Function Follows Form: Oriented Substrate Nanotopography Overrides Neurite-Repulsive Schwann Cell-Astrocyte Barrier Formation in an In Vitro Model of Glial Scarring.

Schwann cell (SC) transplantation represents a promising therapeutic approach for traumatic spinal cord injury but is frustrated by barrier formation, preventing cell migration, and axonal regeneration at the interface between grafted SCs and reactive resident astrocytes (ACs). Although regenerating axons successfully extend into SC grafts, only a few cross the SC-AC interface to re-enter lesioned neuropil. To date, research has focused on identifying and modifying the molecular mechanisms underlying such scarring cell-cell interactions, while the influence of substrate topography remains largely unexplored. Using a recently modified cell confrontation assay to model SC-AC barrier formation in vitro, highly oriented poly(ε-caprolactone) nanofibers were observed to reduce AC reactivity, induce extensive oriented intermingling between SCs and ACs, and ultimately enable substantial neurite outgrowth from the SC compartment into the AC territory. It is anticipated that these findings will have important implications for the future design of biomaterial-based scaffolds for nervous tissue repair.

A Deep-Learning-Computed Cancer Score for the Identification of Human Hepatocellular Carcinoma Area Based on a Six-Colour Multiplex Immunofluorescence Panel.

Liver cancer is one of the most frequently diagnosed and fatal cancers worldwide, with hepatocellular carcinoma (HCC) being the most common primary liver cancer. Hundreds of studies involving thousands of patients have now been analysed across different cancer types, including HCC, regarding the effects of immune infiltrates on the prognosis of cancer patients. However, for these analyses, an unambiguous delineation of the cancer area is paramount, which is difficult due to the strong heterogeneity and considerable inter-operator variability induced by qualitative visual assessment and manual assignment. Nowadays, however, multiplex analyses allow the simultaneous evaluation of multiple protein markers, which, in conjunction with recent machine learning approaches, may offer great potential for the objective, enhanced identification of cancer areas with further in situ analysis of prognostic immune parameters. In this study, we, therefore, used an exemplary five-marker multiplex immunofluorescence panel of commonly studied markers for prognosis (CD3 T, CD4 T helper, CD8 cytotoxic T, FoxP3 regulatory T, and PD-L1) and DAPI to assess which analytical approach is best suited to combine morphological and immunohistochemical data into a cancer score to identify the cancer area that best matches an independent pathologist's assignment. For each cell, a total of 68 individual cell features were determined, which were used as input for 4 different approaches for computing a cancer score: a correlation-based selection of individual cell features, a MANOVA-based selection of features, a multilayer perceptron, and a convolutional neural network (a U-net). Accuracy was used to evaluate performance. With a mean accuracy of 75%, the U-net was best capable of identifying the cancer area. Although individual cell features showed a strong heterogeneity between patients, the spatial representations obtained with the computed cancer scores delineate HCC well from non-cancer liver tissues. Future analyses with larger sample sizes will help to improve the model and enable direct, in-depth investigations of prognostic parameters, ultimately enabling precision medicine.

Quantitative Characterization of Macrophage, Lymphocyte, and Neutrophil Subtypes Within the Foreign Body Granuloma of Human Mesh Explants by 5-Marker Multiplex Fluorescence Microscopy.

Foreign bodies such as fibers of a surgical mesh induce a typical reaction with an inflammatory infiltrate that forms a surrounding granuloma. This infiltrate is dominated by macrophages, lymphocytes, and neutrophils, whereas its extent of collaboration is widely unknown. In this study, we analyzed 12 samples of surgical meshes explanted from humans by multiplex analyses with three different 5-marker panels - 1. macrophage panel: CD68, CD86, CD105, CD163, and CD206; 2. lymphocyte panel: CD3, CD4, CD8, CD20, and CD68; and 3. neutrophil panel: CD15, histone, MPO, NE, and CD68. Measurement of fluorescence intensity within nuclear masks resulting from DAPI nuclear staining allows exact quantification of cells considered "positive" at a user-defined mean intensity threshold of > 100. Obviously, however, there is no natural threshold as a biological criterion for an intensity that separates "positive" stained cells from unstained cells ("negative"). Multiplex staining of 5 markers always reveals a high rate of coexpression for almost all of the 25 possible marker combinations (= 32 combinations, when using 5 markers simultaneously). The present staining results demonstrate that various morphological and functional subtypes of macrophages, lymphocytes, and neutrophils are abundant in the foreign body granuloma (FBG), which were investigated by regions of interest (ROI) with an area of 1 mm2. The widespread coexpression of two or more markers underscores the complex collaboration network of the inflammatory infiltrate. The ability to combine spatial distribution with exact numerical analysis may offer new perspectives for our understanding of the complex interactions in this multidimensional process.

Degradation resistance of PVDF mesh in vivo in comparison to PP mesh.

Mesh implant has been applied in hernia repair and urogynecological reconstruction. Polypropylene (PP) is now the most widely used material for non-resorbable mesh implants. A degradation phenomenon of PP mesh, which is apparent on the mesh surface as cracking, flaking and peeling, was discovered in the 1990's. This phenomenon of mesh implant has drawn attention because of mesh-related litigations. Polyvinylidene fluoride (PVDF), due to its high biocompatible performance, has been used since 2003 as an alternative material for non-resorbable mesh implants. Till now, no such degradation phenomenon of PVDF mesh has been reported, although limited study on PVDF mesh is available. In this paper, we researched the degradation of PVDF meshes taking the degradation of PP mesh as a reference. The meshes analysed in this study were received from a previous animal experiment. To expose the surface of explanted meshes, a tissue removing method with protease was used and the result of this cleaning process was tested by X-ray Photoelectron Spectroscopy (XPS). The morphological condition of the mesh surface was compared using Scanning Electron Microscopy (SEM) and the chemical condition concerning degradation was analysed through Fourier Transform Infrared Spectroscopy (FTIR). The surface condition of PVDF mesh after 3-, 6-, 12- and 24-month implantation was illustrated and compared with two types of PP meshes. XPS revealed an absence of nitrogen, confirming the successful removal of tissue residues using protease. SEM results presented no notable morphological surface change of the PVDF mesh and progressive surface cracking processes over time of both types of PP meshes. FTIR spectra of the implanted PVDF meshes had no considerable difference from the spectrum of the pristine mesh, while FTIR spectra of both types of PP meshes had extra chemical functional groups (carbonyl (CO) and hydroxyl (-OH) groups) increasing with implantation time, indicating progressive degradation. This study highlights the morphological and chemical stability of the PVDF mesh and demonstrates that the PVDF mesh is more resistant to degradation in comparison to the other two types of PP meshes.

A novel in vitro assay for peripheral nerve-related cell migration that preserves both extracellular matrix-derived molecular cues and nanofiber-derived topography.

BACKGROUND: Molecular composition and topography of the extracellular matrix (ECM) influence regenerative cell migration following peripheral nerve injury (PNI). Advanced tissue engineering strategies for the repair of neurotmesis-type PNI include the development of nanofiber-containing implantable scaffolds that mimic features of the ECM to orchestrate regenerative growth. Reliable and quantifiable in vitro assays are required to assess the ability of such substrates to influence migration of the cell types of interest. However, most popular migration assays monitor cell migration into a cell exclusion zone (CEZ) but have dubious abilities to preserve the molecular and topographical cues of the substrate. NEW METHOD: Elastic band spacers (EBS), a simple, economical and standardized technique for the generation of well-defined CEZ based on the use of commercially available elastic bands, are introduced. RESULTS: EBS could sufficiently preserve ECM-derived molecular and poly(ε-caprolactone) (PCL) nanofiber-derived topographical cues. The application of EBS in the absence and presence of nanofiber-derived topographical cues was validated using perineurial cells and Schwann cells, both known to play key roles in peripheral nerve regeneration. COMPARISON WITH EXISTING METHODS: In contrast to EBS, commercial silicone inserts and the popular scratch assay caused substantial ECM substrate disruption, thereby preventing these techniques from being included in further investigations employing deposition of PCL nanofibers and cell migration analysis. CONCLUSIONS: EBS represent a useful addition to the existing repertoire of migration assays offering significant benefits in terms of substrate preservation. The simplicity and economy of the approach make it immediately accessible to research groups at minimal extra expense.

CD68+ macrophages as crucial components of the foreign body reaction demonstrate an unconventional pattern of functional markers quantified by analysis with double fluorescence staining.

Implants like meshes for the reinforcement of tissues implement the formation of a persistent inflammation with an ambient fibrotic reaction. In the inflammatory infiltrate several distinct cell types have been identified, but CD68+ macrophages are supposed to be most important. To investigate the collaboration among the various cell types within the infiltrate we performed at explanted meshes from humans double fluorescence staining with CD68 as a constant marker and a variety of other antibodies as the second marker. The list of second markers includes lymphocytes (CD3, CD4, CD8, CD16, CD56, FoxP3, and CD11b) stem cells (CD34), leucocytes (CD45, CD15), macrophages (CD86, CD105, CD163, and CD206); deposition of EC matrix (collagen-I, collagen-III, MMP2, and MMP8); Ki67 as a marker for proliferation; and the tyrosine-protein kinase receptor AXL. The present study demonstrates within the inflammatory infiltrate the abundant capability of CD68+ cells to co-express a huge variety of other markers, including those of lymphocytes, varying between 5 and 83% of investigated cells. The observation of co-staining was not restricted to a specific polymer but was seen with polypropylene fibers as well as with fibers made of polyvinylidene fluoride, although with differences in co-expression rates. The persisting variability of these cells without the functional reduction toward differentiated mature cell types may favor the lack of healing at the interface of meshes.