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Non-invasive in vitro and in vivo monitoring of degradation of fluorescently labeled hyaluronan hydrogels for tissue engineering applications
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
Politecn Milan, Dept Chem Mat & Chem Engn Giulio Natta, Via Mancinelli, I-20131 Milan, Italy..
IRCCS Ist Ric Farmacol Mario Negri, Dept Neurosci, Via La Masa 19, I-20156 Milan, Italy..
IRCCS Ist Ric Farmacol Mario Negri, Dept Biochem & Mol Pharmacol, Via La Masa 19, I-20156 Milan, Italy..
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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) PublishedText
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.

Place, publisher, year, edition, pages
2016. Vol. 30, 188-198 p.
Keyword [en]
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: urn:nbn:se:uu:diva-277793DOI: 10.1016/j.actbio.2015.11.053ISI: 000368563600017PubMedID: 26621694OAI: oai:DiVA.org:uu-277793DiVA: diva2:905780
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
In thesis
1. Hyaluronan Based Biomaterials with Imaging Capacity for Tissue Engineering
Open this publication in new window or tab >>Hyaluronan Based Biomaterials with Imaging Capacity for Tissue Engineering
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
hyaluronan, hydrogel, scaffold, tissue engineering, imaging, interpenetrating network, MRI, fluorescence imaging, scaffold degradation
National Category
Materials Chemistry Polymer Chemistry
urn:nbn:se:uu:diva-300799 (URN)978-91-554-9649-4 (ISBN)
External cooperation:
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

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