Angiogenesis and full thickness wound repair in a cell sheet-based vascularized skin substitute.

Skin tissue engineering is undergoing tremendous expansion as a result from clinical needs, mandatory replacement of animal models and development of new technologies. Many approaches have been used to produce vascularized skin substitutes for grafting purposes showing the presence of capillary-like structures but with limited analysis of their in vitro maturation and plasticity. Such knowledge is however important for the development of tissue substitutes with improved implantation success as well as for validation of vascularization in vitro models, including as a readout in pharmacological analyses. For optimal interactions of cells with microenvironment and vasculature, we here used a cell sheet approach consisting in the sole production of matrix by the cells. In this context, we limited the density of endothelial cells seeded for self-assembly and rather relied on the stimulation of angiogenesis for the development of an extensive connected microvascular-like network. After detailed characterization of this network, we challenged its plasticity both during and after establishment of the skin substitute. We show that fine tuning of VEGF concentration and time of application differentially affects formation of capillary-like structures and their perivascular coverage. Furthermore, we performed a deep wound assay that displayed tissue repair and angiogenesis with unique characteristics of the physiological process. These studies demonstrate the importance of cell-derived microenvironment for the establishment of mature yet dynamic vascularized skin models allowing a wide range of pharmacological and basic investigations. STATEMENT OF SIGNIFICANCE: The significant advancements in organ-on-chips and tissue engineering call for more relevant models including microvascularization with remodeling potential. While vascularized skin substitutes have been developed for years, focus has primarily been on the impact of microvascularization on implantation rather than on its in vitro characterization. We here developed a cell sheet-based vascularized skin substitute relying on angiogenesis, i.e. growth of vessel-like structures within the 3D model, rather than solely on endothelial cell self-assembly. We then characterized :1/ vascularization after modulation of angiogenic factor VEGF during the substitute construction; -2/ angiogenesis associated to tissue repair after deep mechanical wounding. These studies establish a solid physiologically relevant model for further investigation of skin cell interactions and in vitro wound healing.

Bioactive poly(amino acid)s for multi-modal cancer therapy.

The interplay between the tumor cells and their microenvironments is as inseparable as the relationship between "seeds" and "soil." The tumor microenvironments (TMEs) exacerbate malignancy by enriching malignant cell subclones, generating extracellular matrices, and recruiting immunosuppressive cells, thereby diminishing the efficacy of clinical therapies. Modulating TMEs has emerged as a promising strategy to enhance cancer therapy. However, the existing drugs used in clinical settings do not target the TMEs specifically, underscoring the urgent need for advanced strategies. Bioactive materials present unique opportunities for modulating TMEs. Poly(amino acid)s with precisely controllable structures and properties offer exceptional characteristics, such as diverse structural units, excellent biosafety, ease of modification, sensitive biological responsiveness, and unique secondary structures. These attributes hold significant potential for the modulation of TMEs and clinical applications further. Consequently, developing bioactive poly(amino acid)s capable of modulating the TMEs by elucidating structure-activity relationships and mechanisms is a promising approach for innovative clinical oncology therapy. This review summarizes the recent progress of our research team in developing bioactive poly(amino acid)s for multi-modal tumor therapy. First, a brief overview of poly(amino acid) synthesis and their advantages as nanocarriers is provided. Subsequently, the pioneering research of our research group on synthesizing the biologically responsive, dynamically allosteric, and immunologically effective poly(amino acid)s are highlighted. These poly(amino acid)s are designed to enhance tumor therapy by modulating the intracellular, extracellular matrix, and stromal cell microenvironments. Finally, the future development of poly(amino acid)s is discussed. This review will guide and inspire the construction of bioactive poly(amino acid)s with promising clinical applications in cancer therapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Biology-Inspired Nanomaterials > Peptide-Based Structures.

Role of Emerin in regulating fibroblast differentiation and migration at the substrate of stiffness coupled topology.

In hypertrophic scars, the differentiation and migration of fibroblasts are influenced by the extracellular matrix microenvironment, which includes factors such as stiffness, restraint, and tensile force. These mechanical stresses incite alterations in cell behavior, accompanied by cytoskeletal protein reorganization. However, the role of nucleo-skeletal proteins in this context remains underexplored. In this study, we use a polyacrylamide hydrogel (PAA) to simulate the mechanical stress experienced by cells in scar tissue and investigate the impact of Emerin on cell behavior. We utilize atomic force microscopy (AFM) and RNA interference technology to analyze cell differentiation, migration, and stiffness. Our findings reveal that rigid substrates and cellular restriction elevate Emerin expression and diminish differentiation. Conversely, reducing Emerin expression leads to attenuated cell differentiation, where stiffness and constraining factors exert no notable influence. Furthermore, a softening of cells and an enhanced migration rate are also markedly observed. These observations indicate that variations in nuclear skeletal proteins, prompted by diverse matrix microenvironments, play a pivotal role in the pathogenesis of hypertrophic scars (HSs). This research offers novel insights and a reference point for understanding scar fibrosis formation mechanisms and preventing fibrosis.

Primary cilium-mediated mechanotransduction in cartilage chondrocytes.

Osteoarthritis (OA) is one of the most prevalent joint disorders associated with the degradation of articular cartilage and an abnormal mechanical microenvironment. Mechanical stimuli, including compression, shear stress, stretching strain, osmotic challenge, and the physical properties of the matrix microenvironment, play pivotal roles in the tissue homeostasis of articular cartilage. The primary cilium, as a mechanosensory and chemosensory organelle, is important for detecting and transmitting both mechanical and biochemical signals in chondrocytes within the matrix microenvironment. Growing evidence indicates that primary cilia are critical for chondrocytes signaling transduction and the matrix homeostasis of articular cartilage. Furthermore, the ability of primary cilium to regulate cellular signaling is dynamic and dependent on the cellular matrix microenvironment. In the current review, we aim to elucidate the key mechanisms by which primary cilia mediate chondrocytes sensing and responding to the matrix mechanical microenvironment. This might have potential therapeutic applications in injuries and OA-associated degeneration of articular cartilage.

[Research progress of chondrocyte mechanotransduction mediated by TRPV4 and PIEZOs].

Mechanical signal transduction are crucial for chondrocyte in response to mechanical cues during the growth, development and osteoarthritis (OA) of articular cartilage. Extracellular matrix (ECM) turnover regulates the matrix mechanical microenvironment of chondrocytes. Thus, understanding the mechanotransduction mechanisms during chondrocyte sensing the matrix mechanical microenvironment can develop effective targeted therapy for OA. In recent decades, growing evidences are rapidly advancing our understanding of the mechanical force-dependent cartilage remodeling and injury responses mediated by TRPV4 and PIEZOs. In this review, we highlighted the mechanosensing mechanism mediated by TRPV4 and PIEZOs during chondrocytes sensing mechanical microenvironment of the ECM. Additionally, the latest progress in the regulation of OA by inflammatory signals mediated by TRPV4 and PIEZOs was also introduced. These recent insights provide the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA. This review will shed light on the pathogenesis of articular cartilage, searching clinical targeted therapies, and designing cell-induced biomaterials.

Biological Hyperthermia-Inducing Nanoparticles for Specific Remodeling of the Extracellular Matrix Microenvironment Enhance Pro-Apoptotic Therapy in Fibrosis.

The extracellular matrix (ECM) is a major driver of fibrotic diseases and forms a dense fibrous barrier that impedes nanodrug delivery. Because hyperthermia causes destruction of ECM components, we developed a nanoparticle preparation to induce fibrosis-specific biological hyperthermia (designated as GPQ-EL-DNP) to improve pro-apoptotic therapy against fibrotic diseases based on remodeling of the ECM microenvironment. GPQ-EL-DNP is a matrix metalloproteinase (MMP)-9-responsive peptide, (GPQ)-modified hybrid nanoparticle containing fibroblast-derived exosomes and liposomes (GPQ-EL) and is loaded with a mitochondrial uncoupling agent, 2,4-dinitrophenol (DNP). GPQ-EL-DNP can specifically accumulate and release DNP in the fibrotic focus, inducing collagen denaturation through biological hyperthermia. The preparation was able to remodel the ECM microenvironment, decrease stiffness, and suppress fibroblast activation, which further enhanced GPQ-EL-DNP delivery to fibroblasts and sensitized fibroblasts to simvastatin-induced apoptosis. Therefore, simvastatin-loaded GPQ-EL-DNP achieved an improved therapeutic effect on multiple types of murine fibrosis. Importantly, GPQ-EL-DNP did not induce systemic toxicity to the host. Therefore, the nanoparticle GPQ-EL-DNP for fibrosis-specific hyperthermia can be used as a potential strategy to enhance pro-apoptotic therapy in fibrotic diseases.

Endogenous Repair and Regeneration of Injured Articular Cartilage: A Challenging but Promising Therapeutic Strategy.

Articular cartilage (AC) has a very limited intrinsic repair capacity after injury or disease. Although exogenous cell-based regenerative approaches have obtained acceptable outcomes, they are usually associated with complicated procedures, donor-site morbidities and cell differentiation during ex vivo expansion. In recent years, endogenous regenerative strategy by recruiting resident mesenchymal stem/progenitor cells (MSPCs) into the injured sites, as a promising alternative, has gained considerable attention. It takes full advantage of body's own regenerative potential to repair and regenerate injured tissue while avoiding exogenous regenerative approach-associated limitations. Like most tissues, there are also multiple stem-cell niches in AC and its surrounding tissues. These MSPCs have the potential to migrate into injured sites to produce replacement cells under appropriate stimuli. Traditional microfracture procedure employs the concept of MSPCs recruitment usually fails to regenerate normal hyaline cartilage. The reasons for this failure might be attributed to an inadequate number of recruiting cells and adverse local tissue microenvironment after cartilage injury. A strategy that effectively improves local matrix microenvironment and recruits resident MSPCs may enhance the success of endogenous AC regeneration (EACR). In this review, we focused on the reasons why AC cannot regenerate itself in spite of potential self-repair capacity and summarized the latest developments of the three key components in the field of EACR. In addition, we discussed the challenges facing in the present EACR strategy. This review will provide an increasing understanding of EACR and attract more researchers to participate in this promising research arena.

Cell Derived Matrix Fibulin-1 Associates With Epidermal Growth Factor Receptor to Inhibit Its Activation, Localization and Function in Lung Cancer Calu-1 Cells.

Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC). To test the possible role FBLN1 isoforms could have in regulating EGFR signaling and function in lung cancer, we performed siRNA mediated knockdown of FBLN1C and FBLN1D in NSCLC Calu-1 cells. Their loss significantly increased basal (with serum) and EGF (Epidermal Growth Factor) mediated EGFR activation without affecting net EGFR levels. Overexpression of FBLN1C and FBLN1D also inhibits EGFR activation confirming their regulatory crosstalk. Loss of FBLN1C and FBLN1D promotes EGFR-dependent cell migration, inhibited upon Erlotinib treatment. Mechanistically, both FBLN1 isoforms interact with EGFR, their association not dependent on its activation. Notably, cell-derived matrix (CDM) enriched FBLN1 binds EGFR. Calu-1 cells plated on CDM derived from FBLN1C and FBLN1D knockdown cells show a significant increase in EGF mediated EGFR activation. This promotes cell adhesion and spreading with active EGFR enriched at membrane ruffles. Both adhesion and spreading on CDMs is significantly reduced by Erlotinib treatment. Together, these findings show FBLN1C/1D, as part of the ECM, can bind and regulate EGFR activation and function in NSCLC Calu-1 cells. They further highlight the role tumor ECM composition could have in influencing EGFR dependent lung cancers.

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin.