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Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology has been supported by numerous recent experimental and theoretical investigations in recent years. In this review, we first give a brief introduction to the key ideas of surface curvature in the context of biological systems and discuss the challenges that arise when measuring surface curvature. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, we address the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological and mechanical processes but that curvature acts also as a signal that co-determines these processes. This article is protected by copyright. All rights reserved.

INTRODUCTION: The etiology of lower back pain (LBP) is multifactorial. The cartilage endplate (CEP) is the spinal tissue receiving the least attention in scientific literature. Yet it plays a crucial role in keeping the intervertebral disc (IVD) healthy since it is the main gateway to nutrients and waste in and out of the nucleus pulposus in the avascular IVD1 . It is also suggested that imperfections and weaknesses in the CEP can be a better anticipator of pain than IVD degeneration 2 since chemical changes to the CEP are directly related to the intervertebral disc degeneration3 . Immunohisto-chemical research showed that the decline of CEP permeability due to calcification causes blood vessels and nerve fibers to infiltrate the IVD from the CEP and subchondral bone in an attempt to repair the damage and this infiltration leads to LBP3 . In this study we aim to investigate the mass transport through the CEP in and out of the IVD starting from CT images. METHODS: Given the scarcity of human CEP, a fresh bovine tail was obtained from the local slaughterhouse and 6 CEPs were harvested from it. 8 mm punches were taken from the CEPs. The samples were incubated with 20% Hexabrix, a contrast agent to visualize sulfated glycosaminoglycans. The samples were imaged on a Nanotom® M (GE nanoCT©) using in-house optimized settings for osteochondral tissues. The CT images were exported to Salome and manually curated to generate a high-quality volume mesh of the CEP. OpenFOAM® was used to perform Computational Fluid Dynamics (CFD) Simulations to quantify the mass transport through the CEP. The CEP samples were fitted into the 8 mm silicon tubes of a custom-made mesofluidic setup and fixed in place using clamps. physiological fluid (PBS) and standard growth medium were passed through the specimens separately. The specimens were left to fill then the time to pass 10 ml of solution was recorded for each specimen in the forward and reverse flows to calculate the volumetric flow rate and the permeability using Darcy’s law. RESULTS: The nCT imaging settings used resulted in a good quality image of the scanned samples. The 20% Hexabrix was unable to fully stain the CEP and a higher concentration is currently being tested. Both the CFD simulations and the in vitro diffusion tests are ongoing. Figure 1: nCT-scan of CEP with vertebrate bone attached (sagittal view) DISCUSSION & CONCLUSIONS: Combining image-based in silico modelling with in vitro experiments, as proposed in this study, will allow to quantify the fluid dynamics across the CEP, which is the gateway controlling mass transport in and out of the IVD. ACKNOWLEDGEMENTS: This project received funds from the European Commission (H2020-MSCA-ITN-ETN-2020-955735). REFERENCES: 1 Roberts S, et al. Spine. 1989;14(2):166-174. 2 Lakstins K, et al. J Orthop Res. 2021;39(9):1898-1907. 3 Yao Y, et al. Stem Cell Rep. 2016;7(2):249-262.

Low back pain is the leading cause of morbidity worldwide. Its aetiology is complex and multifactorial with degeneration of the intervertebral disc (IVD) being a key cause. Causality between infection of the IVD and its degenerative process gained great interest. In a systematic review, Granville Smith et al. 1 (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. In 27 studies bacteria were identified. To date, all the studies detecting bacteria found Gram-positive bacteria with Cutibacterium acnes being the most abundant. However, there are too few quantitative studies investigating bacterial infection to show whether bacteria are present in vivo or represent perioperative contamination. This study aims to investigate bacterial presence within the disc and their potential influence. METHODS: Immunohistochemical staining for Gram-positive bacteria was performed on 100 human disc specimens to identify whether bacteria are present. Only samples with an intact annulus fibrosus (AF) were included to minimize the risk of contamination from adjacent tissue. Staining with a second anti Gram-positive bacteria antibody is ongoing. Nucleus pulposus (NP) cells in monolayer were stimulated with lipopolysaccharides (LPS) and peptidoglycans (PPG) (0.1-50 µg/ml) for 24, 48 and 72 hours. Factors associated with disc degeneration were analyzed by qPCR, ELISA and Luminex. Experiments with NP cells resuspended in 1.2 w/v alginate treated with 5 µg/ml and 50 µg/ml LPS respectively PPG are ongoing. RESULTS: Gram-positive bacteria were internalized by human disc cells and influenced the cell and nuclei morphology (Fig.1, a). Luminex data supported by qPCR and ELISA results shows an increase in cytokines such as IL-1β, IL-6, IL-8, TNF-α, IFN-γ, IL-4, IL-17A, matrix degrading enzymes MMP1, MMP9 and neurotrophic and angiogenic factors NGF and VEGF in treated samples compared to the untreated control, with greatest effects seen following 50 µg/ml PPG stimulation. DISCUSSION & CONCLUSIONS: This study demonstrated the presence of bacteria within human IVD tissue samples of patients undergoing spinal surgery. Whether the detected bacteria are representative for the disc microbiome or for an infection is unclear and will be further investigated. Exposure of human NP cells to the bacterial components LPS and PPG showed enhancement of several cytokines, chemokines, matrix degrading enzymes and other factors associated with disc degeneration. Demonstrating the capacity of both LPS and PPG to induce catabolism within disc cells.

Introduction We recently presented ANIMO (Analysis of Networks with Interactive Modeling), a software tool for modeling dynamic molecular networks for use by biologists [1, 2]. We used ANIMO to generate a computational model of articular cartilage. Materials and methods Based on a largescale literature study [3] and our own experiments, we developed ECHO (Executable Chondrocyte), a computational model of the key processes that regulate expression and activity of SOX9 and RUNX2, two master transcriptional regulators of the chondrocyte phenotype. ECHO consists of 93 nodes with 274 interactions that describe the expression and activity of 52 genes and proteins. Simulations in ECHO were performed to investigate the robustness of the chondrocyte network. To validate ECHO predictions, we used FRAP to measure mobility of SOX9 and RUNX2, which we have shown to be a faithful readout of their activity. Results In its unperturbed form, ECHO displays two stable states in which activities of SOX9 and RUNX2 are mutually exclusive. SOX9 rep resents a stable articular cartilage phenotype, while RUNX2 represents transient hypertrophic cartilage. We tested in silico the hypothesis that addition of WNT (performed with a few clicks of the mouse) will change permanent cartilage into transient cartilage by inducing hyper trophy. Indeed, when we add WNT, a known regulator of bone forma tion, the permanent or SOX9 + state changes to a transient or RUNX2 + state in the model. However, it is known that healthy articular cartilage is resistant to hypertrophic differentiation. Our group has previously shown experimentally that this was probably due to the secretion of DKK1, FRZB and GREM1[4, 5]. We therefore added nodes to ECHO representing DKK1, FRZB and GREM1 (fig. 1). GREM1 and DKK1 are able to stabilize the permanent cartilage or SOX9 + state even after addition of WNT in ECHO. We observed that in our model activation of WNT leads to a switch from a SOX9 + state to a RUNX2 + state. To prove that WNT/bcate nin signaling can directly regulate SOX9 function, we investigated the response of SOX9 mobility to WNT3A in live primary chondrocytes. Addition of WNT3A to human chondrocytes transfected with SOX9GFP resulted in a significant decrease of the immobile SOX9 frac tion from 53% to 34% within 15 minutes after addition, indicating a loss of transcriptional activity of SOX9. Discussion and conclusion Using ECHO we predicted the stimuli that prevent hypertrophic differentiation of articular cartilage, and tested this experimentally with FRAP using SOX9 and RUNX2 mobility as a readout.

Purpose: Cartilage homeostasis relies on an intricate balance between anabolic and catabolic processes. In osteoarthritis this balance is shifted towards catabolism, leading to hypertrophy and a gradual degradation of cartilage tissue. So far, drug-based intervention in this process has shown limited progress. We propose to construct a mathematical model of the molecular network that governs key processes in articular cartilage homeostasis. This model can be used as a platform for model expansion by introduction of new experimental findings and hypotheses. Methods: We recently developed ANIMO (Analysis of Networks with Interactive Modeling), an intuitive software tool for modeling molecular networks. Here, we demonstrate a mathematical model of growth plate cartilage using a combination of literature and experimental data. We show how ANIMO allows for intuitive exploration of the model, despite the size and complexity of the model. Results: We constructed a network model of regulatory processes in growth plate chondrocytes. In this model the effects downstream of extracellular growth factors FGF, WNT, IGF-1, PTHrP, Ihh, BMP, and TGF-β are integrated into a cellular response. In silico experiments predict the phenotypic outcome for different inputs and starting states of the model. Osteoarthritic chondrocytes and hypertrophic growth plate chondrocytes show strong parallels in their gene expression profile. We have examined the gene expression profiles of growth plate and articular cartilage. In articular cartilage the expression of the WNT and BMP antagonists DKK1, FRZB and GREM1 is over 10-500 fold higher than in growth plate cartilage. This leads us to think that DKK1, FRZB, GREM1 could act as gatekeepers for preventing hypertrophy. We are currently investigating the range of conditions under which these proteins exert their stabilizing effect on the articular cartilage phenotype in the model. Furthermore, we are interrogating the model to obtain in silico leads to targets for novel combination therapies. Such therapies could be used to intervene in the osteoarthritic state of the network and restore the balanced situation of healthy cartilage. Conclusions: Traditionally, modeling efforts in the realm of molecular cell biology have been the exclusive domain of researchers with a thorough training in mathematics or computer science. We show here that a complex model that is intuitively amenable to exploration and adaptation by biologists is an invaluable asset in cartilage research. Expansion of an existing model with DKK1, FRZB and GREM1 provided evidence for their role in preserving the articular cartilage phenotype.

The term In Silico Trial indicates the use of computer modelling and simulation to evaluate the safety and efficacy of a medical product, whether a drug, a medical device, a diagnostic product or an advanced therapy medicinal product. Predictive models are positioned as new methodologies for the development and the regulatory evaluation of medical products. New methodologies are qualified by regulators such as FDA and EMA through formal processes, where a first step is the definition of the Context of Use (CoU), which is a concise description of how the new methodology is intended to be used in the development and regulatory assessment process. As In Silico Trials are a disruptively innovative class of new methodologies, it is important to have a list of possible CoUs highlighting potential applications for the development of the relative regulatory science. This review paper presents the result of a consensus process that took place in the InSilicoWorld Community of Practice, an online forum for experts in in silico medicine. The experts involved identified 46 descriptions of possible CoUs which were organised into a candidate taxonomy of nine CoU categories. Examples of 31 CoUs were identified in the available literature; the remaining 15 should, for now, be considered speculative.

In Silico Trials methodologies will play a growing and fundamental role in the development and de-risking of new medical devices in the future. While the regulatory pathway for Digital Patient and Personal Health Forecasting solutions is clear, it is more complex for In Silico Trials solutions, and therefore deserves a deeper analysis. In this position paper, we investigate the current state of the art towards the regulatory system for in silico trials applied to medical devices while exploring the European regulatory system toward this topic. We suggest that the European regulatory system should start a process of innovation: in principle to limit distorted quality by different internal processes within notified bodies, hence avoiding that the more innovative and competitive companies focus their attention on the needs of other large markets, like the USA, where the use of such radical innovations is already rapidly developing. ispartof: IEEE JOURNAL OF BIOMEDICAL AND HEALTH INFORMATICS vol:26 issue:11 pages:5282-5286 ispartof: location:United States status: published

The term "In Silico Trial" indicates the use of computer modelling and simulation to evaluate the safety and efficacy of a medical product, whether a drug, a medical device, a diagnostic product or an advanced therapy medicinal product. Predictive models are positioned as new methodologies for the development and the regulatory evaluation of medical products. New methodologies are qualified by regulators such as FDA and EMA through formal processes, where a first step is the definition of the Context of Use (CoU), which is a concise description of how the new methodology is intended to be used in the development and regulatory assessment process. As In Silico Trials are a disruptively innovative class of new methodologies, it is important to have a list of possible CoUs highlighting potential applications for the development of the relative regulatory science. This review paper presents the result of a consensus process that took place in the InSilicoWorld Community of Practice, an online forum for experts in in silico medicine. The experts involved identified 46 descriptions of possible CoUs which were organised into a candidate taxonomy of nine CoU categories. Examples of 31 CoUs were identified in the available literature; the remaining 15 should, for now, be considered speculative., Comment: Submitted to IEEE JOURNAL OF BIOMEDICAL AND HEALTH INFORMATICS