V. L. Workman

The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis

A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction.

Peptide hydrogelin vitronon-inflammatory potential: Peptide hydrogelin vitronon-inflammatory potential

Peptide‐based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a β‐sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL‐β, IL‐6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro‐inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D.

Peptide hydrogel in vitro non-inflammatory potential.

Peptide‐based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a β‐sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL‐β, IL‐6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro‐inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D.

RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed.

Peptide hydrogel in-vitro non-inflammatory potential

Peptide‐based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a β‐sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL‐β, IL‐6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro‐inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D.