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.

Characterization of a Novel Aspect of Tissue Scarring Following Experimental Spinal Cord Injury and the Implantation of Bioengineered Type-I Collagen Scaffolds in the Adult Rat: Involvement of Perineurial-like Cells?

Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant-host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.

Heterozygous POLG variant Ser1181Asn co-segregating in a family with autosomal dominant axonal neuropathy, proximal muscle fatigability, ptosis, and ragged red fibers.

By whole-exome sequencing, we found the heterozygous POLG variant c.3542G>A; p.Ser1181Asn in a family of four affected individuals, presenting with a mixed neuro-myopathic phenotype. The variant is located within the active site of polymerase gamma, in a cluster region associated with an autosomal dominant inheritance. In adolescence, the index developed distal atrophies and weakness, sensory loss, afferent ataxia, double vision, and bilateral ptosis. One older sister presented with Charcot-Marie-Tooth-like symptoms, while the youngest sister and father reported exercise-induced muscle pain and proximal weakness. In none of the individuals, we observed any involvement of the central nervous system. Muscle biopsies obtained from the father and the older sister showed ragged-red fibers, and electron microscopy confirmed mitochondrial damage. We conclude that this novel POLG variant explains this family's phenotype.

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.

Design of Suspended Melt Electrowritten Fiber Arrays for Schwann Cell Migration and Neurite Outgrowth.

In this study, well-defined, 3D arrays of air-suspended melt electrowritten fibers are made from medical grade poly(ɛ-caprolactone) (PCL). Low processing temperatures, lower voltages, lower ambient temperature, increased collector distance, and high collector speeds all aid to direct-write suspended fibers that can span gaps of several millimeters between support structures. Such processing parameters are quantitatively determined using a "wedge-design" melt electrowritten test frame to identify the conditions that increase the suspension probability of long-distance fibers. All the measured parameters impact the probability that a fiber is suspended over multimillimeter distances. The height of the suspended fibers can be controlled by a concurrently fabricated fiber wall and the 3D suspended PCL fiber arrays investigated with early post-natal mouse dorsal root ganglion explants. The resulting Schwann cell and neurite outgrowth extends substantial distances by 21 d, following the orientation of the suspended fibers and the supporting walls, often generating circular whorls of high density Schwann cells between the suspended fibers. This research provides a design perspective and the fundamental parametric basis for suspending individual melt electrowritten fibers into a form that facilitates cell culture.

Dense fibroadhesive scarring and poor blood vessel-maturation hamper the integration of implanted collagen scaffolds in an experimental model of spinal cord injury.

Severe spinal cord injury (SCI) results in permanent functional deficits, which despite pre-clinical advances, remain untreatable. Combinational approaches, including the implantation of bioengineered scaffolds are likely to promote significant tissue repair. However, this critically depends on the extent to which host tissue can integrate with the implant. In the present paper, blood vessel formation and maturation were studied within and around implanted micro-structured type-I collagen scaffolds at 10 weeks post implantation in adult rat mid-cervical spinal cord lateral funiculotomy injuries. Morphometric analysis revealed that blood vessel density within the scaffold was similar to that of the lateral white matter tracts that the implant replaced. However, immunohistochemistry for zonula occludens-1 (ZO-1) and endothelial barrier antigen revealed that scaffold microvessels remained largely immature, suggesting poor blood-spinal cord barrier (BSB) reformation. Furthermore, a band of intense ZO-1-immunoreactive fibroblast-like cells isolated the implant. Spinal cord vessels outside the ZO-1-band demonstrated BSB-formation, while vessels within the scaffold generally did not. The formation of a double-layered fibrotic and astroglial scar around the collagen scaffold might explain the relatively poor implant-host integration and suggests a mechanism for failed microvessel maturation. Targeted strategies that improve implant-host integration for such biomaterials will be vital for future tissue engineering and regenerative medicine approaches for traumatic SCI.

Fibroadhesive scarring of grafted collagen scaffolds interferes with implant-host neural tissue integration and bridging in experimental spinal cord injury.

Severe traumatic spinal cord injury (SCI) results in a devastating and permanent loss of function, and is currently an incurable condition. It is generally accepted that future intervention strategies will require combinational approaches, including bioengineered scaffolds, to support axon growth across tissue scarring and cystic cavitation. Previously, we demonstrated that implantation of a microporous type-I collagen scaffold into an experimental model of SCI was capable of supporting functional recovery in the absence of extensive implant-host neural tissue integration. Here, we demonstrate the reactive host cellular responses that may be detrimental to neural tissue integration after implantation of collagen scaffolds into unilateral resection injuries of the adult rat spinal cord. Immunohistochemistry demonstrated scattered fibroblast-like cell infiltration throughout the scaffolds as well as the presence of variable layers of densely packed cells, the fine processes of which extended along the graft-host interface. Few reactive astroglial or regenerating axonal profiles could be seen traversing this layer. Such encapsulation-type behaviour around bioengineered scaffolds impedes the integration of host neural tissues and reduces the intended bridging role of the implant. Characterization of the cellular and molecular mechanisms underpinning this behaviour will be pivotal in the future design of collagen-based bridging scaffolds intended for regenerative medicine.