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Biomaterials for Promoting Self-Healing of Bone Tissue
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Polymer Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present work addresses poor bone/implant integration and severe bone defects. In both conditions external stimuli is required for new bone to form. A multilayered functional implant coating, comprised of an inner layer of crystalline titanium dioxide (TiO2) and an outer layer of hydroxyapatite (HAP), loaded with bone morphogenetic protein-2 (BMP-2), was proposed as a tool for providing both improved initial bone formation and long-term osseointegration. The in vitro characterization of the implant coatings showed that TiO2 and HAP were more favorable for cell viability, cell morphology and initial cell differentiation, compared to native titanium oxide. Furthermore, significantly higher cell differentiation was observed on surfaces with BMP-2, indicating that a simple soaking process can be used for incorporating bioactive molecules. Moreover, the results suggest that there could be a direct interaction between BMP-2 and HAP, which prolongs the retention of the growth factor, improving its therapeutic effect.

For treating severe bone defects a strategy involving BMP-2 delivery from hyaluronan hydrogels was explored. The hydrogels were prepared from two reactive polymers – an aldehyde-modified hyaluronan and a hydrazide-modified poly(vinyl alcohol). Upon mixing, the two components formed a chemically crosslinked hydrogel. In this work the mixing of the hydrogel components was optimized by rheological measurements. Furthermore, an appropriate buffer was selected for in vitro experiments by studying the swelling of hydrogels in PBS and in cell culture medium. A detection method, based on radioactive labeling of BMP-2 with 125I was used to monitor growth factor release both in vitro and in vivo. The results showed a biphasic release profile of BMP-2, where approximately 16 %  and 3 % of the growth factor remained inside the hydrogel after 4 weeks in vitro and in vivo, respectively. The initial fast release phase corresponded to the early ectopic bone formation observed 8 d after injection of the hydrogel formulation in the thigh muscle of rats. The hydrogel formulation could be improved by incorporation of HAP powder into the hydrogel formulation. Furthermore, bone formation could be increased by pre-incubation of the premixed hydrogel components inside the syringe prior to injection. Crushed hydrogels were also observed to induce more bone formation compared to solid hydrogels, when implanted subcutaneously in rats. This was thought to be due to increased surface area of the hydrogel, which allowed for improved cell infiltration.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 857
Keyword [en]
Osseointegration, implant coating, hyaluronan hydrogel, bone morphogenetic protein-2, hydroxyapatite, bone, delivery system, injectable, mixing, radioactive labeling
National Category
Biomaterials Science Polymer Chemistry
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-158939ISBN: 978-91-554-8168-1 (print)OAI: oai:DiVA.org:uu-158939DiVA: diva2:443492
Public defence
2011-11-11, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2011-10-21 Created: 2011-09-19 Last updated: 2011-11-04Bibliographically approved
List of papers
1. In vitro characterization of bioactive titanium dioxide/hydroxyapatite surfaces functionalized with BMP-2
Open this publication in new window or tab >>In vitro characterization of bioactive titanium dioxide/hydroxyapatite surfaces functionalized with BMP-2
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2009 (English)In: Journal of biomedical materials research. Part B, Applied biomaterials, ISSN 1552-4981, Vol. 91B, no 2, 780-787 p.Article in journal (Refereed) Published
Abstract [en]

Poor implant fixation and bone resorption are two of the major challenges in modern orthopedics and are caused by poor bone/implant integration. In this work, bioactive crystalline titanium dioxide (TiO(2))/hydroxyapatite (HA) surfaces, functionalized with bone morphogenetic protein 2 (BMP-2), were evaluated as potential implant coatings for improved osseointegration. The outer layer consisted of HA, which is known to be osteoconductive, and may promote improved initial bone attachment when functionalized with active molecules such as BMP-2 in a soaking process. The inner layer of crystalline TiO(2) is bioactive and ensures long-term fixation of the implant, once the hydroxyapatite has been resorbed. The in vitro response of mesenchymal stem cells on bioactive crystalline TiO(2)/HA surfaces functionalized with BMP-2 was examined and compared with the cell behavior on nonfunctionalized HA layers, crystalline TiO(2) surfaces, and native titanium oxide surfaces. The crystalline TiO(2) and the HA surfaces showed to be more favorable than the native titanium oxide surface in terms of cell viability and cell morphology as well as initial cell differentiation. Furthermore, cell differentiation on BMP-2-functionalized HA surfaces was found to be significantly higher than on the other surfaces indicating that the simple soaking process can be used for incorporating active molecules, promoting fast bone osseointegration to HA layers.

Keyword
biomimetic hydroxyapatite, growth factors, BMP-2, anatase titanium dioxide, mesenchymal stem cells, differentiation, viability, morphology
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-108680 (URN)10.1002/jbm.b.31456 (DOI)000270868600034 ()19582842 (PubMedID)
Available from: 2009-09-26 Created: 2009-09-26 Last updated: 2016-11-30Bibliographically approved
2. The Effect of Mixing on the Mechanical Properties of Hyaluronan-Based Injectable Hydrogels
Open this publication in new window or tab >>The Effect of Mixing on the Mechanical Properties of Hyaluronan-Based Injectable Hydrogels
2011 (English)In: Macromolecular materials and engineering (Print), ISSN 1438-7492, E-ISSN 1439-2054, Vol. 296, no 10, 944-951 p.Article in journal (Refereed) Published
Abstract [en]

A method for determining the correlation between the mixing of two reactive polymers and the structural and mechanical properties of the formed hydrogels is presented. Rheological measurements show that insufficient mixing gives rise to soft and not fully crosslinked hydrogels while excessive mixing beyond gel point results in weaker hydrogels due to potential breakage of their 3D network. Furthermore, the hydrogels swell significantly more in cell culture medium than in phosphate-buffered saline, attributed to interactions with additional molecules such as proteins. Thus, moderate mixing gives rise to the most homogenous and mechanically stable hydrogels and the choice of medium e.g., for release experiments, should be consistent in order to avoid unnecessary variations in the data caused by different swelling profiles.

Keyword
Hydrogels, mixing, polymer, rheology, swelling
National Category
Polymer Chemistry
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-158952 (URN)10.1002/mame.201100008 (DOI)000296421800008 ()
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
3. Characterization of recombinant human bone morphogenetic protein-2 delivery from injectable hyaluronan-based hydrogels by means of I-125-radiolabelling
Open this publication in new window or tab >>Characterization of recombinant human bone morphogenetic protein-2 delivery from injectable hyaluronan-based hydrogels by means of I-125-radiolabelling
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2014 (English)In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 8, no 10, 821-830 p.Article in journal (Refereed) Published
Abstract [en]

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP-2) from a hyaluronan-based hydrogel system. The in vitro release of BMP-2 from similar hydrogels has previously been studied by enzyme-linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, I-125 radiolabelling was used instead to monitor BMP-2 in vitro and in vivo. To minimize protein loss during handling, I-125-BMP-2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP-2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast-release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP-2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP-2 delivery system.

Keyword
BMP-2 delivery, radioactive labeling, hyaluronan hydrogels, reproducibility, ectopic bone formation, protein adsorption
National Category
Biomaterials Science
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-158961 (URN)10.1002/term.1584 (DOI)000343059700009 ()22927307 (PubMedID)
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
4. Pre-incubation of chemically crosslinked hyaluronan-based hydrogels, loaded with BMP-2 and hydroxyapatite, and its effect on ectopic bone formation
Open this publication in new window or tab >>Pre-incubation of chemically crosslinked hyaluronan-based hydrogels, loaded with BMP-2 and hydroxyapatite, and its effect on ectopic bone formation
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2014 (English)In: Journal of materials science. Materials in medicine, ISSN 0957-4530, E-ISSN 1573-4838, Vol. 25, no 4, 1013-1023 p.Article in journal (Refereed) Published
Abstract [en]

The effects of pre-incubation of hyaluronan hydrogels, for different lengths of time after the initiation of chemical crosslinking and prior to injection, were explored both by investigating the in vitro BMP-2 release kinetics from the hydrogel and by studying the ectopic bone formation in rats. From the curing profile, obtained from rheological analysis, appropriate pre-incubation times (1 min, 5 h and 3 days) were selected, to prepare slightly, moderately and fully cured hydrogels. Comparable release profiles were observed for all three test groups in vitro. Furthermore, radiography, pQCT and histology of the explanted grafts showed cancellous bone formation in all groups after 5 weeks in vivo. However, longer pre-incubation times gave rise to an increase in bone volume, but a decrease in bone density. Moreover, the 5 h and the 3 days grafts appeared to be more ordered and resistant to deformation from the surrounding tissue than the 1 min grafts. The observed variations in mechanical and biological properties could potentially be used to adapt the treatment for a specific indication.

National Category
Biomaterials Science
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-158963 (URN)10.1007/s10856-014-5147-y (DOI)000333093300006 ()
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
5. Improved bone formation through increased surface area of hyaluronan-based hydrogels when used as carriers for BMP-2
Open this publication in new window or tab >>Improved bone formation through increased surface area of hyaluronan-based hydrogels when used as carriers for BMP-2
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(English)Manuscript (preprint) (Other academic)
Keyword
Hyaluronan, hydroxyapatite, BMP, hydrogel, porosity, bone regeneration
National Category
Biomaterials Science
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-158967 (URN)
Available from: 2011-09-26 Created: 2011-09-19 Last updated: 2011-11-04

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