Beyond Encapsulation: Exploring Macrophage-Fibroblast Cross Talk in Implant-Induced Fibrosis.

The foreign body response (FBR) and organ fibrosis are complex biological processes involving the interaction between macrophages and fibroblasts. Understanding the molecular mechanisms underlying macrophage-fibroblast cross talk is crucial for developing strategies to mitigate implant encapsulation, a major cause of implant failure. This article reviews the current knowledge on the role of macrophages and fibroblasts in the FBR and organ fibrosis, highlighting the similarities between these processes. The FBR is characterized by the formation of a fibrotic tissue capsule around the implant, leading to functional impairment. Various factors, including material properties such as surface chemistry, stiffness, and topography, influence the degree of encapsulation. Cross talk between macrophages and fibroblasts plays a critical role in both the FBR and organ fibrosis. However, the precise molecular mechanisms remain poorly understood. Macrophages secrete a wide range of cytokines that modulate fibroblast behavior such as abundant collagen deposition and myofibroblast differentiation. However, the heterogeneity of macrophages and fibroblasts and their dynamic behavior in different tissue environments add complexity to this cross talk. Experimental evidence from in vitro studies demonstrates the impact of material properties on macrophage cytokine secretion and fibroblast physiology. However, the correlation between in vitro response and in vivo encapsulation outcomes is not robust. Adverse outcome pathways (AOPs) offer a potential framework to understand and predict process complexity. AOPs describe causal relationships between measurable events leading to adverse outcomes, providing mechanistic insights for in vitro testing and predictive modeling. However, the development of an AOP for the FBR does require a comprehensive understanding of the molecular initiating events and key event relationships to identify which events are essential. In this article, we describe the current knowledge on macrophage-fibroblast cross talk in the FBR and discuss how targeted research can help build an AOP for implant-related fibrosis.

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.

Improved science-based transformation pathways for the development of safe and sustainable plastics.

Projected plastic production volumes are rising, as is societal and political attention to plastic pollution and possible health impacts. In line with ambitions for climate mitigation and the circular economy, various national and international policies and action plans address the reduction of impacts of plastics. Quantitative scenario analyses show that even if current ambitious targets to reduce plastics are achieved, plastics will remain a source of millions of tons of environmental pollution annually. To achieve a sustainable transformation of the global plastics economy, 'extraordinary effort' and 'coordinated global action' beyond current ambitions are needed. While mapping knowledge gaps for the effects of micro and nano plastics (MNP) is crucial, mapping alone is not enough to achieve the needed transition. In this communication, we propose a scope for the exploration of societal transformation pathways to safe and sustainable plastics. To see which efforts are needed globally we need to advance in the following three areas: (i) embedding risk assessment methodologies in wider cost-benefit and life cycle analyses; (ii) using safe-and-sustainable design strategies that include alternative solutions and look at multiple life cycles, and (iii) reflecting on the societal transformation pathways with stakeholders by using co-created quantitative models. We believe that these practices are crucial in the coming decade to realise the extraordinary effort of defining safe and sustainable plastics.

On the correlation between material-induced cell shape and phenotypical response of human mesenchymal stem cells.

Learning rules by which cell shape impacts cell function would enable control of cell physiology and fate in medical applications, particularly, on the interface of cells and material of the implants. We defined the phenotypic response of human bone marrow-derived mesenchymal stem cells (hMSCs) to 2176 randomly generated surface topographies by probing basic functions such as migration, proliferation, protein synthesis, apoptosis, and differentiation using quantitative image analysis. Clustering the surfaces into 28 archetypical cell shapes, we found a very strict correlation between cell shape and physiological response and selected seven cell shapes to describe the molecular mechanism leading to phenotypic diversity. Transcriptomics analysis revealed a tight link between cell shape, molecular signatures, and phenotype. For instance, proliferation is strongly reduced in cells with limited spreading, resulting in down-regulation of genes involved in the G2/M cycle and subsequent quiescence, whereas cells with large filopodia are related to activation of early response genes and inhibition of the osteogenic process. In this paper we were aiming to identify a universal set of genes that regulate the material-induced phenotypical response of human mesenchymal stem cells. This will allow designing implants that can actively regulate cellular, molecular signalling through cell shape. Here we are proposing an approach to tackle this question.

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

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 5-10 µm 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.

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.

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.

Micro-scaled topographies direct differentiation of human epidermal stem cells.

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. STATEMENT OF SIGNIFICANCE: The epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

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.

The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment.

Mesenchymal stromal cells (MSC) secrete factors that contribute to organ homeostasis and repair in a tissue specific manner. For instance, kidney perivascular mesenchymal stromal cells (kPSCs) can facilitate renal epithelial repair through secretion of hepatocyte growth factor (HGF) while the secretome of bone marrow MSCs gives rise to immunosuppression. Stromal cells function in a complex 3-dimensional (3D) connective tissue architecture that induces conformational adaptation. Here we tested the hypothesis that surface topography and associated cell adaptations dictate stromal cell function through tuning of the cytokines released. To this end, we cultured human bone marrow and kidney perivascular stromal cells in the TopoWell plate, a custom-fabricated multi-well plate containing 76 unique bioactive surface topographies. Using fluorescent imaging, we observed profound changes in cell shape, accompanied by major quantitative changes in the secretory capacity of the MSCs. The cytokine secretion profile was closely related to cell morphology and was stromal cell type specific. Our data demonstrate that stromal cell function is determined by microenvironment structure and can be manipulated in an engineered setting. Our data also have implications for the clinical manufacturing of mesenchymal stromal cell therapy, where surface topography during bioreactor expansion should be taken into account to preserve therapeutic properties.

NanoTopoChip: High-throughput nanotopographical cell instruction.

Surface topography is able to influence cell phenotype in numerous ways and offers opportunities to manipulate cells and tissues. In this work, we develop the Nano-TopoChip and study the cell instructive effects of nanoscale topographies. A combination of deep UV projection lithography and conventional lithography was used to fabricate a library of more than 1200 different defined nanotopographies. To illustrate the cell instructive effects of nanotopography, actin-RFP labeled U2OS osteosarcoma cells were cultured and imaged on the Nano-TopoChip. Automated image analysis shows that of many cell morphological parameters, cell spreading, cell orientation and actin morphology are mostly affected by the nanotopographies. Additionally, by using modeling, the changes of cell morphological parameters could by predicted by several feature shape parameters such as lateral size and spacing. This work overcomes the technological challenges of fabricating high quality defined nanoscale features on unprecedented large surface areas of a material relevant for tissue culture such as PS and the screening system is able to infer nanotopography - cell morphological parameter relationships. Our screening platform provides opportunities to identify and study the effect of nanotopography with beneficial properties for the culture of various cell types. STATEMENT OF SIGNIFICANCE: The nanotopography of biomaterial surfaces can be modified to influence adhering cells with the aim to improve the performance of medical implants and tissue culture substrates. However, the necessary knowledge of the underlying mechanisms remains incomplete. One reason for this is the limited availability of high-resolution nanotopographies on relevant biomaterials, suitable to conduct systematic biological studies. The present study shows the fabrication of a library of nano-sized surface topographies with high fidelity. The potential of this library, called the 'NanoTopoChip' is shown in a proof of principle HTS study which demonstrates how cells are affected by nanotopographies. The large dataset, acquired by quantitative high-content imaging, allowed us to use predictive modeling to describe how feature dimensions affect cell morphology.

Mining for osteogenic surface topographies: In silico design to in vivo osseo-integration.

Stem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships - the relationships between substrate parameters and the phenotypes they induce - have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated.

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.

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 >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, ß-catenin, and FAK could be classified with >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.