Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling.

Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to \u3e 90% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, β-catenin, and FAK could be classified with \u3e 90% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a “fingerprint” of the intracellular mechanotransductive signaling that governs growth factor- and topography-responsive stem cell states

Immune Modulation by Design: Using Topography to Control Human Monocyte Attachment and Macrophage Differentiation

© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Macrophages play a central role in orchestrating immune responses to foreign materials, which are often responsible for the failure of implanted medical devices. Material topography is known to influence macrophage attachment and phenotype, providing opportunities for the rational design of “immune-instructive” topographies to modulate macrophage function and thus foreign body responses to biomaterials. However, no generalizable understanding of the inter-relationship between topography and cell response exists. A high throughput screening approach is therefore utilized to investigate the relationship between topography and human monocyte–derived macrophage attachment and phenotype, using a diverse library of 2176 micropatterns generated by an algorithm. This reveals that micropillars 5–10µm in diameter play a dominant role in driving macrophage attachment compared to the many other topographies screened, an observation that aligns with studies of the interaction of macrophages with particles. Combining the pillar size with the micropillar density is found to be key in modulation of cell phenotype from pro to anti-inflammatory states. Machine learning is used to successfully build a model that correlates cell attachment and phenotype with a selection of descriptors, illustrating that materials can potentially be designed to modulate inflammatory responses for future applications in the fight against foreign body rejection of medical devices

Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells

Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols

Dynamic adaptation of mesenchymal stem cell physiology upon exposure to surface micropatterns

Human mesenchymal stem (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. Even though it is common knowledge that cells behave differently in vitro compared to in vivo, only little is known about the underlying adaptation processes. Here, we used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during this adaptation to their new environment. We used well established techniques to compare hMSCs cultured on flat and topographically enhanced polystyreneand observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro

Understanding public perceptions toward sustainable healthcare through psychological network analysis of material preference and attitudes toward plastic medical devices

Recent and potential future health-care users (i.e., the public) are important stakeholders in the transition toward environmentally sustainable healthcare. However, it remains unclear whether, according to the public, there is room for sustainable innovations in materials for plastic medical devices (PMD). This study explores preferences regarding conventional or bio-based PMD, and psychological mechanisms underlying these preferences. We administered two surveys among Dutch adults from a research panel. Results from the first survey (i.e., open-text survey on attitude elements; NStudy1 = 66) served as input for the second survey (i.e., Likert-scale survey on beliefs, emotions, perceived control, social norms, trust, related to current and bio-based PMD, and health and age; NStudy2 = 1001; Mage = 47.35; 54.4% female). The second survey was completed by 501 participants who, in the last two years, received care in which PMD were used, and 500 participants who did not. Cross-sectional psychological networks were estimated with data from the second study using the EBICglasso method. Results showed that participants preferred bio-based over conventional PMD, and this applied regardless of whether devices are used inside or outside of the body. Results also showed emotions play an important role, with emotions regarding bio-based PMD being strongly related to preference. Furthermore, comparing recent and potential future receivers of PMD revealed differences in preference but comparable relations between preference and other psychological variables. This study shows that receivers’ perspectives should not be seen as potential barriers, but as additional motivation for transitioning toward sustainable healthcare. Recommendations for implementation are discussed

Understanding public perceptions toward sustainable healthcare through psychological network analysis of material preference and attitudes toward plastic medical devices.

Recent and potential future health-care users (i.e., the public) are important stakeholders in the transition toward environmentally sustainable healthcare. However, it remains unclear whether, according to the public, there is room for sustainable innovations in materials for plastic medical devices (PMD). This study explores preferences regarding conventional or bio-based PMD, and psychological mechanisms underlying these preferences. We administered two surveys among Dutch adults from a research panel. Results from the first survey (i.e., open-text survey on attitude elements; NStudy1 = 66) served as input for the second survey (i.e., Likert-scale survey on beliefs, emotions, perceived control, social norms, trust, related to current and bio-based PMD, and health and age; NStudy2 = 1001; Mage = 47.35; 54.4% female). The second survey was completed by 501 participants who, in the last two years, received care in which PMD were used, and 500 participants who did not. Cross-sectional psychological networks were estimated with data from the second study using the EBICglasso method. Results showed that participants preferred bio-based over conventional PMD, and this applied regardless of whether devices are used inside or outside of the body. Results also showed emotions play an important role, with emotions regarding bio-based PMD being strongly related to preference. Furthermore, comparing recent and potential future receivers of PMD revealed differences in preference but comparable relations between preference and other psychological variables. This study shows that receivers' perspectives should not be seen as potential barriers, but as additional motivation for transitioning toward sustainable healthcare. Recommendations for implementation are discussed

TopoWellPlate:a well-plate-based screening platform to study cell-surface topography interactions

The field of biomaterial engineering is increasingly using high‐throughput approaches to investigate cell–material interactions. Because most material libraries are prepared as chips, immunofluorescence‐based read‐outs are used to uniquely image individual materials. This paper proposes to produce libraries of materials using a well‐based strategy in which each material is physically separated, and thus compatible with standard biochemical assays. In this work, the TopoWellPlate, a novel system to study cell–surface topography interaction in high‐throughput is presented. From a larger library of topographies, 87 uniquely defined bioactive surface topographies are identified, which induce a wide variety of cellular morphologies. Topographically enhanced polystyrene films are fabricated in a multistep cleanroom process and served as base for the TopoWellPlate. Thermal bonding of the films to bottomless 96‐well plates results in a cell culture ready, topographically enhanced, 96‐well plate. The overall metabolic activity of bone marrow‐derived human mesenchymal stem cells is measured to show the functionality of the TopoWellPlate as a screening tool, which showed a 2.5‐fold difference range in metabolic activity per cell. TopoWellPlates of this and other topographical designs can be used to analyze cells using the wealth of standardized molecular assays available and thus disclose the mechanisms of biomaterials‐induced mechanotransduction

Identification of topographical architectures supporting the phenotype of rat tenocytes

Tenocytes, the main cell type of the tendon, require mechanical stimuli for their proper function. When the tenocyte environment changes due to tissue damage or by transferring tenocytes from their native environment into cell culture, the signals from the tenocyte niche are lost, leading towards a decline of phenotypic markers. It is known that micro-topographies can influence cell fate by the physical cues they provide. To identify the optimal topography-induced biomechanical niche in vitro, we seeded tenocytes on the TopoChip, a micro-topographical screening platform, and measured expression of the tendon transcription factor Scleraxis. Through machine learning algorithms, we associated elevated Scleraxis levels with topological design parameters. Fabricating micro-topographies with optimal surface characteristics on larger surfaces allowed finding an improved expression of multiple tenogenic markers. However, long-term confluent culture conditions coincided with osteogenic marker expression and the loss of morphological characteristics. In contrast, passaging tenocytes which migrated from the tendon directly on the topography resulted in prolonged elongated morphology and elevated Scleraxis levels. This research provides new insights into how micro-topographies influence tenocyte cell fate, and supports the notion that micro-topographical design can be implemented in a new generation of tissue culture platforms for supporting the phenotype of tenocytes. Statement of Significance: The challenge in controlling in vitro cell behavior lies in controlling the complex culture environment. Here, we present for the first time the use of micro-topographies as a biomechanical niche to support the phenotype of tenocytes. For this, we applied the TopoChip platform, a screening tool with 2176 unique micro-topographies for identifying feature characteristics associated with elevated Scleraxis expression, a tendon related marker. Large area fabrication of micro-topographies with favorable characteristics allowed us to find a beneficial influence on other tenogenic markers as well. Furthermore, passaging cells is more beneficial for Scleraxis marker expression and tenocyte morphology compared to confluent conditions. This study presents important insights for the understanding of tenocyte behavior in vitro, a necessary step towards tendon engineering