiffness mediated by focused blue laser light in a confocal microscope. In this study, we further demonstrate that RCP can induce localized strain stiffening and fiber alignment outside of the selected crosslinked region and induce stiffness anisotropy biased towards the direction of fiber alignment. MDA-MB-231 cells are shown to respond to RCP-induced changes in local ECM architecture and display directional bias towards the direction of fiber alignment, as compared to control cells. Further, the effect of patterned crosslinking on a stiffness landscape is measured using multi-axes optical tweezers active microrheology (AMR) with backscattered laser beam illumination. AMR validates RCP as a suitable tool for creating distinct stiffness anisotropies which promote directed migration of cells, further underscoring the usefulness of RCP in cell-ECM studies. Statement of significanceStudies on cell-ECM interactions in 3D cultures have often been hindered by the lack of available tools to dynamically alter local ECM stiffness and fiber alignment. Here, we present a non-invasive, cell-safe and easily applicable method of patterned photocrosslinking, which can aid in studying biological hypotheses in fibrous 3D hydrogels. Ruthenium-catalyzed crosslinking (RCP) of selected fibrin ECM regions promotes localized increase in stiffness and creates distinct stiffness anisotropies in the presence of the focused blue laser light. Outside of the crosslinked region, RCP causes fiber alignment and strain stiffening in the ECM, verified using multi-axes optical tweezers active microrheology (AMR). Following RCP, human breast cancer MDA-MB-231 exhibit directed cell migration, validating usefulness of this method in cell-ECM studies.

Current opportunities and prospectives of immunotropic therapy in chronic generalized periodontitis

Impairment of immunological reactivity in inflammatory periodontal diseases is well proven. To perform immunomodulatory treatment in domestic dental practice, various medications are used, including natural, chemically modified, recombinant, genetically engineered and synthetic substances, which differ in their effects upon innate and adaptive immune systems. Complex preparations of natural cytokines as well as genetically engineered preparations of IL-1, IL-2, growth factors, IFNα, IFNβ, IFNγ are applied in clinical settings. Clinical implementation of interferon and interferon inducers in combined therapy of generalized periodontitis is shown to increase resistance to viral components of the oral microbiota. Growth factors (platelet growth factor, fibroblast growth factor, endothelial growth factor, etc.) are successfully used for tissue regeneration in periodontics and maxillofacial surgery. Experimental studies have shown that local administration of toll-like receptor-9 and CD40 ligand may reduce periodontal ligature inflammation and bone loss in mice by inducing B-cell proliferation and increasing IL-10 mRNA expression. Promising results in development of new biologically active drugs are obtained with nanotechnology approaches, i.e., production of composite materials of metal nanoparticles with polymers, growth factors, and local application of these products. General limitations of all these growth factors include extremely short periods of biological activity, and adjusted duration of local effective concentrations. Therefore, it is important to develop a drug delivery system using appropriate scaffolding elements thus allowing local effects of the drug for a certain period of time. In experimental models, alginate hydrogels performed well upon local delivery of granulocyte-macrophage colony-stimulating factor and stromal lymphopoietin of the thymus. A new immunomodulatory strategy for alveolar bone regeneration targets macrophages. A biologically functionalized injectable microsphere of heparin-modified gelatin nanofibers that mimic the architecture of the natural bone extracellular matrix, and provide an osteoconductive microenvironment for bone cells includes IL-4, which has heparin-binding domains. These medications represent a component of a comprehensive treatment schedule, and should be evaluated for immune status before and after therapy. Thus, recent advances in studies of innate and acquired immune responses in inflammatory diseases and, in particular, in periodontal disorders, allows us to develop new approaches and methods of treatment in order to improve efficiency of complex therapy in the inflammatory periodontal diseases.

Disease-specific glycosaminoglycan patterns in the extracellular matrix of human lung and brain

A wide variety of diseases throughout the mammalian organism is characterized by abnormal deposition of various components of the extracellular matrix (ECM), including the heterogeneous family of glycosaminoglycans (GAGs), which contribute considerably to the ECM architecture as part of the so-called proteoglycans. The GAG's unique sulfation pattern, derived from highly dynamic and specific modification processes, has a massive impact on critical mediators such as cytokines and growth factors. Due to the strong connection between the specific sulfation pattern and GAG function, slight alterations of this pattern are often associated with enormous changes at the cell as well as at the organ level. This review aims to investigate the connection between modifications of GAG sulfation patterns and the wide range of pathological conditions, mainly focusing on a range of chronic diseases of the central nervous system (CNS) as well as the respiratory tract.

"Raspberry" Hierarchical Topographic Features Regulate Human Mesenchymal Stem Cell Adhesion and Differentiation via Enhanced Mechanosensing.

An understanding of cellular mechanoresponses to well-defined synthetic topographic features is crucial for the fundamental research and biomedical applications of stem cells. Structured biointerfaces, in particular the ones with nanometer and/or micrometer surficial features, have drawn more attention in the past few decades. However, it is still difficult to integrate nanostructures and microstructures onto the synthesized biointerfaces to mimic the hierarchical architecture of the native extracellular matrix (ECM). Herein, a series of "raspberry"-like hierarchical surfaces with well-defined nanofeatures and tunable nano/microfeatures have been achieved via a catecholic polymer coating technique. Cellular responses to these hierarchical interfaces were systemically studied, indicating that the nanofeatures on the raspberry surfaces significantly enhanced the mechanosensing of human mesenchymal stem cells (hMSCs) to interfacial physical cues. Cell mechanotransduction was further investigated by analyzing focal adhesion assembling, cytoskeleton organization, cell nuclear mechanics, and transcriptional activity. The results suggest that nanosize surficial features could increase cellular mechanosensing to environment physical cues. The mechanotransduction and cell fate specification were greatly enhanced by the ECM mimicking nano/microhierarchical biointerfaces but the features should be in an optimized size.

Molecular Imaging of Collagen Destruction of the Spine.

As the leading cause of disability worldwide, low back pain is commonly caused by biomechanical and catabolic disruptions to key structures of the spine, such as intervertebral discs and facet joints. To date, accurate, noninvasive detection of microdestruction within these tissues remains an elusive goal. Here, we report an in vivo imaging approach based on a collagen hybridizing peptide (CHP) that specifically targets disruption to the extracellular matrix architecture at the molecular scale─the denatured collagen molecules. Utilizing fluorescently labeled CHPs, live animal imaging, and light sheet fluorescence microscopy, we mapped collagen destruction in the lumbar spines in 3D, revealing that under normal conditions collagen destruction was localized to load-bearing anatomical structures including annulus fibrosus of the disc and the facet joints, where aging, tensile force (hindlimb suspension), and disc degeneration (needle puncture) escalated the CHP-binding in specific mouse models. We showed that targeting denatured collagen molecules allowed for an accurate, quantifiable interrogation of the structural integrity of these spinal matrixes with a greater sensitivity than anatomical imaging and histology. Finally, we demonstrated CHP's binding to degenerated human discs, suggesting exciting potentials for applying CHP for diagnosing, monitoring, and treating various spinal disorders, including intervertebral disc degeneration, facet joint osteoarthritis, and ankylosing spondylitis.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Hemicentin-1 is an essential extracellular matrix component of the dermal\u2013epidermal and myotendinous junctions

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal–epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1−/− mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1−/− mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Development of Cell-Derived Matrices for Three-Dimensional In Vitro Cancer Cell Models.

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice

ABSTRACT In the current study, a decellularized ovary scaffold was used for the encapsulation and development of preantral follicles. Maturation and the expression of the related genes were evaluated and compared against in vivo conditions. Mature rat ovaries were decellularized and decellularization was confirmed using hematoxylin/eosin and Hoechst stains. Collagen and glycosaminoglycan (GAG) preservation and the extracellular matrix (ECM) architecture were evaluated by Masson’s trichrome staining, Alcian blue staining, and scanning electron microscopy, respectively. Raman confocal microscopy and DNA quantification were performed to show DNA and cell debris depletion and ECM retention. Preantral follicles of mice were cultured for 12 days in the decellularized (3D) and two-dimensional (2D) conditions. Finally, the survival rate, antrum formation rate and diameters of follicles, levels of estradiol (E2) and progesterone (P4), and expression of the ZP2, Gdf9, Bmp6, and Bmp15 genes were evaluated in the 2D and 3D culture systems. On day 13 of culture, oocyte maturation was induced by hCG. Although histochemical tests showed partial retention of ECM, Raman confocal microscopy showed some extent of ECM washing out along with the depletion of DNA and cell debris. However, SEM showed the preservation of the ovarian ECM’s ultrastructure. The survival rate of follicles was similar, but the antrum formation rate, concentrations of E2 and P4, and oocyte maturation rate were significantly higher in 3D compared with 2D. The level of ZP2, Gdf9, Bmp6, and Bmp15 gene expression in follicles matured in 2D, 3D, and in vivo was statistically similar. In all matured follicles, their gene expression levels significantly declined relative to preantral follicles. Ovarian follicles that matured in the decellularized ovaries demonstrated the same follicular growth and maturation gene expression levels as those that grew in the natural ovary. This is consistent with using the decellularized ovary as a scaffold suitable for ovarian reconstruction. Abbreviations GAG: glycosaminoglycan; ECM: extracellular matrix; 2D: two-dimensional; E2: estradiol; P4: progesterone; BMP15: bone morphogenetic protein 15; GDF9: growth differentiation factor 9; ZP2: zona pellucida 2; Gdf9: growth/differentiation factor-9; Bmp6: bone morphogenetic protein 6; Bmp15: bone morphogenetic protein 15.

Survey of the extracellular matrix architecture across the rat arterial tree

ObjectiveTo understand arterial remodeling and the pathophysiology of arterial diseases, it is necessary to understand the baseline qualities and variations in arterial structure. Arteries could differ in wall thickness, laminar structure, and laminar fenestration depending on their position within the arterial tree. We endeavored to evaluate and compare the extracellular matrix structure of different arteries throughout the arterial tree, from the aorta to the adductor muscle arteriole, with a particular focus on the internal elastic lamina (IEL). MethodsArterial segments were harvested from male Sprague-Dawley rats and imaged using multiple modalities. En face scans by multiphoton microscopy were used to compare native-state adventitial collagen undulation and IEL fenestration. ResultsCollagen undulation was similar across most examined arteries but straighter in the skeletal muscle arterioles (P < .05). The elastic lamellae showed several differences. The IEL fenestrae were similar in average size among abdominal aorta and celiac, renal, common iliac, and common femoral arteries (range, 14-24 μm2), with wide within-vessel variance (square of the standard deviation, 462-1904 μm4). However, they tended to be smaller (9.08 μm2) and less variable (square of the standard deviation, 88.3 μm4) in the popliteal artery. Fenestrae were greater in number in the superior mesenteric artery (SMA; 6686/mm2; P < .05) and profunda femoris artery (PFA; 11,042/mm2; P < .05) compared with the other examined vessels, which ranged in surface density from 3143/mm2 to 4362/mm2. The SMA and PFA also showed greater total fenestration as a proportion of the IEL surface area (SMA, 15.04%; P < .05; PFA, 24.11%; P < .001) than the other examined arteries (range of means, 4.7%-9.4%). The arteriolar IEL was structurally distinct, comparable to a low-density wireframe. Other structural differences were also noted, including differences in the number of medial lamellae along the arterial tree. ConclusionsWe found that vessels at different locations along the arterial tree differ in structure. The SMA, PFA, and intramuscular arterioles have fundamental differences in the extracellular matrix structure compared with other arteries. Location-specific features such as the medial lamellae number and elastic laminar structure might have relevance to physiology and confer vulnerabilities to the development of pathology.

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4h or 1% SLES solution for 6h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.

In vitro and in vivo advancement of multifunctional electrospun nanofiber scaffolds in wound healing applications: Innovative nanofiber designs, stem cell approaches, and future perspectives.

The skin is one of the most essential tissues in the human body, interacting with the outside environment and shielding the body from diseases and excessive water loss. Hydrogels, decellularized porcine dermal matrix, and lyophilized polymer scaffolds have all been used in studies of skin wound repair, wound dressing, and skin tissue engineering, however, these materials cannot replicate the nanofibrous architecture of the skin's native extracellular matrix (ECM). Electrospun nanofibers are a fascinating new form of nanomaterials with tremendous potential across a broad spectrum of applications in the biomedical field, including wound dressings, wound healing scaffolds, regenerative medicine, bioengineering of skin tissue, and multifaceted drug delivery. This article reviews recent in vitro and in vivo developments in multifunctional electrospun nanofibers (MENs) for wound healing. This review begins with an introduction to the electrospinning process, its principle, and the processing parameters which have a significant impact on the nanofiber properties. It then discusses the various geometries and advantages of MEN scaffolds produced by different innovative electrospinning techniques for wound healing applications when used in combination with stem cells. This review also discusses some of the possible future nanofiber-based models that could be used. Finally, we conclude with potential perspectives and conclusions in this area.

Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities.

Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself.

Alternatives to Conventional Antibiotic Therapy: Potential Therapeutic Strategies of Combating Antimicrobial-Resistance and Biofilm-Related Infections.

Antibiotics have been denoted as the orthodox therapeutic agents for fighting bacteria-related infections in clinical practices for decades. Nevertheless, overuse of antibiotics has led to the upsurge of species with antimicrobial resistance (AMR) or multi-drug resistance. Bacteria can also grow into the biofilm, which accounts for at least two-thirds of infections. Distinct gene expression and self-produced heterogeneous hydrated extracellular polymeric substance matrix architecture of biofilm contribute to their tolerance and externally manifest as antibiotic resistance. In this review, the difficulties in combating biofilm formation and AMR are introduced, and novel alternatives to antibiotics such as metal nanoparticles and quaternary ammonium compounds, chitosan and its derivatives, antimicrobial peptides, stimuli-responsive materials, phage therapy and other therapeutic strategies, from compounds to hydrogel, from inorganic to biological, are discussed. We expect to provide useful information for the readers who are seeking for solutions to the problem of AMR and biofilm-related infections.

3: Multimodal Molecular Analysis Reveals Divergent Trajectories Of Wound Regeneration Versus Fibrosis

Purpose:Scarring in the mouse dorsal dermis is mediated by pro-fibrotic, Engrailed-1 lineage-positive fibroblasts (EPFs). We recently showed that mechanotransduction blockade (YAP inhibition, using the drug verteporfin), results in complete wound regeneration, with full recovery of normal dermal appendages (hair follicles, glands), extracellular matrix (ECM) architecture, and tensile strength. This regenerative outcome following verteporfin treatment is mediated by Engrailed-1 lineage-negative fibroblasts (ENFs). The complex milieu of cell types and molecular signals involved in wound repair makes it difficult to study using any single data modality. Thus, we sought to use a holistic approach, incorporating multiple high-throughput, high-dimensional analyses, to define the divergent molecular events distinguishing typical scarring healing from verteporfin-induced wound regeneration.Methods:C57BL/6J mice underwent dorsal splinted excisional wounding per standard protocol. Wounds were treated with local injection of either verteporfin or vehicle control (PBS) on POD 0. We harvested unwounded skin and wounds at POD 2, 7, 14, and 30 (n=5 mice/timepoint and treatment) and subjected wound cells to three analyses: single-cell RNA-sequencing (scRNA-seq, using 10X Genomics Chromium); timsTOF, a recently-developed, high-throughput proteomic sequencing platform; and a novel machine learning algorithm for quantitatively comparing ECM ultrastructure.Results:Pseudotime analysis (Monocle3) of pooled scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (En-1 lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (En-1 lineage-negative; Rspo1, Dkk2/3, Trps1). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and fibrotic/scar ECM features. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were associated with a “basket-weave” ECM pattern quantitatively indistinguishable from that of unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed in situ hybridization (RNAScope) for Rspo1 (Wnt agonist), Trps1 (master hair follicle regulator), Ank1 (YAP target gene), and Dpp4 (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low Dpp4) in YAP-inhibited (low Ank1) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high Rpos1, Trps1). These data suggest that YAP inhibition unlocks wound regeneration via Wnt-active, En-1 lineage-negative fibroblasts.Conclusion:By studying regenerating (verteporfin-treated) versus scarring wounds across multiple healing timepoints and high-dimensional data modalities, we were able to profile fibrotic versus regenerative healing at unprecedented depth. Our integrated analysis revealed that dermal fibroblasts in these two wound settings exhibit distinct molecular trajectories defined by divergent transcriptomic, proteomic, and ultrastructural properties. Further, we found that wound regeneration in the context of verteporfin treatment is associated with suppression of mechanical signaling and activation of key Wnt pathway members including Trps1 (a gene with known hair follicle developmental roles). These results could have important implications for both the fundamental study of wound healing and potential anti-scarring therapeutic avenues

A Signaling View into the Inflammatory Tumor Microenvironment

The development of tumors requires an initiator event, usually exposure to DNA damaging agents that cause genetic alterations such as gene mutations or chromosomal abnormalities, leading to deregulated cell proliferation. Although the mere stochastic accumulation of further mutations may cause tumor progression, it is now clear that an inflammatory microenvironment has a major tumor-promoting influence on initiated cells, in particular when a chronic inflammatory reaction already existed before the initiated tumor cell was formed. Moreover, inflammatory cells become mobilized in response to signals emanating from tumor cells. In both cases, the microenvironment provides signals that initiated tumor cells perceive by membrane receptors and transduce via downstream kinase cascades to modulate multiple cellular processes and respond with changes in cell gene expression, metabolism, and morphology. Cytokines, chemokines, and growth factors are examples of major signals secreted by immune cells, fibroblast, and endothelial cells and mediate an intricate cell-cell crosstalk in an inflammatory microenvironment, which contributes to increased cancer cell survival, phenotypic plasticity and adaptation to surrounding tissue conditions. Eventually, consequent changes in extracellular matrix stiffness and architecture, coupled with additional genetic alterations, further fortify the malignant progression of tumor cells, priming them for invasion and metastasis. Here, we provide an overview of the current knowledge on the composition of the inflammatory tumor microenvironment, with an emphasis on the major signals and signal-transducing events mediating different aspects of stromal cell-tumor cell communication that ultimately lead to malignant progression.

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment.

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy.

Curcuminoid-Tailored Interfacial Free Energy of Hydrophobic Fibers for Enhanced Biological Properties.

The ability of mimicking the extracellular matrix architecture has gained electrospun scaffolds a prominent space into the tissue engineering field. The high surface-to-volume aspect ratio of nanofibers increases their bioactivity while enhancing the bonding strength with the host tissue. Over the years, numerous polyesters, such as poly(lactic acid) (PLA), have been consolidated as excellent matrices for biomedical applications. However, this class of polymers usually has a high hydrophobic character, which limits cell attachment and proliferation, and therefore decreases biological interactions. In this way, functionalization of polyester-based materials is often performed in order to modify their interfacial free energy and achieve more hydrophilic surfaces. Herein, we report the preparation, characterization, and in vitro assessment of electrospun PLA fibers with low contents (0.1 wt %) of different curcuminoids featuring π-conjugated systems, and a central β-diketone unit, including curcumin itself. We evaluated the potential of these materials for photochemical and biomedical purposes. For this, we investigated their optical properties, water contact angle, and surface features while assessing their in vitro behavior using SH-SY5Y cells. Our results demonstrate the successful generation of homogeneous and defect-free fluorescent fibers, which are noncytotoxic, exhibit enhanced hydrophilicity, and as such greater cell adhesion and proliferation toward neuroblastoma cells. The unexpected tailoring of the scaffolds’ interfacial free energy has been associated with the strong interactions between the PLA hydrophobic sites and the nonpolar groups from curcuminoids, which indicate its role for releasing hydrophilic sites from both parts. This investigation reveals a straightforward approach to produce photoluminescent 3D-scaffolds with enhanced biological properties by using a polymer that is essentially hydrophobic combined with the low contents of photoactive and multifunctional curcuminoids