Cross talk between fibroblasts and keratinocytes, which maintains skin homeostasis, is disrupted in chronic wounds. For venous leg ulcers and diabetic foot ulcers, a bilayered living cellular construct (BLCC), containing both fibroblasts and keratinocytes that participate in cross talk, is a safe and effective product in healing chronic wounds. To show the importance of both cell types in BLCC, constructs were generated containing only fibroblasts or only keratinocytes and compared directly to BLCC via histology, mechanical testing, gene/protein analysis, and angiogenesis assays. BLCC contained a fully differentiated epithelium and showed greater tensile strength compared with one-cell-type constructs, most likely due to formation of intact basement membrane and well-established stratum corneum in BLCC. Furthermore, expression of important wound healing genes, cytokines, and growth factors was modulated by the cells in BLCC compared with constructs containing only one cell type. Finally, conditioned medium from BLCC promoted greater endothelial network formation compared with media from one-cell-type constructs. Overall, this study characterized a commercially available wound healing product and showed that the presence of both fibroblasts and keratinocytes in BLCC contributed to epithelial stratification, greater tensile strength, modulation of cytokine and growth factor expression, and increased angiogenic properties compared with constructs containing fibroblasts or keratinocytes alone.
The value of in silico methods in drug development and evaluation has been demonstrated repeatedly and convincingly. While their benefits are now unanimously recognized, international standards for their evaluation, accepted by all stakeholders involved, are still to be established.In this white paper, we propose a risk-informed evaluation framework for mechanistic model credibility evaluation. To properly frame the proposed verification and validation activities, concepts such as context of use, regulatory impact and risk-based analysis are discussed. To ensure common understanding between all stakeholders, an overview is provided of relevant in silico terminology used throughout this paper.To illustrate the feasibility of the proposed approach, we have applied it to three real case examples in the context of drug development, using a credibility matrix currently being tested as a quick-start tool by
Accepted ArticleThis article is protected by copyright. All rights reserved regulators. Altogether, this white paper provides a practical approach to model evaluation, applicable in both scientific and regulatory evaluation contexts.
This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.
Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.
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