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

Relationship Between Particle Properties and Immunotoxicological Effects of Environmentally-Sourced Microplastics

Background: Concerns on microplastics (MPs) in food are increasing because of our increased awareness of daily exposure and our knowledge gap on their potential adverse health effects. When particles are ingested, macrophages play an important role in scavenging them, potentially leading to an unwanted immune response. To elucidate the adverse effects of MPs on human health, insights in the immunotoxicity of MPs are essential. Objectives: To assess the effect of environmentally collected ocean and land weathered MP particles on the immunological response of macrophages using a state-of-the art in vitro immunotoxicity assay specifically designed for measuring particle toxicity. Methods: Environmentally-weathered macroplastic samples were collected from the North Pacific Subtropical Gyre and from the French coastal environment. Macroplastics were identified using (micro)Raman-spectrometry, FT-IR and Py-GC-MS and cryo-milled to obtain size-fractionated samples up to 300 μm. Physiochemical MP properties were characterized using phase contrast microscopy, gel-permeation chromatography, nuclear magnetic resonance, and differential scanning colorimetry. Macrophages (differentiated THP-1 cells) were exposed to particles (<300 μm) for 48 h before assessment of cell viability and cytokine release. Using both the physiochemical particle properties and biological data, we performed multi-dimensional data analysis to explore relationships between particle properties and immunotoxicological effects. Results: We investigated land-derived polyethylene, polypropylene, polystyrene, and polyethylene terephthalate, water-derived polypropylene macroplastics, and virgin polyethylene fibers and nylon MPs. The different plastic polymeric compositions and MP size classes induced distinct cytokine responses. Macrophages had the largest response to polyethylene terephthalate-particle exposure, including a dose-related increase in IL-1β, IL-8, and TNF-α secretion. Smaller MPs induced cytokine production at lower concentrations. Additionally, a relationship between both physical and chemical particle properties and the inflammatory response of macrophages was found. Discussion: This research shows that MP exposure could lead to an inflammatory response in vitro, depending on MP material and size. Whether this implies a risk to human health needs to be further explored