Impaired wound healing and the resulting chronic wounds may cause significant morbidity and mortality. In these pathogenic wound environments, the ratio of inflammatory and anti-inflammatory cytokines is highly biased to the pro-inflammatory side. While the inflammatory process is an essential step in healthy wound healing, chronic wounds remain in a constant self-sustaining state of inflammation. Thus, decreased cell proliferation, reduced matrix deposition and delayed wound closure are the results. Although various cytokine-based therapies have shown promising results on skin regeneration in preliminary studies, their overall clinical use has been considerably limited by the short half-life time of the signaling molecules due to rapid dilution and degradation in the protease-rich chronic wound environment.
In this work, we explored the ability of starPoly(ethylene glycol)-GAG hydrogels to modulate the hallmarks of chronic wound development, such as the prolonged inflammation, increased cell influx and delayed proliferative phase. Therefore, different strategies were developed to shape the cytokine levels in the wound towards a more pro-regenerative direction, finally promoting the natural repair process in chronic skin wounds. By biomimetically utilizing the interactions between cytokines and the tissue ECM in a GAG-based biohybrid hydrogel, we could engineer the concentrations of various signaling factors involved in the regulation of the repair process.
More in detail, we utilized customized functionalized starPEG-GAG hydrogels to (1) reduce the extensive levels of inflammatory chemokines by scavenging them via GAG component of the hydrogel and thus diminish immune cell influx in a mouse wound model; (2) locally deliver the immunomodulatory IL-4 and IL-10 to shift the signaling balance into the pro-regenerative direction and thus resolve inflammation and (3) administer pre-conjugated TGF-β to enhance myofibroblast differentiation and extracellular matrix deposition.
We believe that the presented hydrogel platform may become a promising tool in the management of cytokines in regenerative applications, which can be translated towards the clinical use for the treatment of chronic wounds and other diseases characterized by uncontrolled inflammation.:1 introduction
1.1 Motivation
1.2 Current state of biomaterial-based concepts in dermal wound healing
1.3 Objective
2 fundamentals
2.1 The physiological process of wound healing
2.1.1 The role of macrophages in wound healing
2.1.2 The role of fibroblasts in wound healing
2.1.3 The role of cytokines and their interaction with the ECM
2.2 The pathophysiology of chronic wounds
2.3 Strategies for treatment of chronic wounds
2.4 Biomaterials in medicine
2.4.1 Polymers in medicine
2.4.2 Mechanical properties
2.4.3 Cellular adhesion
2.4.4 Interaction with cytokines
2.4.5 Scaffold degradability
2.4.6 StarPEG-GAG hydrogels as potential material in wound healing
3 materials & methods
3.1 Preparation of hydrogels
3.1.1 Functionalization of glass surfaces
3.1.2 Hydrogel formation with EDC - NHS chemistry
3.1.3 Hydrogel formation with thiol - maleimide chemistry
3.1.4 Rheometric measurement of hydrogel discs
3.1.5 Characterization of cytokine uptake and release
3.2 Culture of human & murine cells
3.2.1 Isolation and differentiation of murine dermal fibroblasts
3.2.2 Isolation & differentiation of murine macrophages
3.2.3 Culture of human & murine cell lines
3.3 In vitro methods
3.3.1 Enzyme-linked immunosorbent assay (ELISA)
3.3.2 Bead-based multiplex immunoassay
3.3.3 Live/Dead Staining
3.3.4 Crystal violet staining
3.3.5 Cell proliferation assay
3.3.6 RNA extraction & analysis
3.3.7 cDNA synthesis
3.3.8 Quantitative real time rt-PCR
3.4 Statistical analysis
3.5 Software use
4 scavenging inflammatory chemokines to control immune cell influx in the wound
4.1 Results
4.1.1 Engineering heparin-based hydrogels to scavenge chemokines
4.1.2 Heparin-based hydrogels reduce migration of immune cells
4.1.3 Heparin-based hydrogels decrease wound immune cell influx and inflammatory signaling
4.2 Discussion
5 promotion of regenerative macrophage polarizationin inflammatory environments
5.1 Results
5.1.1 Reversible complexation of IL-4 & IL-10 to starPEG-heparin gels
5.1.2 Stabilizing effects of starPEG-heparin gels on IL-4
5.1.3 IL-4 & IL-10-laden starPEG-heparin hydrogels modulate macrophage polarization
5.1.4 IL-4-laden starPEG-heparin induce collagen deposition in dermal fibroblasts
5.2 Discussion
6 modulation of human dermal fibroblast proliferation and differentiation
6.1 Results
6.1.1 Reversible complexation of TGF-b to starPEG heparin gels
6.1.2 Cell attachment, spreading and proliferation
6.1.3 Matrix deposition by fibroblasts grown on starPEG-heparin hydrogels
6.1.4 Degradation of starPEG-heparin hydrogels
6.1.5 TGF-b-laden starPEG-heparin that efficiently induces myofibroblast differentiation
6.2 Discussion
7 general discussion
7.1 Summary and conclusion
7.2 Future perspective
Appendix
8 supplementary materials & methods
9 declaration of authorship
10 publications and conference contributions
bibliography
list of figures
list of tables
nomenclature
selbstständigkeitserklärung
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:28961 |
| Date | 04 November 2020 |
| Creators | Schirmer, Lucas |
| Contributors | Werner, Carsten, Eming, Sabine, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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