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Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. (Ytbioteknik)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. (Ytbioteknik)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. (Ytbioteknik)
2009 (English)In: Journal of Biomedical Materials Research Part A, ISSN (printed): 1549-3296. (electronic): 1552-4965., Vol. 91A, no 3, 762-772 p.Article in journal (Refereed) Published
Abstract [en]

Previously, we presented evidence that mucins have potential as   biomaterial coatings. Here, we reveal substantial batch-to-batch   variations for a frequently used commercial bovine salivary mucin   preparation (BSM) and stress the importance of standardizing mucins   intended for comparative purposes. "Mild" fractionation strategies,   aiming at preserving natural mucin functions, were used to prepare two   more defined BSM fractions as well as three mucin fractions from   porcine gastric (PGM) and human salivary (MG1) sources. While the BSM   and PGM were highly purified and mainly adopted random coil   conformations in solution, the MG1 contained mucin-bound components   (1.6 wt% albumin) and appeared compact. Average molar masses and   root-mean-square radii for the predominant BSM, PGM, and MG1 species   spanned 0.8-4.2 MDa and 46-86 nm, respectively. An ellipsometric   evaluation, using hydrophilic and hydrophobic silica, showed the mucin   adsorption to be slow and related to mucin charge, size, conformation,   and compositional complexity. The mass uptakes on hydrophobic silica   averaged 2.6, 2.6, and 5.0 mg/m(2), for BSM, PGM, and MG1,   respectively. Finally, we find that stable mucin coatings can be formed   on polymers of different wettability. The reported mucin preparations   serve as platforms for a series of studies on the biocompatibility of  mucin coatings.

Place, publisher, year, edition, pages
2009. Vol. 91A, no 3, 762-772 p.
Keyword [en]
mucin fractionation, mucin-associated components, size-exclusion chromatograpliy-multiangle light scattering-refractometry (SEC-MALS-RI), biomaterial coating, MUC5B
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97899DOI: 10.1002/jbm.a.32266ISI: 000271588800014PubMedID: 19051309OAI: oai:DiVA.org:uu-97899DiVA: diva2:173004
Available from: 2008-11-28 Created: 2008-11-28 Last updated: 2012-08-01Bibliographically approved
In thesis
1. On the Development of Mucin-based Biomaterial Coatings
Open this publication in new window or tab >>On the Development of Mucin-based Biomaterial Coatings
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications.

In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces.

To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described.

It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance.

Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 83 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 583
Keyword
Mucin, Biomaterial, Surface-exposed protein, XPS, Neutrophil, Cell morphology, SEM, QCM-D, Viscoelasticity, Protein-stabilization, HNL, Coating, MG1, BSM, PGM, SEC-MALS-RI, Mucin quantification, Protein adsorption, Ellipsometry
National Category
Dentistry
Identifiers
urn:nbn:se:uu:diva-9439 (URN)978-91-554-7368-6 (ISBN)
Public defence
2008-12-19, B21, BMC, Husargatan 3, 75123, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-11-28 Created: 2008-11-28 Last updated: 2010-12-14Bibliographically approved
2. Purification Processes for Complex Biomacromolecules
Open this publication in new window or tab >>Purification Processes for Complex Biomacromolecules
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis details various techniques and considerations for the purification of complex biomacromolecules.

 

Initially an α-mannosidase from babaco fruit was purified using anion exchange-, lectin affinity- and size exclusion chromatography.  The enzyme was approximately 260-280 kDa in size with an apparent an unusual octagonal stoichiometry and displayed properties similar to other known plant α-mannosidases.

 

Mucins were fractionated by ion exchange and size exclusion chromatography to assess the properties that govern the mucin surface coating interactions in biomaterial research.  Commercially available mucins, of bovine and porcine origin, as wells as crude human mucin were tested. All showed to consist of a population of molecules which differ in size, charge and composition.

 

The third part of the thesis concerns different aspects of plasmid DNA purification processes.

A two-step method for analysis of plasmid DNA consisting of size exclusion followed by thiophilic adsorption chromatography was evaluated. It allowed determination of the supercoiled plasmid DNA concentration in all process steps without requirement for extensive sample preparation. This method was shown to be fully comparable in terms of accuracy to capillary gel electrophoresis, considered as the industry standard.

Purification of plasmid DNA generally involves bacterial cell alkaline lysis, which creates a solution with flocculate material which needs to be removed prior to further processing. The addition of ammonium hydrogen carbonate to the suspension was evaluated to clarify the solution. The released carbon dioxide and ammonium lifts the flocculate to the surface and allows draining of a clear solution. The method is fully scalable, does not affect the plasmid DNA quality and requires no special equipment.

Thiophilic adsorption chromatography was evaluated for simplification of an existing commercial large scale purification process and was shown to increase both product purity and yields of several tested plasmids. Also, implementation of this step significantly reduced overall production process time.

 

 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 928
Keyword
Chromatography, enzymes, mucins, fractionation, plasmid DNA, analysis, clarification, process design
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Surface Biotechnology
Identifiers
urn:nbn:se:uu:diva-172892 (URN)978-91-554-8353-1 (ISBN)
Public defence
2012-05-25, BMC B7:101, Husargatan 3, Uppsala, 13:15 (English)
Supervisors
Available from: 2012-05-04 Created: 2012-04-17 Last updated: 2012-08-01Bibliographically approved

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