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Tuning biomaterial pH for regulating BMP-2 stability and bioactivity in vitro and in vivo
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
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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.  

Keyword [en]
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: urn:nbn:se:uu:diva-308444OAI: oai:DiVA.org:uu-308444DiVA: diva2:1049884
Available from: 2016-11-25 Created: 2016-11-25 Last updated: 2016-11-27
In thesis
1. New insights into principles of scaffolds design for bone application
Open this publication in new window or tab >>New insights into principles of scaffolds design for bone application
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
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. 87 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1459
Keyword
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:nbn:se:uu:diva-308318 (URN)978-91-554-9767-5 (ISBN)
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

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