Cell-matrix Interactions Research Articles

Three-Dimensional Organotypic Systems for Modelling and Understanding Molecular Regulation of Oral Dentogingival Tissues

Three-dimensional organotypic models benefit from the ability to mimic physiological cell–cell or cell–matrix interactions and therefore offer superior models for studying pathological or physiological conditions compared to 2D cultures. Organotypic models consisting of keratinocytes supported by fibroblasts embedded in collagen matrices have been utilised for the study of oral conditions. However, the provision of a suitable model for investigating the pathogenesis of periodontitis has been more challenging. Part of the complexity relates to the different regional epithelial specificities and connective tissue phenotypes. Recently, it was confirmed, using 3D organotypic models, that distinct fibroblast populations were implicated in the provision of specific inductive and directive influences on the overlying epithelia. This paper presents the organotypic model of the dentogingival junction (DGJ) constructed to demonstrate the differential fibroblast influences on the maintenance of regionally specific epithelial phenotypes. Therefore, the review aims are (1) to provide the biological basis underlying 3D organotypic cultures and (2) to comprehensively detail the experimental protocol for the construction of the organotypic cultures and the unique setup for the DGJ model. The latter is the first organotypic culture model used for the reconstruction of the DGJ and is recommended as a useful tool for future periodontal research.

MicroRNA expression profiles in plasma exosomes of late pregnant giant pandas.

MicroRNAs can regulate various biological functions including cell proliferation, differentiation, embryo formation, and implantation. The giant panda exhibits embryonic diapause, with embryo development resuming in late pregnancy. However, the changes in microRNAs during late pregnancy remain poorly understand. After mating, plasma samples were collected on day 40 of early pregnancy (EP; n = 3) and 30 days before delivery of late pregnancy (LP; n = 3). Following microRNAs screening, a total of 120 microRNAs were detected in the plasma exosomes of pregnant pandas. Nine differentially expressed microRNAs (DEmicroRNAs) were identified in LP compared to EP, including three that were upregulated and six that were downregulated. Notably, miR-25b and miR-47 were significantly downregulated in LP group. All DEmicroRNAs were predicted to target a total of 2,675 genes. Pathway enrichment analysis of these target genes revealed significant enrichment in the MAPK and Rap1 signaling pathways, which are closely related to cell proliferation, differentiation, and cell-cell and cell-matrix interactions. Analysis of protein-protein interaction networks showed that most of the hub genes (five out of eight), including Fgfr1, Fgf2, Fgf18, Erbb4, and Kras within the MAPK and Rap1 pathways are associated with the cell proliferation and differentiation. Significantly, Erbb4 was regulated by significantly differentially expressed miRNA-47. We suggest that plasma exosomal microRNAs are involved in cell proliferation and differentiation during embryonic development by regulating key hub genes within MAPK and Rap1 pathways. These findings provided new insights into the development of giant panda embryos.

Neodymium-Facilitated Visualization of Extreme Phosphate Accumulation in Fibroblast Filopodia: Implications for Intercellular and Cell–Matrix Interactions

A comprehensive understanding of intercellular and cell–matrix interactions is essential for advancing our knowledge of cell biology. Existing techniques, such as fluorescence microscopy and electron microscopy, face limitations in resolution and sample preparation. Supravital lanthanoid staining provides new opportunities for detailed visualization of cellular metabolism and intercellular interactions. This study aims to describe the structure, elemental chemical, and probable origin of zones of extreme lanthanoid (neodymium) accumulation that form during preparation for scanning electron microscopy (SEM) analysis in corneal fibroblasts filopodia. The results identified three morphological patterns of neodymium staining in fibroblast filopodia, each exhibiting asymmetric staining within a thin, sharp, and extremely bright barrier zone, located perpendicular to the filopodia axis. Semi-quantitative chemical analyses showed neodymium-labeled non-linear phosphorus distribution within filopodia, potentially indicating varying phosphate anion concentrations and extreme phosphate accumulation at a physical or physicochemical barrier. Phosphorus zones labeled with neodymium did not correspond to mitochondrial clusters. During apoptosis, the number of filopodia with extreme and asymmetric phosphorus accumulation increases. Supravital lanthanoid staining coupled with SEM allows detailed visualization of intercellular and cell–matrix interactions with high contrast and resolution. These results enhance our understanding of phosphate anion accumulation and transfer mechanisms in cells under normal conditions and during apoptosis.

RGDSP-functionalized peptide hydrogel stimulates growth factor secretion via integrin αv/PI3K/AKT axis for improved wound healing by human amniotic mesenchymal stem cells.

The wound healing process involves communication among growth factors, cytokines, signaling pathways, and cells in the extracellular matrix, with growth factors acting as key regulators. Although stem cells can promote wound healing by secreting diverse growth factors, their therapeutic potential is hindered by poor survival and engraftment. Mimicking the stem cell-matrix interactions can improve stem cell survival, regulate their fate, and even enhance their paracrine effects. This study investigated the use of composite RGDmix hydrogel, which can support the survival and proliferation of human amniotic mesenchymal stem cells (hAMSCs), and effectively increase the expression of various growth factors, thereby promoting wound re-epithelialization, angiogenesis, and epidermal maturation. At last, the specific role of integrin αv and PI3K/AKT signaling pathways in the secretion of growth factors were examined by silencing them in vitro and in vivo. Results suggested that the RGDmix hydrogel improved the secretion of growth factors by hAMSCs through the RGDSP/integrin αv/PI3K/AKT axis, thereby enhancing the therapeutic effect in wound healing.

Microvascular Engineering for the Development of a Nonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do Not Lose Their Edge.

Microvascular engineering seeks to exploit known cell-cell and cell-matrix interactions in the context of vasculogenesis to restore homeostasis or disease development of reliable capillary models in vitro. However, current systems generally focus on recapitulating microvessels embedded in thick gels of extracellular matrix, overlooking the significance of discontinuous capillaries, which play a vital role in tissue-blood exchanges particularly in organs like the liver. In this work, we introduce a novel method to stimulate the spontaneous organization of endothelial cells into nonembedded microvessels. By creating an anisotropic micropattern at the edge of a development-like matrix dome using Marangoni flow, we achieved a long, nonrandom orientation of endothelial cells, laying a premise for stable lumenized microvessels. Our findings revealed a distinctive morphogenetic process leading to mature lumenized capillaries, demonstrated with both murine and human immortalized liver sinusoidal endothelial cell lines (LSECs). The progression of cell migration, proliferation, and polarization was clearly guided by the pattern, initiating the formation of a multicellular cord that caused a deformation spanning extensive regions and generated a wave-like folding of the gel, hinged at a laminin-depleted zone, enveloping the cord with gel proteins. This event marked the onset of lumenogenesis, regulated by the gradual apico-basal polarization of the wrapped cells, leading to the maturation of vessel tight junctions, matrix remodeling, and ultimately the formation of a lumen─recapitulating the development of vessels in vivo. Furthermore, we demonstrate that the process strongly relies on the initial gel edge topography, while the geometry of the vessels can be tuned from a curved to a straight structure. We believe that our facile engineering method, guiding an autonomous self-organization of vessels without the need for supporting cells or complex prefabricated scaffolds, holds promise for future integration into microphysiological systems featuring discontinuous, fenestrated capillaries.

Integrating N -glycan and CODEX imaging reveal cell-specific protein glycosylation in healthy human lung.

N -linked glycosylation, the major post-translational modification of cellular proteins, is important for proper lung functioning, serving to fold, traffic, and stabilize protein structures and to mediate various cell-cell recognition events. Identifying cell-specific N -glycan structures in human lungs is critical for understanding the chemistry and mechanisms that guide cell-cell and cell-matrix interactions and determining nuanced functions of specific N -glycosylation. Our study, which used matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combined with co-detection by indexing (CODEX) to reveal the cellular origin of N -glycans, is a significant step in this direction. This innovative technological combination enabled us to detect and differentiate N -glycans located in the vicinity of cells surrounding airways and blood vessels, parenchyma, submucosal glands, cartilage, and smooth muscles. The potential impact of our findings on future research is immense. For instance, our algorithm for grouping N -glycans based on their functional chemical features, combined with identifying group niches, paves the way for targeted studies. We found that fucosylated N -glycans are dominant around immune cells, tetra antennary N -glycans in the cartilage, high-mannose N -glycans surrounding the bronchus originate from associated collagenous structures, complex fucosylated-tetra antennary-polylactosamine N -glycans are spread over smooth muscle structures and in epithelial cells surrounding arteries, and N -glycans with Hex:6 HexNAc:6 compositions, which, according to our algorithm, can be ascribed to either tetra antennary or bisecting N -glycan, are highly abundant in the parenchyma. The findings suggest cell or region-specific functions for these localized glycan structures.

Potential Oncogenic and Immunosuppressive Roles of Multiple EGF-Like Domains 6 (MEGF6) in Colon Cancer: Insights from Bioinformatic Analysis

Multiple epidermal growth factor-like domains protein 6 (MEGF6) is a high molecular weight protein with several EGF-like domains. Although it has been demonstrated to promote metastasis and chemoresistance in colon cancer cells, its specific oncogenic and immunologic functions remain largely unexplored. This study aims to comprehensively analyze publicly available datasets to define the role of MEGF6 in colon cancer development and immune response modulation. The expression of MEGF6 in colon adenocarcinoma (COAD) and normal tissues at both mRNA and protein levels was assessed using the GEPIA2 and HPA databases, respectively. The UALCAN database was employed to examine the association between MEGF6 expression and clinicopathological outcomes in COAD patients, and survival analysis was conducted using the KM plotter database. Functional enrichment analysis of MEGF6-correlated genes was performed using the ShinyGO online tool. Additionally, the correlation of MEGF6 with immune cell infiltration and immunosuppressive molecules was explored through the TIMER and TISIDB databases. Our analysis revealed significantly higher mRNA and protein expressions of MEGF6 in COAD tissues compared to normal tissues, with associations to cancer stage, nodal metastasis status, and histological subtype. High MEGF6 expression was correlated with poorer survival outcomes, and genes correlated with MEGF6 were enriched in biological processes related to cellular component organization, cell adhesion, and extracellular matrix interaction. Furthermore, MEGF6 expression demonstrated a significant correlation with immune cell infiltration and the expression of immunosuppressive molecules, particularly CTLA-4, PD-1, PD-L1, TGF-β, and IL-10. In conclusion, our findings suggest that MEGF6 may play an oncogenic role by influencing cell and extracellular matrix interactions. Its overexpression may indicate immunosuppressive properties within the cancer microenvironment. Therefore, MEGF6 holds promise as a potential biomarker for predicting both prognosis and therapeutic responses in colon cancer. HIGHLIGHTS MEGF6 has been shown to involve colon cancer growth and chemotherapeutic resistance. Bioinformatics confirmed a prognostic significance of MEGF6 in colon cancer. MEGF6 may exert its oncogenic effects by influencing cell-matrix interactions. MEGF6 expression is correlated with immunosuppressive properties of cancers. GRAPHICAL ABSTRACT

Droplet Microfluidics Powered Hydrogel Microparticles for Stem Cell-Mediated Biomedical Applications.

Stem cell-related therapeutic technologies have garnered significant attention of the research community for their multi-faceted applications. To promote the therapeutic effects of stem cells, the strategies for cell microencapsulation in hydrogel microparticles have been widely explored, as the hydrogel microparticles have the potential to facilitate oxygen diffusion and nutrient transport alongside their ability to promote crucial cell-cell and cell-matrix interactions. Despite their significant promise, there is an acute shortage of automated, standardized, and reproducible platforms to further stem cell-related research. Microfluidics offers an intriguing platform to produce stem cell-laden hydrogel microparticles (SCHMs) owing to its ability to manipulate the fluids at the micrometer scale as well as precisely control the structure and composition of microparticles. In this review, the typical biomaterials and crosslinking methods for microfluidic encapsulation of stem cells as well as the progress in droplet-based microfluidics for the fabrication of SCHMs are outlined. Moreover, the important biomedical applications of SCHMs are highlighted, including regenerative medicine, tissue engineering, scale-up production of stem cells, and microenvironmental simulation for fundamental cell studies. Overall, microfluidics holds tremendous potential for enabling the production of diverse hydrogel microparticles and is worthy for various stem cell-related biomedical applications.

Dense Collagen I as a Biomimetic Material to Track Matrix Remodelling in Renal Carcinomas.

Aims: Renal tissue is a dynamic biophysical microenvironment, regulating healthy function and influencing tumor development. Matrix remodelling is an iterative process and aberrant tissue repair is prominent in kidney fibrosis and cancer. Biomimetic 3D models recapitulating the collagen composition and mechanical fidelity of native renal tissue were developed to investigate cell-matrix interactions in renal carcinomas. Methods: Collagen I and laminin hydrogels were engineered with renal cancer cells (ACHN and 786-O), which underwent plastic compression to generate dense matrices. Mechanical properties were determined using shear rheology and qPCR determined the gene expression of matrix markers. Results: The shear modulus and phase angle of acellular dense collagen I gels (474 Pa and 10.7) are similar to human kidney samples (1410 Pa and 10.5). After 21 days, 786-O cells softened the dense matrix (∼155 Pa), with collagen IV downregulation and upregulation of matrix metalloproteinases (MMP7 and MMP8). ACHN cells were found to be less invasive and stiffened the matrix to ∼1.25 kPa, with gene upregulation of collagen IV and the cross-linking enzyme LOX. Conclusions: Renal cancer cells remodel their biophysical environment, altering the material properties of tissue stroma in 3D models. These models can generate physiologically relevant stiffness to investigate the different matrix remodelling mechanisms utilized by cancer cells.

Hydrogels with Independently Controlled Adhesion Ligand Mobility and Viscoelasticity Increase Cell Adhesion and Spreading.

A primary objective in designing hydrogels for cell culture is recreating the cell-matrix interactions found within human tissues. Identifying the most important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands and the ability of these ligands to support cellular forces. In this work we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tailor the storage modulus of the hydrogel by altering the concentration and composition of each network, and we can tune the stress relaxation half-life through the non-covalent bonding in the PA network. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the RGD cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. Interestingly, our findings show that endothelial cell adhesion formation and spreading is maximized in soft, viscoelastic gels in which RGD adhesion ligands are present on both the covalent PEG and non-covalent PA networks. The dynamic nature of cell adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or are capable of withstanding significant forces are needed mimic different aspects of complex cell-matrix adhesions. By demonstrating how different presentations of RGD ligands affect cell behavior independently of viscoelastic properties, these results contribute to the rational design of hydrogels that facilitate desired cell-matrix interactions, with the potential of improving in vitro models and regenerative therapies.

Fibrosis uncovered: ADAMTS12 cuts to the core of extracellular matrix drama.

Fibrosis is a common manifestation of most progressive and degenerative diseases, with myofibroblast activation and matrix accumulation playing a key role. In this issue of the JCI, Hoeft et al. identify the important role of ADAMTS12 in fibroblast activation. ADAMTS12, a secreted protein, is involved in extracellular matrix (ECM) remodeling, cell signaling, and inflammation. ADAMTS12 facilitates proteolysis by cleaving various substrates such as ECM components, which are vital for cellular signaling and remodeling. Additionally, it modulates cell-matrix interactions, influencing cell adhesion and migration, and plays an important role in the inflammatory processes. Understanding the role of ADAMTS12 offers potential therapeutic insights for targeting fibrosis in progressive diseases.

Stimuli-Responsive Substrates to Control the Immunomodulatory Potential of Stromal Cells.

Mesenchymal stromal cells (MSCs) have broad immunomodulatory properties that range from regulation, proliferation, differentiation, and immune cell activation to secreting bioactive molecules that inhibit inflammation and regulate immune response. These properties provide MSCs with high therapeutic potency that has been shown to be relevant to tissue engineering and regenerative medicine. Hence, researchers have explored diverse strategies to control the immunomodulatory potential of stromal cells using polymeric substrates or scaffolds. These substrates alter the immunomodulatory response of MSCs, especially through biophysical cues such as matrix mechanical properties. To leverage these cell-matrix interactions as a strategy for priming MSCs, emerging studies have explored the use of stimuli-responsive substrates to enhance the therapeutic value of stromal cells. This review highlights how stimuli-responsive materials, including chemo-responsive, microenvironment-responsive, magneto-responsive, mechano-responsive, and photo-responsive substrates, have specifically been used to promote the immunomodulatory potential of stromal cells by controlling their secretory activity.

Abstract B075: Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant cancer- associated fibroblasts (CAFs) which contribute to a tumor-promoting, chemotherapy-resistant and immunosuppressive tumor microenvironment (TME). However, CAF-ablating therapeutic strategies have thus far failed in clinical trials. One potential reason is that PDAC CAFs are heterogeneous and plastic. It was recently discovered that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) can originate from pancreatic stellate cells (PSCs) via TGF-β and IL-1 signaling, respectively. However, their functional heterogeneity remains to be elucidated. We hypothesize that distinct CAF subtypes exhibit different tumor-promoting roles mediated by targetable signaling mechanisms. To this end, we aimed at characterizing the functions of TGF- β-dependent myCAFs in shaping the TME and influencing PDAC progression. Methods: We generated novel myCAF-depleted PDAC mouse models by orthotopically injecting PDAC organoids derived from KPC (Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre) mice into Fap-Tgfbr2 knockout (Fap-tTA; Rosa26 LSLtdTom/+ ; tet-O-Cre; Tgfbr2 fl/fl ) mice. The pancreatic tumors were analyzed by flow cytometry, histology and single-cell RNA-sequencing (scRNAseq). In a complementary approach, we generated myCAF-enriched PDAC mouse models by orthotopically co-injecting KPC PDAC organoids and novel TGF-β-driven ‘myCAF-locked’ murine PSCs. In addition, to further dissect myCAF-malignant cell signaling crosstalk, KPC PDAC organoids and myCAF-locked PSCs were co-cultured and then flow-sorted for bulk RNA- sequencing. Results: MyCAF-depleted PDAC tumors from Fap-Tgfbr2 knockout mice had a significant decrease in myCAFs, and a significant expansion of iCAFs, compared to control mice. Additionally, Masson’s trichrome staining indicated significant reduction in collagen content in the TME upon myCAF depletion. Moreover, scRNAseq analysis revealed that myCAF depletion shaped the composition of innate immune cells, including macrophages and neutrophils. Notably, although primary tumor growth was not altered, liver and lung metastases decreased, while diaphragm metastases and ascites increased, in myCAF-depleted PDAC mice. Conversely, liver and lung metastases increased, while diaphragm metastases and ascites decreased in myCAF-enriched PDAC mouse models, suggesting that TGF-β-dependent myCAFs play a role in promoting distant metastases. In line with this, transcriptomic analysis of PDAC organoids co-cultured with myCAF-locked PSCs showed enrichment of pathways in epithelial-mesenchymal transition, hypoxia, VEGF, WNT and cell-matrix interactions. Conclusion: Our study demonstrates that TGF-β-dependent CAFs are not only largely responsible for collagen deposition in PDAC, but also possess immune-modulating properties. Moreover, our study supports a role of TGF-β-dependent CAFs in promoting PDAC metastasis to the liver and the lungs, potentially through upregulating pro-metastatic pathways in the malignant cells. Citation Format: Priscilla S.W. Cheng, Muntadher Jihad, Judhell S. Manansala, Weike Luo, Gianluca Mucciolo, Sara Pinto Teles, Giulia Biffi. Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B075.

Abstract A054: Collagen fiber architecture modulates PDAC cell and organoid phenotype

Abstract Introduction Collagen plays a significant role in the development of pancreatic ductal adenocarcinoma (PDAC). Disease progression is mediated by a dense collagenous stroma, which acts as a barrier to drug delivery and immune cell access. Current in vitro models do not well represent the collagenous stroma seen in PDAC. The aim of this study is to develop a fibrillar collagen-based hydrogel system which mimics the tumour microenvironment seen in PDAC and assess cell-mediated collagen remodeling. Methods Metastatic SUIT2-007 pancreatic cancer cells, non-metastatic BxPC3 pancreatic cancer cells, and human pancreatic stellate cells (hPSCs) were encapsulated in bovine type I collagen gels of various concentrations. KPC mouse derived tumour organoids were also encapsulated in bovine type I collagen gels of 2 mg/ml and 6 mg/ml concentrations, basement membrane extract (BME), and a combination of BME and collagen gel. Cell viability and proliferation was assessed using Live/Dead imaging and CellTitre-Glo 3D respectively. Cell mediated collagen remodeling was assessed via monitoring of gel contraction on the mm scale, and confocal reflectance microscopy on the cellular scale. Collagen contraction around cells was assessed by measuring the pixel intensity of collagen fibrils within the vicinity of the cell. Cell phenotype was assessed via immunofluorescent staining and qPCR. Results Collagen concentration alters the hydrogel structure and stiffness, with 3 mg/ml collagen resulting in straighter, shorter and thicker fibrils. Metastatic SUIT-007 cells contracted collagen gels more rapidly than non-metastatic BxPC3 cells, with both achieving similar contraction by day 7. SUIT2-007 cells remodel and align soft 1 mg/ml gels while 3 mg/ml gels proved to be sufficiently stiff to resist remodeling. Cellular collagen association was increased in more elongated SUIT-007 cells than in BxPC3 cells. Organoid proliferation varies with hydrogel collagen concentration. Conclusion Metastatic SUIT2-007 cells were able to remodel collagen more rapidly and to a greater extent than non-metastatic BxPC3 cells. Collagen supports an invasive organoid phenotype. Collagen gels combined with confocal reflectance microscopy provide a platform for the quantitative assessment of cell-matrix interactions in PDAC. Acknowledgements This project was funded by a Medical Traineeship from UCD School of Medicine, and summer student scholarships from Irish Cancer Society and Breakthrough Cancer Research. We acknowledge the Conway Imaging Core Facility for assistance with microscopy. Citation Format: Ciara L Doyle, Shanu Xavier, Jarren Y.S Lu, Leah Fallon, Anwesha Sarkar, Jan J Baguyo, Maela Gars, Stephen D Thorpe. Collagen fiber architecture modulates PDAC cell and organoid phenotype [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A054.

Optimizing miRNA transfection for screening in precision cut lung slices.

Precision cut lung slices(PCLS) are complex 3D lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules(e.g. miRNA). The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. 4mm diameter PCLS were generated using agarose-filled rodent lungs and a vibratome. TYE665 labelled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualised using live high content fluorescence microscopy, followed by higher resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using WST-1 and lactate dehydrogenase(LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2TM, we established that cellular membranes in PCLS are permeable in the initial 24 hours of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 hours after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared to other LNPs, with detection possible as low as 25nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.

Influence of dexamethasone-induced matrices on the TM transcriptome

Pathologic bidirectional interactions between the extracellular matrix (ECM) and cells within the human trabecular meshwork (hTM) contribute to ocular hypertension. An in vitro model is needed to study these cell-matrix interactions and their effect on outflow homeostasis. This study aimed to determine whether pathogenic ECM derived from dexamethasone (DEX)-treated hTM cultures induces clinically relevant glaucoma-like changes in healthy hTM cells at the transcriptional level. Corneoscleral rims from non-glaucoma donors were used to isolate primary hTM cells after validation according to the consensus recommendations for TM culture. Normal hTM cells (n = 5) were plated on a coverslip and treated with 100 nM DEX or ethanol for four weeks. These cultures were then decellularized, plated with primary hTM cells, and allowed to grow for another 72 h. RNA was extracted from these hTM cells for stranded total RNA-Seq. Sequencing libraries prepared using the Zymo-Seq RiboFree Total RNA library kit were pooled and sequenced using Illumina NovaSeq 6000. After quality control, sequence reads were aligned to the human genome build hg19. Differential expression (DE) analyses were performed using paired multi-factorial ANOVA. The expression of several DE genes associated with glaucoma (ANGPTL2, PDE7B, C22orf23, COL4A1, ADAM12, IFT122, SEMA6C) was validated using EvaGreen-based Droplet Digital PCR (ddPCR) assays. Gene ontology analyses of the DE genes were performed using the PANTHER and NDEx IQA databases, and functional analyses were performed with the DAVID Bioinformatics software. Using a cutoff of p-value <0.05 and fold change ≥2.0, our differential analysis identified 267 up- and 135 down-regulated genes in DEX-induced ECM-treated cells compared to the control. These differentially expressed genes were found to play a significant role in pathways such as cytokine and oxidative stress-induced inflammation, integrin signaling, matrix remodeling, and angiogenesis. These findings were further supported by previously performed proteomics studies using the same model. Using ddPCR, we validated the expression of seven genes associated with the risk of primary open-angle glaucoma. These results not only provide support for the pathogenic ECM model of steroid-induced glaucoma, but also demonstrate that the pathologic changes induced by this model are indeed found at the transcriptional level. These findings further demonstrate that matrix changes significantly influence cell expression profiles, which enable further understanding of the molecular mechanisms underlying glaucomatous changes in the TM. However, future studies with a larger and more diverse set of samples and longer time points are needed to confirm the utility of this model for mechanistic studies.

Plasticity variable collagen-PEG interpenetrating networks modulate cell spreading

The extracellular matrix protein collagen I has been used extensively in the field of biomaterials due to its inherent biocompatibility and unique viscoelastic and mechanical properties. Collagen I self-assembly into fibers and networks is environmentally sensitive to gelation conditions such as temperature, resulting in gels with distinct network architectures and mechanical properties. Despite this, collagen gels are not suitable for many applications given their relatively low storage modulus. We have prepared collagen-poly(ethylene glycol) [PEG] interpenetrating network (IPN) hydrogels to reinforce the collagen network, which also induces changes to network plasticity, a recent focus of study in cell-matrix interactions. Here, we prepare collagen/PEG IPNs, varying collagen concentration and collagen gelation temperature to assess changes in microarchitecture and mechanical properties of these networks. By tuning these parameters, IPNs with a range of stiffness, plasticity and pore size are obtained. Cell studies suggest that matrix plasticity is a key determinant of cell behavior, including cell elongation, on these gels. This work presents a natural/synthetic biocompatible matrix that retains the unique structural properties of collagen networks with increased storage modulus and tunable plasticity. The described IPN materials will be of use for applications in which control of cell spreading is desirable, as only minimal changes in sample preparation lead to changes in cell spreading and circularity. Additionally, this study contributes to our understanding of the connection between collagen self-assembly conditions and matrix structural and mechanical properties and presents them as useful tools for the design of other collagen based biomaterials. Statement of significanceWe developed a collagen-poly(ethylene glycol) interpenetrating network (IPN) platform that retains native collagen architecture and biocompatibility but provides higher stiffness and tunable plasticity. With minor changes in collagen gelation temperature or concentration, IPN gels with a range of plasticity, storage modulus, and pore size can be obtained. The tunable plasticity of the gels is shown to modulate cell spreading, with a greater proportion of elongated cells on the most plastic of IPNs, supporting the assertion that matrix plasticity is a key determinant of cell spreading. The material can be of use for situations where control of cell spreading is desired with minimal intervention, and the findings herein may be used to develop similar collagen based IPN platforms.

Enhancing Cell Aggregation and Migration via Double-Click Cross-Linking with Azide-Modified Hyaluronic Acid.

We present a novel approach to the formation of cell aggregates by employing click chemistry with water-soluble zwitterionic dibenzo cyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N3) as a linker to facilitate rapid and stable cell aggregation. By optimizing the concentrations of HA-N3 and WS-CODY, we achieved efficient cross-linking between azide-modified cell surfaces and HA-N3, generating cell aggregates within 10 min, and the resulting aggregates remained stable for up to 5 days, with cell viability maintained at approximately 80%. Systematic experiments revealed that a stoichiometric balance between HA-N3 and WS-CODY is important for effective cross-linking, highlighting the roles of both cell-surface azide modification and HA in the aggregate formation. We also investigated the genetic basis of altered cell behavior within these aggregates. Transcriptome analysis (RNA-seq) of aggregates postcultivation revealed a marked fluctuation of genes associated with 'cell migration' and 'cell adhesion', including notable changes in the expression of HYAL1, ICAM-1, CEACAM5 and RHOB. These findings suggest that HA-N3-mediated cell aggregation can induce intrinsic cellular responses that not only facilitate cell aggregate formation but also modulate cell-matrix interactions. We term this phenomenon 'chemo-resilience', The simplicity and efficacy of this click chemistry-based approach suggest it may have broad applicability for forming cell aggregates and modulating cell-matrix interactions in tissue engineering and regenerative medicine.

Engineered 3D Periodontal Ligament Model with Magnetic Tensile Loading

In vitro models are invaluable tools for deconstructing the biological complexity of the periodontal ligament (PDL). Model systems that closely reproduce the 3-dimensional (3D) configuration of cell–cell and cell–matrix interactions in native tissue can deliver physiologically relevant insights. However, 3D models of the PDL that incorporate mechanical loading are currently lacking. Hence, we developed a model where periodontal tissue constructs (PTCs) are made by casting PDL cells in a collagen gel suspended between a pair of slender, silicone posts for magnetic tensile loading. Specifically, one of the posts was rigid and the other was flexible with a magnet embedded in its tip so that PTCs could be subjected to tensile loading with an external magnet. Additionally, the deflection of the flexible post could be used to measure the contractile force of PDL cells in the PTCs. Prior to tensile loading, second harmonics generation analysis of collagen fibers in PTCs revealed that incorporation of PDL cells resulted in collagen remodeling. Biomechanical testing of PTCs by tensile loading revealed an elastic response at 4 h, permanent deformation by 1 d, and creep elongation by 1 wk. Subsequently, contractile forces of PDL cells were substantially lower for PTCs under tensile loading. Immunofluorescence analysis revealed that tensile loading caused PDL cells to increase in number, express higher levels of F-actin and α–smooth muscle actin, and become aligned to the tensile axis. Second harmonics generation analysis indicated that collagen fibers in PTCs progressively remodeled over time with tensile loading. Gene expression analysis also confirmed tension-mediated upregulation of the F-actin/Rho pathway and osteogenic genes. Our model is novel in demonstrating the mechanobiological behavior that results in cell-mediated remodeling of the PDL tissue in a 3D context. Hence, it can be a valuable tool to develop therapeutics for periodontitis, periodontal regeneration, and orthodontics.

Multiphoton excited polymerized biomimetic models of collagen fiber morphology to study single cell and collective migration dynamics in pancreatic cancer

The respective roles of aligned collagen fiber morphology found in the extracellular matrix (ECM) of pancreatic cancer patients and cellular migration dynamics have been gaining attention because of their connection with increased aggressive phenotypes and poor prognosis. To better understand how collagen fiber morphology influences cell-matrix interactions associated with metastasis, we used Second Harmonic Generation (SHG) images from patient biopsies with Pancreatic ductal adenocarcinoma (PDAC) as models to fabricate collagen scaffolds to investigate processes associated with motility. Using the PDAC BxPC-3 metastatic cell line, we investigated single and collective cell dynamics on scaffolds of varying collagen alignment. Collective or clustered cells grown on the scaffolds with the highest collagen fiber alignment had increased E-cadherin expression and larger focal adhesion sites compared to single cells, consistent with metastatic behavior. Analysis of single cell motility revealed that the dynamics were characterized by random walk on all substrates. However, examining collective motility over different time points showed that the migration was super-diffusive and enhanced on highly aligned fibers, whereas it was hindered and sub-diffusive on un-patterned substrates. This was further supported by the more elongated morphology observed in collectively migrating cells on aligned collagen fibers. Overall, this approach allows the decoupling of single and collective cell behavior as a function of collagen alignment and shows the relative importance of collective cell behavior as well as fiber morphology in PDAC metastasis. We suggest these scaffolds can be used for further investigations of PDAC cell biology. Statement of significancePancreatic ductal adenocarcinoma (PDAC) has a high mortality rate, where aligned collagen has been associated with poor prognosis. Biomimetic models representing this architecture are needed to understand complex cellular interactions. The SHG image-based models based on stromal collagen from human biopsies afford the measurements of cell morphology, cadherin and focal adhesion expression as well as detailed motility dynamics. Using a metastatic cell line, we decoupled the roles of single cell and collective cell behavior as well as that arising from aligned collagen. Our data suggests that metastatic characteristics are enhanced by increased collagen alignment and that collective cell behavior is more relevant to metastatic processes. These scaffolds provide new insight in this disease and can be a platform for further experiments such as testing drug efficacy.