Introduction



-215901278890McDonnell, E.1, 2, Buckley, C. T.1, 2, 3, 4 1 Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, 2 School of Engineering, Trinity College Dublin, 3 AMBER Centre, RCSI & Trinity College Dublin, 4 Department of Anatomy and Regenerative Medicine, RCSIEmail: mcdonne5@tcd.ie00McDonnell, E.1, 2, Buckley, C. T.1, 2, 3, 4 1 Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, 2 School of Engineering, Trinity College Dublin, 3 AMBER Centre, RCSI & Trinity College Dublin, 4 Department of Anatomy and Regenerative Medicine, RCSIEmail: mcdonne5@tcd.ie0922020Elucidating and Optimising the Distribution of Metabolites within Ex Vivo Disc Organ Culture00Elucidating and Optimising the Distribution of Metabolites within Ex Vivo Disc Organ CultureIntroductionIn recent years, there has been significant interest in the development of cell-based therapies for the treatment of intervertebral disc (IVD) degeneration. These exciting therapies aim to repopulate the nucleus pulposus (NP) and augment tissue repair. Ex vivo disc culture systems have become a valuable tool during development and preclinical testing, as they provide an important platform between cell culture and in vivo studies. Bovine caudal discs are often selected as the most suitable model 1, yet there remains variation in disc isolation technique and culturing conditions. To ensure normal cellular function and successful tissue regeneration, it is critical to assess these cell therapies in the local physicochemical microenvironment experienced in vivo 2. However, it remains to be elucidated whether the metabolite concentrations within ex vivo cultures are physiologically relevant or comparable to human degeneration. This work aims to create a validated computational model which can be used to predict the metabolite gradients generated in ex vivo culture systems based on variations on disc isolation techniques and culturing conditions. Materials and methodsFinite element models of caudal discs in culture were created using COMSOL Multiphysics? ver5.4. These models were governed by coupled reaction-diffusion equations, taking into account geometrical differences, cell viability, cellular metabolism and nutrient diffusion through the different tissue domains. Cell density and viability criteria were evaluated using the MTT assay with DAPI counterstaining. Diffusion parameters through the tissue were obtained from the literature 3-6. Cellular metabolism (glucose and oxygen consumption, and lactate production) were dependent on local oxygen and pH levels by employing equations derived previously 7, 8. Experimental verification of these models was performed by measuring the metabolite concentrations in discs cultured for 7 days, in a custom-made static compression bioreactor, with culturing conditions corresponding to the in silico boundary conditions.resultsThe diffusion distance across the NP thickness was found to be significantly different between caudal discs location in the bovine tail (n = 6, P<0.05). There was an initial cell viability of 85% and 83% for the NP and annulus fibrosus (AF), respectively. Therefore, the cell density employed in the model was 5,578 ± 801 cells/mm3 for the NP (n = 3) and 14,465 ± 3,937 cells/mm3 for the AF (n = 3). The cell densities were assumed to remain constant as no significant difference was found in the viability of the NP at day 7. The glucose concentration predicted in the centre of one disc cultured in 25 mM media was 6.22 mM, which was within the standard deviation (SD) of the experimentally measured concentration, 5.35 ± 1.47 mM (n = 3). The lactate concentration predicted in the disc centre, under the same conditions, was 10.44 mM, which also lay within the SD of the experimentally measured concentration, 9.64 ± 1.49 mM (n = 3). The experimental pH level decreased from 7.3 ± 0.1 in the media to 6.4 ± 0.1 in the disc centre (n = 8). While the predicted pH in the disc centre ranged from 6.3 to 6.6 depending on disc size.Fig. 1 Schematic of study methodology and analysed results.DiscussionTo the best of our knowledge, this work presents the first experimentally verified predictive model of metabolite distribution within ex vivo disc cultures. Not only does it advance the knowledge of the nutrient microenvironment within these systems, but also highlights that there is a pressing need for standardisation between isolation technique and culturing conditions. Ultimately, it is imperative that the critical metabolite values (minimum glucose, oxygen & pH values) are matched to those at a stage of human IVD degeneration, where regenerative cell-therapy is an appropriate strategy, to realise successful clinical translation based on nutritional demands.References[1] Haglund (et al.) Tissue Eng. Part C: Methods. 17:1011-1019; 2011 [2] Buckley (et al.) JOR Spine. 1:e1029; 2018 [3] Yuan (et al.) J. Biomech. Eng. 131; 2009 [4] Galbusera (et al.) Comput. Methods Biomech. Biomed. Eng. 16:328-337; 2013 [5] Wu (et al.) J. Biomech. 49:2756-2762; 2016 [6] Jackson (et al.) Ann. Biomed. Eng. 40:1-8; 2012 [7] Bibby (et al.) Spine. 30:487-496; 2005 [8] Huang & Gu J. Biomech. 41:1184-1196; 2008 ................
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