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Hyaluronan Based Biomaterials with Imaging Capacity for Tissue Engineering
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström. (Polymer group)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents the preparation of hyaluronan-based biomaterials with imaging capability and their application as scaffolds in tissue engineering. First, we have synthesized HA derivatives functionalized with different chemoselective groups. Then, functional ligands with capacities for hydrophobic drug loading, imaging, and metal ion coordination were chemically conjugated to HA by chemoselective reactions with these groups. An injectable in situ forming HA hydrogel was prepared by hydrazone cross-linking between hybrid iron-oxide nanogel and HA-aldehyde (paper-I). The degradation of this hydrogel could be monitored by MRI and UV-vis spectroscopy since it contained iron oxide as a contrast agent and pyrene as a fluorescent probe. Additionally, this hydrogel has a potential for a delivery of hydrophobic drugs due to its pyrene hydrophobic domains. The degradation study showed that degradability of the hydrogel was correlated with its structure. Based on the obtained results, disulfide cross-linked and fluorescently labeled hydrogels with different HA concentration were established as a model to study the relationship between the structure of the hydrogel and its degradability (paper-II). We demonstrated that disulfide cross-linked HA hydrogel could be tracked non-invasively by fluorescence imaging. It was proved that the in vivo degradation behavior of the hydrogel is predictable basing on its in vitro degradation study. In paper-III, we developed a disulfide cross-linked HA hydrogel for three-dimensional (3D) cell culture. In order to improve cell viability and adhesion to the matrix, HA derivatives were cross-linked in the presence of fibrinogen undergoing polymerization upon the action of thrombin. It led to the formation of an interpenetrating double network (IPN) of HA and fibrin. The results of 3D cell culture experiments revealed that the IPN hydrogel provides the cells with a more stable environment for proliferation. The results of the cellular studies were also supported by in vitro degradation of IPN monitored by fluorescence measurements of the degraded products. In paper-IV, the effect of biomineralization on hydrogel degradation was evaluated in a non-invasive manner in vitro. For this purpose, two types of fluorescently labeled hydrogels with the different ability for biomineralization were prepared. Fluorescence spectroscopy was applied to monitor degradation of the hydrogels in vitro under two different conditions in longitudinal studies. Under the supply of Ca2+ ions, the BP-modified hydrogel showed the tendency to bio-mineralization and reduction of the rate of degradation. Altogether, the performed studies showed the importance of imaging of hydrogel biomaterials in the design of optimized scaffolds for tissue engineering.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1402
Keyword [en]
hyaluronan, hydrogel, scaffold, tissue engineering, imaging, interpenetrating network, MRI, fluorescence imaging, scaffold degradation
National Category
Materials Chemistry Polymer Chemistry
URN: urn:nbn:se:uu:diva-300799ISBN: 978-91-554-9649-4OAI: oai:DiVA.org:uu-300799DiVA: diva2:952466
Public defence
2016-09-29, 80101, Lägerhyddsvägen 1, Uppsala, 09:00 (English)
Available from: 2016-09-06 Created: 2016-08-14 Last updated: 2016-09-13
List of papers
1. Injectable In Situ Forming Hybrid Iron Oxide-Hyaluronic Acid Hydrogel for Magnetic Resonance Imaging and Drug Delivery
Open this publication in new window or tab >>Injectable In Situ Forming Hybrid Iron Oxide-Hyaluronic Acid Hydrogel for Magnetic Resonance Imaging and Drug Delivery
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2014 (English)In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 14, no 9, 1249-1259 p.Article in journal (Refereed) Published
Abstract [en]

The development of multimodal in situ cross-linkable hyaluronic acid nanogels hybridized with iron oxide nanoparticles is reported. Utilizing a chemoselective hydrazone coupling reaction, the nanogels are converted to a macroscopic hybrid hydrogel without any additional reagent. Hydrophobic cargos remain encapsulated in the hydrophobic domains of the hybrid hydrogel without leakage. However, hydrogel degradation with hyaluronidase liberates iron oxide nanoparticles. This allows the utilization of imaging agents as tracers of the hydrogel degradation. UV-vis spectrometry and MRI studies reveal that the degradability of the hydrogels correlates with their structure. The hydrogels presented here are very promising theranostic tools for hyaluronidase-mediated delivery of hydrophobic drugs, as well as imaging of hydrogel degradation and tracking of degradation products in vivo.

hybrid organic-inorganic nanogels, hyaluronan hydrogels, in situ cross-linking, iron oxide nanoparticles, magnetic resonance imaging
National Category
Polymer Chemistry Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-236090 (URN)10.1002/mabi.201400117 (DOI)000342921900005 ()
Available from: 2014-11-17 Created: 2014-11-12 Last updated: 2016-09-02Bibliographically approved
2. Non-invasive in vitro and in vivo monitoring of degradation of fluorescently labeled hyaluronan hydrogels for tissue engineering applications
Open this publication in new window or tab >>Non-invasive in vitro and in vivo monitoring of degradation of fluorescently labeled hyaluronan hydrogels for tissue engineering applications
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2016 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 30, 188-198 p.Article in journal (Refereed) Published
Abstract [en]

Tracking of degradation of hydrogels-based biomaterials in vivo is very important for rational design of tissue engineering scaffolds that act as delivery carriers for bioactive factors. During the process of tissue development, an ideal scaffold should remodel at a rate matching with scaffold degradation. To reduce amount of animals sacrificed, non-invasive in vivo imaging of biomaterials is required which relies on using of biocompatible and in situ gel forming compounds carrying suitable imaging agents. In this study we developed a method of in situ fabrication of fluorescently labeled and injectable hyaluronan (HA) hydrogel based on one pot sequential use of Michael addition and thiol-disulfide exchange reactions for the macromolecules labeling and cross-linking respectively. Hydrogels with different content of HA were prepared and their enzymatic degradation was followed in vitro and in vivo using fluorescence multispectral imaging. First, we confirmed that the absorbance of the matrix-linked near-IR fluorescent IRDye (R) 800CW agent released due to the matrix enzymatic degradation in vitro matched the amount of the degraded hydrogel measured by classical gravimetric method. Secondly, the rate of degradation was inversely proportional to the hydrogel concentration and this structure-degradation relationship was similar for both in vitro and in vivo studies. It implies that the degradation of this disulfide cross-linked hyaluronan hydrogel in vivo can be predicted basing on the results of its in vitro degradation studies. The compliance of in vitro and in vivo methods is also promising for the future development of predictive in vitro tissue engineering models. Statement of significance The need for engineered hydrogel scaffolds that deliver bioactive factors to endogenous progenitor cells in vivo via gradual matrix resorption and thus facilitate tissue regeneration is increasing with the aging population. Importantly, scaffold should degrade at a modest rate that will not be too fast to support tissue growth nor too slow to provide space for tissue development. The present work is devoted to longitudinal tracking of a hydrogel material in vivo from the time of its implantation to the time of complete resorption without sacrificing animals. The method demonstrates correlation of resorption rates in vivo and in vitro for hydrogels with varied structural parameters. It opens the possibility to develop predictive in vitro models for tissue engineered scaffolds and reduce animal studies.

Hyaluronan, Hydrogel degradation, Non-invasive imaging, Spinal cord, Tissue engineering
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:uu:diva-277793 (URN)10.1016/j.actbio.2015.11.053 (DOI)000368563600017 ()26621694 (PubMedID)
EU, FP7, Seventh Framework Programme

De två första författarna delar förstaförfattarskapet.

Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2016-09-02Bibliographically approved
3. Hyaluronic acid-fibrin interpenetrating double network hydrogel prepared in situ by orthogonal disulfide cross-linking reaction for biomedical applications
Open this publication in new window or tab >>Hyaluronic acid-fibrin interpenetrating double network hydrogel prepared in situ by orthogonal disulfide cross-linking reaction for biomedical applications
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2016 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 38, 23-32 p.Article in journal (Refereed) Published
Abstract [en]

To strengthen the mechanical properties of a fibrin gel and improve its applicability as a scaffold for tissue engineering (TE) applications, a strategy for the in situ preparation of the interpenetrating network (IPN) of fibrin and hyaluronic acid (HA) was developed on the basis of simultaneous and orthogonal fibrinogenesis and disulfide cross-linking. The synthetic pathway included the preparation of mutually reactive HA derivatives bearing thiol and 2-dithiopyridyl groups. Combining thiol-derivatized HA with thrombin and 2-dithiopyridyl-modified HA with fibrinogen and then mixing the obtained liquid formulations afforded IPNs with fibrin-resembling fibrillar architectures at different ratios between fibrin and HA networks. The formation of two networks was confirmed by conducting reference experiments with the compositions lacking one of the four components. The composition of 2% (w/v) fibrin and 1% (w/v) HA showed the highest storage modulus (G'), as compared with the single network counterparts. The degradation of fibrin in IPN hydrogels was slower than that in pure fibrin gels both during incubation of the hydrogels in a fibrin-cleaving nattokinase solution and during the culturing of cells after their encapsulation in the hydrogels. Together with the persistence of HA network, it permitted longer cell culturing time in the IPN. Moreover, the proliferation and spreading of MG63 cells that express the hyaluronan receptor CD44 in IPN hydrogel was increased, as compared with its single network analogues. These results are promising for tunable ECM-based materials for TE and regenerative medicine. Statement of Significance The present work is devoted to in situ fabrication of injectable extracellular matrix hydrogels through simultaneous generation of networks of fibrin and hyaluronic acid (HA) that interpenetrate each other. This is accomplished by combination of enzymatic fibrin cross-linking with orthogonal disulphide cross-linking of HA. High hydrophilicity of HA prevents compaction of the fibrin network, while fibrin provides an adhesive environment for in situ encapsulated cells. The interpenetrating network hydrogel shows an increased stiffness along with a lower degradation rate of fibrin in comparison to the single fibrin network. As a result, the cells have sufficient time for the remodelling of the scaffold. This new approach can be applied for modular construction of in vitro tissue models and tissue engineering scaffolds in vivo.

Interpenetrating hydrogel, Fibrin, Hyaluronic acid, Biodegradation, 3D cell culture
National Category
Polymer Chemistry Bio Materials
urn:nbn:se:uu:diva-300044 (URN)10.1016/j.actbio.2016.04.041 (DOI)000378963800003 ()27134013 (PubMedID)
EU, European Research Council
Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-09-02Bibliographically approved
4. Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio-mineralization
Open this publication in new window or tab >>Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio-mineralization
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences Polymer Chemistry
urn:nbn:se:uu:diva-300791 (URN)
Available from: 2016-08-14 Created: 2016-08-13 Last updated: 2016-09-02

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