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

Enzymes and subcritical water can be used for the extraction of hemicelluloses from cereal by-products, making the processes eco-friendly. The polysaccharides extracted from cereal by-products can be used as matrices for development of materials for various applications. This includes bio-based materials such as films and hydrogels, which offer alternatives to existing materials produced from petrochemicals. The polymeric structure of cereal hemicelluloses contains functional groups which enable the modification of their structure by cross-linking, resulting in the formation of hydrogels. This project aims to use subcritical water extraction (SWE) to extract arabinoxylans (AXs) from corn and rye bran meanwhile the enzymatic treatment is done for purifying the samples during both pre- and post-treatment. AXs were further crosslinked by enzyme (laccase) for hydrogel preparation. During the whole project, the characterization included moisture and yield determination, starch and protein content which were tested using a spectrophotometer, monosaccharide content was analyzed by high performance anion exchange chromatography followed by pulsed amperometric detection (HPAEC-PAD) and phenolic acid content was quantified by high performance liquid chromatography (HPLC). The pretreatment for destarching and SWE process was successful. The result showed that arabinoxylans form corn bran were having higher content of arabino substituents, arabino toxylans ratio and ferulic acid content than rye samples. The enzymatic crosslinking could form strong gels in the condition that the AXs had high ferulic acid content. In terms of forming strong hydrogels or to improving the properties of AXs gel, the pre- and post-treatment should be optimized to increase the purity of the extracted feruloylated AX content. / Enzymer och subkritiskt vatten kan användas för extraktion av hemicellulosa från spannmålsbiprodukter, vilket gör extraktionen miljövänlig. Polysackariderna extraherade från spannmålsprodukter kan användas som matriser för utveckling av material för diverse applikationer. Detta inkluderar biobaserade material som filmer och hydrogeler, där petrokemikalier kan ersättas som råvara. Den polymera strukturen hos spannmålshemicelluloser innehåller funktionella grupper som möjliggör formation av tvärbindningar vilket resulterar i bildandet av hydrogeler. Syftet med detta projekt är extraktion av arbinoxylaner (AXs) från majs och rågkli genom att använda subkritiskt vatten-extraktion (SWE) där rening under för- och efterbehandling utförs enzymatiskt. AX modifierades därefter enzymatiskt (laccas) med tvärbindningar för hydrogelframställning. Under hela projektet karakteriserades hydrogelen utifrån fuktinnehåll, bestämmelse av utbyte, stärkelse och proteininnehåll som testades med en spektrofotometer, monosackaridhalten analyserades med högpresterande anjonsutbyteskromatografi följt avpuls-amperometrisk detektion (HPAEC-PAD), samt kvantifierades fenolsyrahalten med högupplöst vätskekromatografi (HPLC). Resultatet visade att arabinoxylaner från majskli hade högre innehåll av arabinosubstituenter, där förhållandet mellan arabino och xylans, samt arabino och ferulsyra innehållet var högre än för rågproverna. Den enzymatiska tvärbindningen kunde bilda starka geler i det tillståndet där AX hade en hög ferulsyrahalt. När det gäller att bilda starka hydrogeler eller att förbättra egenskaperna hos AXs-gel, bör för-och efterbehandlingen optimeras för att öka renheten fördet extraherade feruloylerade AX-innehållet.

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Doctor of Philosophy / Department of Grain Science and Industry / Xiuzhi Susan Sun / Om Prakash / Rational design of peptide molecules to undergo spontaneous organization as a higher-ordered supramolecular structure is an attractive and fast-growing field for developing new functional biomaterials. Hydrogel, with its high water content and three-dimensional architecture, is formed by a self-assembling peptide and has great potential for broad biomedical applications. The key challenge in controlling the functional properties of final biomaterials can be met by designing the peptide primary structure carefully at the beginning and developing a comprehensive understanding of peptide self-assembly pathways. In this study, we first designed a Ca2+ responsive peptide (eD2) using identified functional native domains from a spider flagelliform silk protein and the Ca2+ binding domain of lipase Lip A from Serratia marcescens. Instead of directly linking the two peptide sequences, we rationally inserted the ion-binding motif into the silk structure sequence and made the new peptide inherit the physical characteristics of both model sequences and assemble into nanofibers when triggered by Ca2+. Next, we introduced the amphiphilic property to the eD2 peptide by conjugating its N-terminus with a strong hydrophobic sequence from a trans-membrane segment of human muscle L-type calcium channel. This self-assembly peptide, called h9e, was responsive to Ca2+, solution pH, and selected proteins for hydrogel formation. Interestingly, the turning segment GSII of h9e was considered to play a critical role in construction of the finial matrix. This hypothesis was further demonstrated by exploiting a series of amphiphilic diblock model peptides with different conformational flexibility. The kinetic rate of peptide assembly was suggested as one of the key influences for peptide supramolecular assembly morphology. To better understand the peptide self-assembly process during hydrogel formation, the conformational, morphological, and mechanical properties of h9e molecules in different dimethylsulfoxide/H2O solutions were monitored by 1D and 2D proton nuclear magnetic resonance (NMR), electron microscopy, and a rheometer. The h9e peptide hydrogel formed with Ca2+ and albumin exhibited superior physiological and specific injectable properties, which provides a more realistic tool for 3D cell culture and drug delivery. This study generates new knowledge and contributes to the field by leading to a better understanding the self-assembly hydrogel formation and designing peptides with unique properties for biomedical applications such as cell culture, drug delivery, and tissue engineering.

This study investigates the feasibility of using TTO and Ag+ alone and in combination either as free or liposome encapsulated agents. Based on the minimum lethal concentration (MLC), the fractional lethal concentration index (FLCI) showed that treatment with unencapsulated combinations of TTO and Ag+ exerted a synergistic effect against P. aeruginosa (FLCI = 0.263) and indifferent effects against S. aureus and C. albicans (0.663 and 0.880, respectively). Using polyvinyl alcohol (PVA) emulsified agents in combination, showed synergistic effects against P. aeruginosa and S. aureus (FLCI = 0.325 and 0.375, respectively), but C. albicans remained indifferent (FLCI = 0.733). Time kill experiments revealed that the combined agent concentrations and elimination time (to the lowest limit of detection, LOD) are as follows: C. albicans: 0.12%v/vTTO:2.5x10-4Ag+:1.5hrs, P. aeruginosa: 1%v/vTTO:3.2x10-4Ag+:15mins and S. aureus: 1.2%v/vTTO:3.2x10-4Ag+:30mins. Repeating these experiments with emulsified TTO encapsulated in liposomes (lipo-TTO:PVA30-70kDa) against P. aeruginosa and S. aureus reduced the effective amount of TTO required (compared to free TTO). However, this was not observed in C. albicans. The required effective concentration of Ag+ from liposome encapsulated Ag+ (lipo-Ag+) was shown to remain the same as free Ag+. The effective concentration and elimination time of liposomal agents in combination are as follows: C. albicans: 0.05%v/vTTO:PVA:8.9x10-5Ag:PVA:2.0hrs, P. aeruginosa: 0.25%v/vTTO:PVA:3.2x10-4Ag:PVA:30mins and S. aureus: 0.05%v/vTTO:PVA:6.0x10-4Ag:PVA:1.5hrs. These results showed the potential of using TTO and Ag+ in combination, along with liposome delivery systems to effectively lower the MLC. Scanning electron micrographs of microorganisms exposed to Ag+ showed a reduction in cell size when compared to untreated cells. Transmission electron micrograph of C. albicans showed the cell surface damaging potential of Ag+. Furthermore, this investigation also demonstrated the feasibility of using chitosan hydrogels as an alternative delivery system for TTO and/or Ag+. The development of these controlled release systems to deliver alternative antimicrobial agents may allow sustained targeted delivery at microbiocidal concentrations.

L'utilisation du méthacrylate de 2-isocyanatoéthyle (IEM), dérivé commercial, a permis le développement de trois architectures polymères fonctionnalisées par un isocyanate bloqué. Un hydrogel fonctionnalisé par des groupements -NH-C(=O)-SO3- a été synthétisé à partir de l'IEM et du métabisulfite de potassium. Cet hydrogel présente un dégré de fonctionnalisation important, un bon comportement au stockage sous voie sèche ainsi qu'une très bonne capacité au gonflement dans l'eau. Le recours à un agent de blocage alternatif a permis la protection de l'IEM et l'obtention d'un monomère hydrosoluble qui a été polymérisé par voie radicalaire dans l'eau. La réactivité de ce polymère vis-à-vis d'amines aliphatiques dans l'eau a été démontrée. Enfin, le blocage de l'IEM par le phénol ou l'acétone oxime a permis l'obtention de microsphères à base de divinylbenzène, fonctionnalisées par un isocyanate bloqué.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

Cell-based therapies involving adipose-derived stem cells (ASCs) have shown promise in stimulating cardiovascular regeneration, including in the treatment of myocardial infarction (MI) and ischemic heart disease. However, previous studies involving the delivery of ASCs following MI have indicated that therapeutic efficacy has been limited by low survival and/or poor retention of the transplanted cells at the site of injury. To address these limitations, the goal of this thesis was to develop a more effective delivery strategy incorporating an injectable biomaterial combined with chemotactic growth factor delivery to enhance ASC retention within the gel. Working towards future in vivo analysis in a rat model, multilineage characterization studies confirmed that ASCs isolated from the epididymal fat pad of male Wistar rats could differentiate in vitro along the adipogenic, osteogenic, and chondrogenic lineages. Subsequently, the chemotactic response of the rat ASCs (rASCs) to varying concentrations of stromal derived factor-1 α (SDF-1α) and hepatocyte growth factor (HGF) was analyzed using a modified Boyden chamber assay. The results demonstrated that SDF-1α and HGF, at 20, 50, and 100 ng/mL elicited significant migratory responses under normoxic (21%) and hypoxic (5%) culture conditions. RT-PCR analysis was conducted to assess the expression of the two chemotactic growth factors and their associated receptors in the rASCs, and secreted SDF-1α protein expression was quantified by ELISA. Moving towards the development of the biomaterials-based delivery approach, the viability of rASCs encapsulated by photopolymerization in methacrylated glycol chitosan (MGC) hydrogels modified with various degrees of arginine-glycine-aspartic acid (RGD)-peptide modification was examined. More specifically, rASCs were encapsulated in MGC hydrogels with 0%, 4%, and 7% RGD modification and cultured for up to 14 days. Viability staining results indicated that rASC viability was enhanced in the 4% and 7% RGD-modified MGC hydrogels in comparison to the MGC hydrogels with no peptide modification. Pre-loading the gels with 50 ng/mL of SDF-1α had no significant effects on cell viability over 14 days. Overall, the results demonstrate that peptide modification to promote cell adhesion within the MGC hydrogels is key to improving cell viability and thereby improving the therapeutic potential of ASCs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-10-24 23:54:37.126

In this paper a novel stimuli responsive hydrogel material, methacrylated sodium alginate beta-cyclodextrin (Alg-MA-β-CD), was used in combination with a microfluidic device to create microspheres. Currently there is no reliable method for fabricating homogeneous stimuli-responsive microspheres, in-house microfluidic devices are not reliable in manufacture quality or long-term use. Alginate hydrogels have many attractive characteristics for bioengineering applications and are commonly used to mimic the features and properties of the extracellular matrix (ECM). Human mesenchymal stem cells (hMSCs) are of top interest to tissue engineers. hMSCs are widely available and can be harvested and cultured directly out of human bone marrow. hMSCs have the ability to differentiate into osteoblasts, adipocytes, chondrocytes, muscle cells, and stromal fibroblasts depending on mechanical signals transmitted through surrounding ECM. The biomechanical properties of alginate based stimuli-responsive hydrogels can be tuned to match those of different types of tissues. When trying to transport and control the differentiation of hMSCs into generating new tissues or regenerating damaged tissues, it is highly beneficial to encapsulate the cells inside a microsphere made from these hydrogels. The proposed research objectives are: 1) To optimize fabrication techniques and create functional microfluidic devices; 2) Analyze the effects of flow parameters on microsphere production; and 3) Encapsulate viable hMSCs inside multi-stimuli responsive alginate microspheres using the fabricated microfluidic devices (MFDs). In this study, photolithography microfabrication methods were used to create flow-focusing style MFDs. The hydrogel materials were characterized via rheological methods. Syringe pumps controlled flow rates of fluids through the devices. Active droplets formation was monitored through a camera attached to an inverted microscope, where images were analyzed. Microsphere production was analyzed optically and characterized. Alg-MA-β-CD polymer solutions containing hMSCs were encapsulated, and a live/dead florescence assay was preformed to verify cell viability. Using a modified fabrication process it was possible to manufacture Alg-MA-β-CD microspheres and encapsulate and maintain viable hMSCs inside.

Heart failure (HF) is one of the leading causes of death and impacts millions of people throughout the world. Recently, injectable hydrogels have been developed as a potential new therapy to treat myocardium infarction (MI). This dissertation is focused on two main topics: 1) to gain a better understanding the transmural contractility in the healthy left ventricle (LV) wall and 2) investigate the efficacy of the hydrogel injection treatment on LV wall stress and function. The results indicate that a non-uniform distribution of myocardial contractility in the LV wall provide a better representation of normal LV function. The other important study explored the influence altering the stiffness of the biomaterial hydrogel injections. These results show that a larger volume and higher stiffness injection reduce myofiber stress the most and maintaining the wall thickness during loading. The computational approach developed in this dissertation could be used in the future to evaluate the optimal properties of the hydrogel. The last study used a combination of MRI, catheterization, finite element (FE) modeling to investigate the effects of hydrogel injection on borderzone (BZ) contractility after MI. The results indicate that the treatment with hydrogel injection significantly improved BZ function and reduce LV remodeling, via altered MI properties. Additionally, the wall thickness in the infarct and BZ regions were significantly higher in the treated case. Conclusion: hydrogel injection could be a valuable clinical therapy for treating MI.