Extracellular Matrix Substrates Research Articles

Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal β-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

The metabolic characteristics and changes of chondrocytes in vivo and in vitro in osteoarthritis

Osteoarthritis (OA) is an intricate pathological condition that primarily affects the entire synovial joint, especially the hip, hand, and knee joints. This results in inflammation in the synovium and osteochondral injuries, ultimately causing functional limitations and joint dysfunction. The key mechanism responsible for maintaining articular cartilage function is chondrocyte metabolism, which involves energy generation through glycolysis, oxidative phosphorylation, and other metabolic pathways. Some studies have shown that chondrocytes in OA exhibit increased glycolytic activity, leading to elevated lactate production and decreased cartilage matrix synthesis. In OA cartilage, chondrocytes display alterations in mitochondrial activity, such as decreased ATP generation and increased oxidative stress, which can contribute to cartilage deterioration. Chondrocyte metabolism also involves anabolic processes for extracellular matrix substrate production and energy generation. During OA, chondrocytes undergo considerable metabolic changes in different aspects, leading to articular cartilage homeostasis deterioration. Numerous studies have been carried out to provide tangible therapies for OA by using various models in vivo and in vitro targeting chondrocyte metabolism, although there are still certain limitations. With growing evidence indicating the essential role of chondrocyte metabolism in disease etiology, this literature review explores the metabolic characteristics and changes of chondrocytes in the presence of OA, both in vivo and in vitro. To provide insight into the complex metabolic reprogramming crucial in chondrocytes during OA progression, we investigate the dynamic interaction between metabolic pathways, such as glycolysis, lipid metabolism, and mitochondrial function. In addition, this review highlights prospective future research directions for novel approaches to diagnosis and treatment. Adopting a multifaceted strategy, our review aims to offer a comprehensive understanding of the metabolic intricacies within chondrocytes in OA, with the ultimate goal of identifying therapeutic targets capable of modulating chondrocyte metabolism for the treatment of OA.

Inhaled nintedanib nanoparticles for enhanced efficacy in idiopathic pulmonary fibrosis (IPF) treatment – Evidence in disease-relevant in-vitro models

Idiopathic pulmonary fibrosis (IPF) is a fatal and progressive interstitial lung disease with a high mortality rate due to limited prognosis. According to the National Institutes of Health (NIH), approximately 100,000 people in the United States suffer from IPF, and around 50% likelihood of dying within 2–3 years. Currently, nintedanib (Nint) is one of the only 2 FDA-approved drugs for the treatment of IPF, with anti-fibrotic and anti-inflammatory activities. However, Nint oral capsules have some undesirable limitations such as low bioavailability and high dose requirement for sufficient therapeutic efficacy, leading to various side effects associated with oral delivery. To address these shortcomings and enhance the therapeutic potential, Nint was loaded into PLGA nanoparticles (Nint NPs) for localized pulmonary delivery. In-vitro cell culture studies indicated that nano-sized particles (179 ± 3 nm) of Nint NPs enhanced cellular uptake in airway epithelial cells and normal human lung fibroblasts, while readily avoiding macrophage phagocytosis. Transforming growth factor-beta (TGF-β) (10 ng/mL) was selected to establish IPF in cells, which resulted in proliferation and rapid wound healing, whereas Nint NPs showed superior ability in inhibiting these pathological phenomena compared to free Nint. Upon further evaluation of pathological mechanisms of IPF, Nint NPs remarkably attenuated epithelial-to-mesenchymal transition (EMT) by elevating epithelial marker E-cadherin level and reducing extracellular matrix (ECM) deposition by evidence of reduced collagen production and fibroblast-to-myofibroblast differentiation marker, α-SMA. Nint NPs also decreased expression of inflammatory cytokine IL-17A and indicated autophagy as a separate anti-fibrotic mechanism. Finally, Nint NPs proved their superior efficacy in a novel IPF cell model using diseased lung ECM substrates. Hence, inhalation therapy of Nint NPs is a promising alternative tool for oral capsules in IPF therapy.

Abstract 7561: Standardized conditions for growth of lung cancer tumoroids cultured reproducibly in 3D-cultures

Abstract Background: Lung cancer is the most common cause of cancer-related death in Sweden with an estimated mortality of 3500 persons in 2021. Late diagnosis and heterogeneity of the disease lead to difficulty in predicting optimal treatment regime for each case. Further development of personalized therapy would be facilitated by reliable and efficient cultivation of cancer cell samples from patients in a clinical setting. A promising method is to grow the cells as 3D structures known as tumoroids, though the choice of extracellular matrix substrate or solid support most suitable for this purpose should be elucidated. Aim: To assess which extracellular matrix substrates or solid supports which enable lung cancer tumoroid generation reproducibly without affecting the status or detection of genetic alterations and protein expression, using lung cancer cell lines. Methods: Eight lung cancer cell lines, where of three adenocarcinoma and five squamous cell carcinomas, were included in this project. Each cell line was characterized with immunohistochemistry (IHC) for commonly expressed antigens in the two lung cancer types. Also, protein detection with Western Blots was performed. Cell lines found not expressing relevant antigens associated with the specific lung cancer variant were discarded. The remaining cell lines were cultivated in BIOFLOAT TC-Platte 96 well plate, Nunclon Sphera flasks, or GrowDex-T hydrogel, later transferred to a Corning Elplasia 6 well plate, and monitored for tumoroid generation. Results: The lung squamous cell carcinomas HCC-95 and HCC-1588 were both positive for CK5 and p40, while H1703 was negative for both markers. The adenocarcinoma H1975 was positive for TTF-1, also, unexpectedly, seen for NCI-H1703, earlier defined as squamous cell carcinoma. The tested cell lines were successfully cultured as tumoroids in the BIOFLOAT TC- 96 well plate, Nunclon Sphera flasks, and GrowDex-T hydrogel. The mutational status of the tumoroids as compared to the original tumors is currently under evaluation. Conclusion: We here show successful 3D culturing of cell lines with alternative substrates. Further fine-tuning of the chosen reproducible system's simplicity and cost is required before culturing tumoroids from lung cancer patients can be launched. Citation Format: Karina Malmros, Daniel Carlsén, Hans Brunnström. Standardized conditions for growth of lung cancer tumoroids cultured reproducibly in 3D-cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7561.

Abstract 1492: Unveiling the re-programming of choroid plexus and leptomeningeal metastasis

Abstract Leptomeningeal metastasis (LM) is an increasingly common, fatal complication of breast and lung cancer. Despite aggressive treatment, neurologic deficits accumulate rapidly, and patients generally succumb to LM within months. We must expand our limited mechanistic knowledge of this disease. Under homeostatic conditions, choroid plexus (ChP), highly vascularized structures within the brain ventricles, restrict the entry of macromolecules and cells into the leptomeninges; however, select cancer cells can cross this barrier and grow within the leptomeningeal space. We hypothesize that interactions between cancer and ChP niche cells alter both the niche and the cancer cells, ultimately supporting LM.During neurogenesis, the ChP display substantial extracellular matrix (ECM) remodeling, which is elegantly modulated by dynamic spatiotemporal regulation of MMPs, Wnt signaling, among other effector proteins. Similarly, 2-photon imaging of ChP collected from mice harboring LM revealed a profound ECM remodeling of the ChP when compared with naïve mice. This remodeling was accompanied by increased levels of MMP2 and MMP9 in LM mice. Upon pharmacological inhibition of MMP2 and MMP9 in vivo, we observed a decrease in LM burden and increase in overall survival, which correlates with a decrease in ChP permeability. Interestingly, human CSF from patients with leptomeningeal metastases is also enriched in collagen 1 fragments, MMP2, MMP9 and TIMP1. TIMP1, a MMP9 natural inhibitor, is not present in complex with MMP9, suggesting a role of TIMP1 as a cytokine in LM progression. TIMP1 neutralization and knockout from the cancer cells leads to a decrease in LM burden and increase in overall survival. Together, these data suggest a provocative link between proteolysis and inflammation in LM progression. The use of clinically-annotated human samples and mouse models of LM, and the integration of transcriptomics and proteomics enabled us to identify key signaling pathways, and capture the re-programming of ChP niche and cancer cells that unlock the leptomeningeal space to cancer cells. The timeframe for this evolution will be further revealed by analysis of mouse and human samples at different stages of cancer progression. Citation Format: Ana Rita Nobre, Helen Wang, Rachel Estrera, Isaiah Osei-Gyening, Adrienne Boire. Unveiling the re-programming of choroid plexus and leptomeningeal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1492.

Influence of Remodeled ECM and Co-culture with iPSC-Derived Cardiac Fibroblasts on the Mechanical Function of Micropatterned iPSC-Derived Cardiomyocytes.

In native heart tissue, functions of cardiac fibroblasts (CFs) include synthesis, remodeling, and degradation of the extracellular matrix (ECM) as well as secreting factors that regulate cardiomyocyte (CM) function. The influence of direct co-culture and CF-derived ECM on CM mechanical function are not fully understood. Here we use an engineered culture platform that provides control over ECM geometry and substrate stiffness to evaluate the influence of iPSC-CFs, and the ECM they produce, on the mechanical function of iPSC-CMs. Mechanical analysis was performed using digital image correlation to quantify maximum contractile strain, spontaneous contraction rate, and full-field organization of the contractions. When cultured alone, iPSC-CFs produce and remodel the ECM into fibers following the underlying 15° chevron patterned ECM. The substrates were decellularized and confirmed to have highly aligned fibers that covered a large fraction of the pattern area before reseeding with iPSC-CMs, alone or in co-culture with iPSC-CFs. When seeded on decellularized ECM, larger maximum contractile strains were observed in the co-culture condition compared to the CM Only condition. No significant difference was found in contractile strain between the Matrigel and decellularized ECM conditions; however, the spontaneous contraction rate was lower in the decellularized ECM condition. A methodology for quantifying alignment of cell contraction across the entire field of view was developed based on trajectories approximating the cell displacements during contraction. Trajectory alignment was unaltered by changes in culture or ECM conditions. These combined observations highlight the important role CFs play in vivo and the need for models that enable a quantitative approach to examine interactions between the CFs and CMs, as well as the interactions of these cells with the ECM.

Abstract B023: Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion

Abstract New therapies targeting metastatic spread are greatly needed to improve breast cancer patient survival. Recently, obscurin, an ~800kDa protein localized to the cell membrane, has risen to the spotlight as an overlooked metastasis suppressor commonly lost in breast cancer. Specifically, breast epithelial cells lacking obscurin undergo epithelial-mesenchymal transition (EMT) and demonstrate increased migration and invasion. Biochemical studies in our lab have linked breast epithelial obscurin loss to increased activation of the pro-metastatic PI3K/Akt signaling pathway. Mechanistically, the obscurin pleckstrin homology (PH)-domain directly binds the SH3-domain of the p85 regulatory subunit of PI3K. Thus far, due to obscurin’s massive size, gain of function/rescue experiments of obscurin have not been feasible. To combat this challenge, we have generated a mini-obscurin protein construct composed of an obscurin core functional unit: a myristilated obscurin PH-domain (Myr-PH). We hypothesized that expression of Myr-PH in obscurin-low breast cancer cells will reduce PI3K/Akt pathway activity and suppress metastasis. Remarkably, we report that ectopic expression of Myr-PH in the obscurin-low MDA-MB-231 (triple negative) and SKBr3 (HER2 positive) breast cancer cell lines diminishes PI3K/Akt pathway activity. Furthermore, Myr-PH sequesters p85 to the cell membrane, anchoring actin at the cell periphery. Functionally, we demonstrate that expression of Myr-PH in both cell line receptor subtypes: 1) reduces cellular migration collectively through decreased scratch assay wound closure and decreased transwell assay 8um pore migration or at the single cell level, through reduced 3um diameter microchannel migration, 2) diminishes cell seeding on multiple extracellular matrix (ECM) substrates known to populate metastatic microenvironments, and 3) decreases cell dissemination and tumorsphere invasion into a 3D collagen-1 matrix. Underlying these functional changes, we show that Myr-PH inhibits actin-dependent filopodia and invadopodia formation and decreases the expression of downstream PI3K/Akt target matrix metalloproteinases, all of which are potent drivers of metastatic dissemination. To this end, we are currently delivering the obscurin-PH domain in animals via lipid nanoparticles, aiming to examine its anti-metastatic efficacy in vivo. If successful, this work would pioneer the first gene therapy-based, targeted use of a single metastasis suppressor protein domain to block PI3K signaling and inhibit breast cancer metastasis. Citation Format: Matthew K Eason, Se-Jong Lee, Poornima Dubey, Anthony Kim, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B023.

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

Artificial keloid skin models: understanding the pathophysiological mechanisms and application in therapeutic studies.

Keloid is a type of scar formed by the overexpression of extracellular matrix substances from fibroblasts following inflammation after trauma. The existing keloid treatment methods include drug injection, surgical intervention, light exposure, cryotherapy, etc. However, these methods have limitations such as recurrence, low treatment efficacy, and side effects. Consequently, studies are being conducted on the treatment of keloids from the perspective of inflammatory mechanisms. In this study, keloid models are created to understand inflammatory mechanisms and explore treatment methods to address them. While previous studies have used animal models with gene mutations, chemical treatments, and keloid tissue transplantation, there are limitations in fully reproducing the characteristics of keloids unique to humans, and ethical issues related to animal welfare pose additional challenges. Consequently, studies are underway to create in vitro artificial skin models to simulate keloid disease and apply them to the development of treatments for skin diseases. In particular, herein, scaffold technologies that implement three-dimensional (3D) full-thickness keloid models are introduced to enhance mechanical properties as well as biological properties of tissues, such as cell proliferation, differentiation, and cellular interactions. It is anticipated that applying these technologies to the production of artificial skin for keloid simulation could contribute to the development of inflammatory keloid treatment techniques in the future.

The contribution of reticular basement membrane proteins to basal airway epithelial attachment, spreading and barrier formation: implications for airway remodeling in asthma.

In the healthy lung, the pseudostratified conducting airway epithelium is anchored to the reticular basement membrane (RBM) via hemidesmosome junction complexes formed between basal cells and the extracellular matrix (ECM). The RBM within the healthy lung is composed of the ECM proteins laminin and collagen-IV. In patients with asthma, the RBM is remodeled with collagen-I, -III and fibronectin deposition. The goal of this study was to assess the effect of RBM ECM proteins on basal airway epithelial cell attachment, spreading and barrier formation using real-time electrical cell-substrate impedance sensing (ECIS). ECIS 8-well arrays were coated with 50 μg/mL of fibronectin, collagen-I, collagen-III, collagen-IV, or laminin and compared to bovine serum albumin (BSA) or uncoated controls. The airway epithelial cell line (1HAEo-) was seeded 40, 50, 60, and 70 k cells/well and continuously monitored over 70 h to assess cell attachment, spreading and barrier formation using high (64 k Hz) and low (500 Hz) frequency resistance and capacitance. Data were analyzed using a one-phase decay model from which half-life (time cells cover half of the electrode area) and rate-constant (cell-spreading rate/h) were determined and a generalized additive mixed effect model (GAMM) was used to assess ECM proteins over the entire experiment. High-frequency (64 kHz) capacitance measures demonstrated the half-life for 1HAEo-cells to attach was fastest when grown on fibronectin (6.5 h), followed by collagen-I (7.2 h) and collagen-III (8.1 h), compared to collagen-IV (11.3 h), then laminin (13.2 h) compared to BSA (12.4 h) and uncoated (13.9 h) controls. High-frequency (64 kHz) resistance measures demonstrated that the rate of 1HAEo- cell spreading was significantly faster on fibronectin and collagen-I compared to collagen-III, collagen-IV, laminin, BSA and the uncoated control. Low-frequency (500 Hz) resistance measures demonstrated that 1HAEo-cells formed a functional barrier fastest when grown on fibronectin and collagen-I, compared to the other ECM conditions. Lastly, the distance of 1HAEo-cells from the ECM substrates was the smallest when grown on fibronectin reflecting high cell-matrix adhesion. Airway epithelial cells attach, spread and form a barrier fastest on fibronectin, and collagen-I and these reticular basement membrane ECM proteins may play a protective role in preserving the epithelial barrier during airway remodeling in asthma.

Retinal Progenitor Cells Exhibit Cadherin-Dependent Chemotaxis across Transplantable Extracellular Matrix of In Vitro Developmental and Adult Models

Retinal degeneration is an escalating public health challenge, as diseases such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa cause irreversible vision loss in millions of adults each year. Regenerative medicine has pioneered the development of stem cell replacement therapies, which treat degeneration by replacing damaged retinal neurons with transplanted stem-like cells (SCs). While the collective migration of SCs plays critical roles during retinal development, our understanding of collective SC behaviors within biomaterials transplanted into adult tissue remains understudied. This project examines the potential therapeutic impacts of collective SC migration during transplantation by correlating the expression of cadherin, cell-cell cohesion molecules that maintain intercellular communication during development, with receptor proteins of chemoattractant molecules prevalent in degenerated adult tissue. Experiments examine these well-conserved biomechanisms by using two different model organisms: Drosophila melanogaster, a seminal model for retinal development, and Mus, an important preclinical model for transplantation. Results indicate that SCs from both animal models significantly upregulate cadherin expression to achieve more directed collective migration towards species-specific chemoattractants and exhibit longer distance motility upon different extracellular matrix substrates.

BSLD-06 UNVEILING THE RE-PROGRAMMING OF CHOROID PLEXUS AND LEPTOMENINGEAL METASTASIS

Abstract Leptomeningeal metastasis (LM) is an increasingly common, fatal complication of breast and lung cancer. Despite aggressive treatment, neurologic deficits accumulate rapidly, and patients generally succumb to LM within months. We must expand our limited mechanistic knowledge of this disease. Under homeostatic conditions, choroid plexus (ChP), highly vascularized structures within the brain ventricles, restrict the entry of macromolecules and cells into the leptomeninges; however, select cancer cells can cross this barrier and grow within the leptomeningeal space. We hypothesize that interactions between cancer and ChP niche cells alter both the niche and the cancer cells, ultimately supporting LM. During neurogenesis, the ChP display substantial extracellular matrix (ECM) remodeling, which is elegantly modulated by dynamic spatiotemporal regulation of MMPs, Wnt signaling, among other effector proteins. Similarly, 2-photon imaging of ChP collected from mice harboring LM revealed a profound ECM remodeling of the ChP when compared with naïve mice. This remodeling was accompanied by increased levels of MMP2 and MMP9 in the CSF. Furthermore, single-cell-RNA-sequencing of ChP reveals that Wnt is the top differentially expressed pathway in metastatic ChP in comparison with normal ChP, supporting a role for developmental signaling within the ChP in LM. Interestingly, SPARC, a major ECM modulator in tissue repair and an essential component of the stem cell niche chemoattractant factors secreted by the subventricular zone (SVZ), is enriched in metastatic mouse ChP suggesting that SVZ- and ChP-secreted signals are key in LM progression. The use of clinically-annotated human samples and mouse models of LM, and the integration of transcriptomics and proteomics enabled us to identify key signaling pathways, and capture the re-programming of ChP niche and cancer cells that unlock the leptomeningeal space to cancer cells. The timeframe for this evolution will be further revealed by analysis of mouse and human samples at different stages of cancer progression.

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM-polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms.

Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.

Innovative Approaches and Advances for Hair Follicle Regeneration.

Pathological hair loss (also known as alopecia) and shortage of hair follicle (HF) donors have posed an urgent requirement for HF regeneration. With the revelation of mechanisms in tissue engineering, the proliferation of HFs in vitro has achieved more promising trust for the treatments of alopecia and other skin impairments. Theoretically, HF organoids have great potential to develop into native HFs and attachments such as sweat glands after transplantation. However, since the rich extracellular matrix (ECM) deficiency, the induction characteristics of skin-derived cells gradually fade away along with their trichogenic capacity after continuous cell passaging in vitro. Therefore, ECM-mimicking support is an essential prelude before HF transplantation is implemented. This review summarizes the status of providing various epidermal and dermal cells with a three-dimensional (3D) scaffold to support the cell homeostasis and better mimic in vivo environments for the sake of HF regeneration. HF-relevant cells including dermal papilla cells (DPCs), hair follicle stem cells (HFSCs), and mesenchymal stem cells (MSCs) are able to be induced to form HF organoids in the vitro culture system. The niche microenvironment simulated by different forms of biomaterial scaffold can offer the cells a network of ordered growth environment to alleviate inductivity loss and promote the expression of functional proteins. The scaffolds often play the role of ECM substrates and bring about epithelial-mesenchymal interaction (EMI) through coculture to ensure the functional preservation of HF cells during in vitro passage. Functional HF organoids can be formed either before or after transplantation into the dermis layer. Here, we review and emphasize the importance of 3D culture in HF regeneration in vitro. Finally, the latest progress in treatment trials and critical analysis of the properties and benefits of different emerging biomaterials for HF regeneration along with the main challenges and prospects of HF regenerative approaches are discussed.

2023 TERMIS – AMERICAS Conference &amp; Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference &amp; Exhibition Boston Marriott Copley Place April 11–14, 2023

Heparin and β2-microglobulin amyloidogenesis

β2-Microglobulin (β2M) occurs as a precursor protein in dialysis-related amyloidosis (DRA), which is a major complication in lives of patients undergoing dialysis. However, the underlying mechanism by which a native β2M transform into an amyloid β2M remain unclear. This disease has developed exclusively in interstitial tissues, which suggest possible implication of extracellular matrix substances in amyloidogenesis of this precursor protein. By using our monoclonal antibody specific for amyloid β2M, we investigated the function of heparin, that is, one of the glycosaminoglycans, as associated with amyloidogenic conversion of the β2M molecule. We confirmed that heparin induced a dose-dependent and time-dependent unfolding at the C-terminal region of the β2M molecule, which led to amyloidogenic transformation of the β2M molecule and brought about intermolecular interactions between β2M and substances in the interstitial matrix such as GAGs and collagen.

The role of extracellular matrix proteins on placental angiogenesis in severe fetal growth restriction

Impaired vascular development is a central finding in severe fetal growth restriction (FGR) with abnormal Doppler velocimetry. Angiogenesis involves complex interactions between endothelial cells (ECs) and extracellular matrix (ECM). Our laboratory previously found that FGR ECs exhibit deficient angiogenic properties, a deficit that is further worsened in the presence of ECM generated from FGR placentae but partially rescued by control ECM. Our objective was to identify the effects of individual ECM substrates on EC function to better characterize the impaired angiogenic pathway characteristic of FGR. ECs were isolated from term control (n=8) or severe FGR (n=8) human placentae and plated on six individual ECM proteins: fibronectin; collagens I, III, and IV; laminin; and thrombospondin. Adhesion was assessed by colorimetric staining. Live cell microscopy was used to determine proliferation. Migration was analyzed via relative wound density following wound scratch. Apoptosis was evaluated by immunoblotting for cleaved caspase 3. T tests, ANOVAs, and nonlinear regressions were performed. There was no difference between adhesion of control and FGR ECs to individual ECM substrates. Control ECs proliferated more rapidly than FGR ECs (p< 0.01; figure 1). Deficient growth of FGR ECs was not rescued by any ECM substrate. Migration of FGR ECs was impaired relative to control ECs when accounting for all substrates (p=0.02). This finding persisted specifically in the presence of collagen I (p=0.03; figure 2) but not other substrates. Apoptosis was not detected for either type of ECs on any substrate. ECs from placentae affected by severe FGR display impaired proliferation and migration. This growth deficit appears to be independent of individual ECM substrates. FGR migratory defects persist on collagen I but not other substrates. Our data suggest that no singular ECM protein is responsible for the aberrant angiogenesis seen in severe FGR and rather there are likely multiple mechanisms underlying this pathology.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. We identified ADAMTS3 as a potential therapeutic target for GBM.