GEORG SCHMORL PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2018: combined inflammatory and mechanical stress weakens the annulus fibrosus: evidences from a loaded bovine AF organ culture.

PURPOSE: The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae. METHODS: A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1ß (1 ng/mL) culture medium supplementation. AF-OCs were analysed for PGE2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound. RESULTS: The combination of CTS + IL-1ß led to a significant increase in PGE2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1ß group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1ß group compared to Control (p < 0.05). CONCLUSION: Our findings suggest that CTS and IL-1ß act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure-function relationship and is a platform to test AF-focused therapeutics. These slides can be retrieved under Electronic Supplementary Material.

A Microfluidic High-Capacity Screening Platform for Neurological Disorders.

Compartmentalized cell cultures (CCCs) provide the possibility to study mechanisms of neurodegenerative diseases, such as spreading of misfolded proteins in Alzheimer's or Parkinson's disease or functional changes in, e.g., chronic pain, in vitro. However, many CCC devices do not provide the necessary capacity for identifying novel mechanisms, targets, or drugs in a drug discovery context. Here, we present a high-capacity cell culture microtiter microfluidic plate compliant with American National Standard Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) standards that allows to parallelize up to 96 CCCs/experimental units, where each experimental unit comprises three microchannel-connected compartments. The plate design allows the specific treatment of cells in individual compartments through the application of a fluidic barrier. Moreover, the compatibility of the plate with neuronal cultures was confirmed with rodent primary as well as human-induced pluripotent stem cell-derived neurons of the central or peripheral nervous system for up to 14 days in culture. Using immunocytochemistry, we demonstrated that the plate design restricts neuronal soma to individual compartments, while axons, but not dendrites, can grow through the connecting microchannels to neighboring compartments. In addition, we show that neurons are spontaneously active and, as deemed by the appearance of synchronous depolarizations in neighboring compartments, are synaptically coupled. In summary, the design of the microfluidic plate allows for both morphological and functional studies of neurological in vitro cultures with increased capacity to support identification of novel mechanisms, targets, or drugs.

3D micro-organisation printing of mammalian cells to generate biological tissues.

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.