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New insights into principles of scaffolds design for bone application
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry. (polymer chemistry)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules.

Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7.

During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence.  The different inflammatory responses to HA hydrogels were observed (Appendix).

Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo.

Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model.

Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over a long term at a closer physiological level. Yet, efficient gene delivery reagents are needed. In paper IV, two novel gene delivery nanoplexes were developed by post coating DNA-nanoplexes with chondroitin sulfate (CS). To ensure the stability, aldehyde-modified CS (CS-CHO) reacted with free amines of pDNA/PEI complexes. We provided first evidence that CS-CHO coated nanoplexes controlled the release from endosomes, which is essential for higher transfection efficiency.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , p. 87
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1459
Keywords [en]
Chondroitin sulfate, hyaluronic acid, pH, cross-linking chemistry, bone morphogenetic protein, lithium, mesenchymal stem cell, in vivo.
National Category
Medical Materials
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
URN: urn:nbn:se:uu:diva-308318ISBN: 978-91-554-9767-5 (print)OAI: oai:DiVA.org:uu-308318DiVA, id: diva2:1049983
Public defence
2017-01-17, Room 80121, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2016-12-20 Created: 2016-11-24 Last updated: 2016-12-20
List of papers
1. Tuning biomaterial pH for regulating BMP-2 stability and bioactivity in vitro and in vivo
Open this publication in new window or tab >>Tuning biomaterial pH for regulating BMP-2 stability and bioactivity in vitro and in vivo
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

The poor affinity of rhBMP-2 to the scaffolds leads to high dose administration requirement resulted in massive side effects has been the hurdle for successful clinic translation for treating delayed unions or remained non-union at bone defect. Optimizing the scaffolds with the purpose of obtaining optimal BMP2 dose and release have been addressed as critical for BMP-2 administration, however, the results are contradictory concerning whether bone formation is more beneficial from burst or controlled release of BMP2. While this might be due to these studies incorporated other bioactive molecules onto scaffolds to immobilize BMP-2.  In this study, we report the affinities of rhBMP-2 to the scaffolds can be improved by only tuning the pH of hyaluronic acid (HA) hydrazone crosslinking hydrogel without addition of other molecules. Neo bone induced by BMP-2 showed significantly higher volume with more impact structure and vascularization in pH 4.5 HA hydrogel compared to that in pH7 HA hydrogel. The mechanisms were demonstrated by In vitro BMP-2 release followed by diffusion quantitative calculation and computational simulation methods. Initial burst release of BMP-2 from pH 7 HA hydrogels with the fitting of Fickian behavior while sustained release from pH 4.5 HA hydrogel was observed. Computational stimulation revealed this is due to the protonation state of BMP2 at pH 4.5 resulted in stronger electrostatic interaction with negatively charged groups along the backbone of hyaluronic acid molecules compared to at pH 7. This study gives new direction to scaffolds designing for basic bioactive protein applications in future.  

Keywords
RhBMP-2, Hyaluronic acid hydrogel, pH, in vivo bone formation.
National Category
Medical Materials
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-308444 (URN)
Available from: 2016-11-25 Created: 2016-11-25 Last updated: 2016-11-27
2. The choice of crosslinking chemistry for hydrogel development influences BMP-2 stability and bioactivity in vivo
Open this publication in new window or tab >>The choice of crosslinking chemistry for hydrogel development influences BMP-2 stability and bioactivity in vivo
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Site-specific administration of bone morphogenetic protein-2 (BMP-2) at the site of a bone fracture via scaffolds can minimise systemic side-effects and exhibit sustained biological effects. While this method requires sufficient scaffolds to preserve the BMP-2 structure and tuned release patterns, the incorporation of thiol-acrylate chemistry has shown great success in scaffold synthesis. However, thiolates attack the sulphur atoms of disulphide bonds, displacing the other sulphur atom and forming a new disulphide bond, hence at physiological conditions, thiol-modified scaffold components could potentially attack inter-chain disulphide bonds of BMP-2 by thiol-exchange reactions. This therefore led us to compare hyaluronic acid (HA) hydrogels synthesised via thiol-acrylate (HA-S) and hydrazone crosslinking chemistry (HA-H) formed BMP-2 carriers. The study revealed the integrity of BMP-2 dimer structures can be disrupted and reveals the osteogenic capacity of BMP-2 by HA derivatives (HA-SH). BMP-2 bioactivity released from HA-S hydrogels are decreased when compared to HA-H hydrogels. This was further confirmed via the rat ectopic bone model, showing that bone volume was significantly higher when induced by HA-H hydrogels with BMP-2 than compared to HA-S hydrogel with BMP-2. This study gives new insights into scaffolds synthesis, showing that biomolecule bioactivity needs to be considered when choosing a chemistry for scaffolds synthesis. 

Keywords
Hyaluronic acid, in vivo, bone morphogenetic protein, thiol-acrylate chemistry.
National Category
Medical Materials
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-307686 (URN)
Available from: 2016-11-25 Created: 2016-11-20 Last updated: 2016-11-27
3. Lithium hyaluronate hydrogels enhance osteogenesis in vitro and ex vivo
Open this publication in new window or tab >>Lithium hyaluronate hydrogels enhance osteogenesis in vitro and ex vivo
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Lithium is a clinical drug for bipolar disorders and can enhance bone mass, promote osteogenesis of MSCs through inhibiting the Wnt/β-catenin signalling inhibitor GSK 3β. However, the systemic administration of lithium can trigger severe side-effects. Local administration has been attempted in the treatment of bone defects in animal models with positive outcomes. In this study, we explored a pre-manufactured hydrogel system containing the Li ion (Li-gel) in bone applications. Human MSCs cultured in this Li-gel exhibited enhanced osteogenic differentiation. Furthermore, this Li-gel was used to treat chick embryo chorioallantoic membrane (CAM) femur defects and enhanced the bone healing process. 

Keywords
Lithium, mesenchymal stem cells, bone morphogenetic protein, CAM model.
National Category
Medical Materials
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-307689 (URN)
Available from: 2016-11-25 Created: 2016-11-20 Last updated: 2016-12-02
4. Chondroitin Sulfate-Coated DNA-Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection
Open this publication in new window or tab >>Chondroitin Sulfate-Coated DNA-Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection
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2015 (English)In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 25, no 25, p. 3907-3915Article in journal (Refereed) Published
Abstract [en]

Degradation of plasmid DNA (pDNA) in the endosome compartment and its release to the cytosol are the major hurdles for efficient gene transfection. This is generally addressed by using transfection reagents that can overcome these limitations. In this article, the first report is presented which suggests that controlling the release of pDNA from endosome is the key for achieving efficient transfection. In this study, chondroitin sulfate (CS)-coated DNA-nanoplexes are developed using a modular approach where CS is coated post-pDNA/PEI nanoplex formation. To ensure good stability of the nanoplexes, imine/enamine chemistry is exploited by reacting aldehyde-modified chondroitin sulfate (CS-CHO) with free amines of pDNA/PEI complex. This supramolecular nanocarrier system displays efficient cellular uptake, and controlled endosomal pDNA release without eliciting any cytotoxicity. On the contrary, burst release of pDNA from endosome (using chloroqine) results in significant reduction in gene expression. Unlike pDNA/PEI-based transfection, the nanoparticle design presented here shows exceptional stability and gene transfection efficiency in different cell lines such as human colorectal cancer cells (HCT116), human embryonic kidney cells (HEK293), and mouse skin-derived mesenchymal stem cells (MSCs) using luciferase protein as a reporter gene. This new insight will be valuable in designing next generation of transfection reagents.

Keywords
chondroitin sulfate, endosomal escape, gene delivery, mesenchymal stem cells, nanoparticles
National Category
Physical Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-259095 (URN)10.1002/adfm.201500695 (DOI)000357268900013 ()
Funder
EU, FP7, Seventh Framework Programme
Available from: 2015-07-28 Created: 2015-07-27 Last updated: 2017-12-04Bibliographically approved

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