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Developing a Fibrosis Targeting and Tissue Reparative (FiTTeR) Therapy for the Infarcted Myocardium via an Injectable Functionalized Extracellular Matrix Hydrogel

Periodic Reporting for period 1 - FiTTeR (Developing a Fibrosis Targeting and Tissue Reparative (FiTTeR) Therapy for the Infarcted Myocardium via an Injectable Functionalized Extracellular Matrix Hydrogel)

Reporting period: 2017-08-21 to 2019-08-20

The overall objective of the FiTTeR project was to develop characterize, and determine the efficacy of a biomaterial platform to target pathologic fibrosis and promote tissue repair. All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosisis a process that stands diametrically opposed to functional tissue repair and regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. However, no specific and localized therapies currently exist for targeting fibrosis. Poor pre-clinical and/or clinical outcomes have been reported for the inhibition of multifunctional molecules like exogenous MMP inhibitors which have failed due to detrimental off-target side effects. In the FiTTeR project, an extracellular matrix (ECM) hydrogel system was developed and validated, epithelial- and macrophage-based model systems for fibrosis were developed, and the feasibility of clinical application of ECM systems was assessed. Together, these results have provided a proof-of-concept demonstration that ECM hydrogel technologies can be used in applications to enhance tissue healing outcomes. As an MSCA fellow, I presented my research at TERMIS World Congress (Kyoto, Japan), Biologic Scaffolds Symposium (Napa Valley, USA), and TERMIS North America (Charlotte, NC). I have also co-authored publications in Advanced Materials and Advanced Drug Delivery Reviews and have a number of publications in preparation.

This ambitious project was possible due to the placement within the world-renowned Stevens Group at Imperial College London. Because of the interdisciplinary nature of the research plan, this diverse research group was the ideal host for this project. I benefited greatly from working alongside experts in chemistry, materials science, cell biology, and nanoparticle technologies.
The following summarizes the main research tasks and results from this fellowship:
1. The components required to fabricate an extracellular matrix (ECM) hydrogel were prepared and characterized. The effect of each component of the hydrogel (i.e. ECM and recombinant protein fragment) on epithelial cells was assessed. Raman spectroscopy was used to characterize the processing of the ECM and to validate the efficacy of decellularization. This permitted the non-destructive assessment of the ECM and validated the removal of residual cells.
2. Modular bioconjugation strategies were developed for linking components to ECM via amine or cysteine residues. Malemide-activated proteins can be linked via thiols contained within the ECM but may not always be the optimal strategy when due to non-specificity and potential protein aggregation. Thus, we developed a second strategy which involved added a strained-alkyne via a cysteine residue on the protein component and adding an azide to the hydrogel. After separately modifying each component, the protein can be ‘clicked’ on to the ECM hydrogel.
3. The adhesive properties of the ECM alone and after bioconjugation were characterized in various model tissues. To facilitate quantification and visualization of the material, fluorescent nanoparticles were incorporated in the hydrogel. These data, in addition to rheological testing, showed that ECM hydrogels are adhesive to epithelial tissues and are robust enough to withstand agitation and several washes.
4. The effect of ECM hydrogel +/- recombinant protein fragment on epithelial cells was assessed via morphologic and immunofluorescent analysis. Epithelial cells were shown to exhibit and augmented epithelial-mesenchymal transition (EMT) when exposed to ECM. However, no additional or synergistic response was noted with the ECM was combined with a recombinant laminin fragment.
5. The innate immune response to ECM hydrogel +/- recombinant protein was characterized using an in-vitro assay with macrophages. By combining morphologic analysis, protein expression, gene expression, and Raman spectroscopy, it was possible to gain an increased understanding of how biologic scaffolds affect immune cell activation. Interestingly, different sources of ECM were found to uniquely alter immune cells.
6. The stiffness of ECM hydrogels was controlled by varying the hydrogel formulation (i.e. increasing the concentration of ECM). The tissue adhesive properties to various epithelial tissues was verified by ex vivo experiments. The results from this work package showed that the hydrogel formulation is tunable and adhesive to tissues as a versatile platform for various applications of tissue healing. This work serves as preparation for the next stage of in vivo experiments to test the efficacy of the hydrogel platform for enhancing tissue healing / mitigating scarring.

The key findings from this project were disseminated at international research conferences including TERMIS-World Congress 2018 in Japan, 2018 Biologic Scaffold Symposium in USA, and 2019 Tissue Cell and Engineering Society in UK. I have also co-authored publications in Advanced Materials and Advanced Drug Delivery Reviews and have 2 other publications in preparation. During the MSCA fellowship period, I also mentored 16 students ranging from undergraduate to PhD-level.
The results from this MSCA-funded project have progressed the state of the art in several research areas. First, a new bioconjugation strategy was developed to “click” linkers onto biologic hydrogels using cysteine-selective techniques. This development should be very useful in enabling more researchers to use these types of cross-linkers in their work to functionalize biologic and synthetic hydrogel with desired bioactivity. Typically, this type of chemistry is only implemented by research groups that have strong expertise in chemical synthesis. Second, immune cell activation in response to biomaterials was analysed by Raman spectroscopy. This analysis, which has potential for screening biomaterials based on the host response has allowed for identification of unique immune cell “activation signatures” based upon the source tissue from which ECM is derived. In addition, we have developed sprayable formulations of ECM materials that have a wide potential for varied poorly or unmet clinical applications of wound healing. The proof-of-concept validation of this ECM delivery strategy should enhance the therapeutic efficacy ECM-based treatments in the future. This is expected to have several positive impacts on society as system for delivering ECM in a minimally invasive manner has great promise for use in a range of clinical applications that have a major global impact.
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