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Nanoscale Osseointegration: Characterization of Biomaterials and their Interfaces with Electron Tomography
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences. (Materials in Medicine)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bone response is one of the key determining factors in the overall success of biomaterials intended for bone regeneration and osseointegration. Understanding the formation of bone at an implant surface may lead to the improved design of biomaterials for the future. However, due to the inhomogeneity of bone tissue at an interface, two-dimensional images often lack detail on the interfacial complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar nano length scale.

While current analysis methods, such as X-ray tomography, transmission electron microscopy, focused ion beam microscopy and scanning electron microscopy, provide a basis for analysing biomaterials and biointerfaces, they are incapable of doing so with both nanometre resolution and three-dimensional clarity. In contrast, electron tomography may be used to characterize the three-dimensional structure of biomaterials and their interfaces to bone with nanometre resolution.

In this work, hydroxyapatite scaffolds, and laser-modified titanium and Ti6Al4V implants were studied in contact with human or rabbit bone. Z-contrast electron tomography revealed that the orientation of collagen in bone apposing hydroxyapatite, titanium and Ti6Al4V implants is consistently parallel to the implant surface, where the bioactive layer that precipitates on HA is oriented perpendicular to the implant surface. With this method, complete three-dimensional nanoscale osseointegration of titanium-based implants was also established.

The extension of this technique from interfacial analyses to the design of biomaterials provided an understanding of the pore structure of mesoporous titania. In further investigations, the open three-dimensional pore network, as revealed by electron tomography, showed promise as a coating that improves implant osseointegration and enables site-specific drug-delivery from an implant surface.

In summary, it was demonstrated that two-dimensional characterization techniques were insufficient for the investigation of nanostructured biomaterials, as well as their interfaces to bone. Visualizing biointerfaces and biomaterials with nanometre precision in three-dimensions can expose new fundamental information on materials properties and bone response, enabling better design of biomaterials for the future.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 962
Keywords [en]
Electron tomography, transmission electron microscopy, hydroxyapatite, titanium, titania, bone, implant, osseointegration, interface, mesoporous
National Category
Materials Engineering
Research subject
Materials Science
Identifiers
URN: urn:nbn:se:uu:diva-179445ISBN: 978-91-554-8441-5 (print)OAI: oai:DiVA.org:uu-179445DiVA, id: diva2:544694
Public defence
2012-10-05, Polhemsalen, Ångström Laboratory, Lägerhyddsvägen 1, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-08-15 Last updated: 2013-01-22Bibliographically approved
List of papers
1. Bone response to free form fabricated hydroxyapatite and zirconia scaffolds: a transmission electron microscopy study in the human maxilla
Open this publication in new window or tab >>Bone response to free form fabricated hydroxyapatite and zirconia scaffolds: a transmission electron microscopy study in the human maxilla
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2012 (English)In: Clinical Implant Dentistry and Related Research, ISSN 1523-0899, E-ISSN 1708-8208, Vol. 14, no 3, p. 461-469Article in journal (Refereed) Published
Abstract [en]

Background: Understanding the interfacial reactions to synthetic bone regenerative scaffolds in vivo is fundamental for improving osseointegration and osteogenesis. Using transmission electron microscopy, it is possible to study the biological response of hydroxyapatite (HA) and zirconia (ZrO2) scaffolds at the nanometer scale.

Purpose: In this study, the bone-bonding abilities of HA and ZrO2 scaffolds produced by free-form fabrication were evaluated in the human maxilla at 3 months and 7 months.

Materials and Methods: HA and ZrO2 scaffolds (ø: 3 mm) were implanted in the human maxilla, removed with surrounding bone, embedded in resin, and sectioned. A novel focused ion beam (FIB) sample preparation technique enabled the production of thin lamellae for study by scanning transmission electron microscopy.

Results: Interface regions were investigated using high-angle annular dark-field imaging and energy-dispersive X-ray spectroscopy analysis. Interfacial apatite layers of 80 nm and 50 nm thickness were noted in the 3- and 7-month HA samples, respectively, and bone growth was discovered in micropores up to 10 µm into the samples.

Conclusions: The absence of an interfacial layer in the ZrO2 samples suggest the formation of a direct contact with bone, while HA, which bonds through an apatite layer, shows indications of resorption with increasing implantation time. This study demonstrates the potential of HA and ZrO2 scaffolds for use as bone regenerative materials.

Keywords
FIB, free-form fabrication, hydroxyapatite, scaffolds, TEM, zirconia
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Microsystems Technology; Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-122088 (URN)10.1111/j.1708-8208.2009.00270.x (DOI)000304759300019 ()
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2018-02-08Bibliographically approved
2. Visualizing biointerfaces in three dimensions: electron tomography of the bone-hydroxyapatite interface
Open this publication in new window or tab >>Visualizing biointerfaces in three dimensions: electron tomography of the bone-hydroxyapatite interface
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2010 (English)In: Journal of the Royal Society Interface, ISSN 1742-5689, E-ISSN 1742-5662, Vol. 7, no 51, p. 1497-501Article in journal (Refereed) Published
Abstract [en]

A positive interaction between human bone tissue and synthetics is crucial for the success of bone-regenerative materials. A greater understanding of the mechanisms governing bone-bonding is often gained via visualization of the bone-implant interface. Interfaces to bone have long been imaged with light, X-rays and electrons. Most of these techniques, however, only provide low-resolution or two-dimensional information. With the advances in modern day transmission electron microscopy, including new hardware and increased software computational speeds, the high-resolution visualization and analysis of three-dimensional structures is possible via electron tomography. We report, for the first time, a three-dimensional reconstruction of the interface between human bone and a hydroxyapatite implant using Z-contrast electron tomography. Viewing this structure in three dimensions enabled us to observe the nanometre differences in the orientation of hydroxyapatite crystals precipitated on the implant surface in vivo versus those in the collagen matrix of bone. Insight into the morphology of biointerfaces is considerably enhanced with three-dimensional techniques. In this regard, electron tomography may revolutionize the approach to high-resolution biointerface characterization.

Keywords
hydroxyapatite, bone, transmission, electron microscopy, electron tomography
National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-133206 (URN)10.1098/rsif.2010.0213 (DOI)000281281000010 ()20534599 (PubMedID)
Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2018-02-08Bibliographically approved
3. High-resolution three-dimensional probes of biomaterials and their interfaces
Open this publication in new window or tab >>High-resolution three-dimensional probes of biomaterials and their interfaces
2012 (English)In: Philosophical Transactions. Series A: Mathematical, physical, and engineering science, ISSN 1364-503X, E-ISSN 1471-2962, Vol. 370, no 1963, p. 1337-1351Article in journal (Refereed) Published
Abstract [en]

Interfacial relationships between biomaterials and tissues strongly influence the success of implant materials and their long-term functionality. Owing to the inhomogeneity of biological tissues at an interface, in particular bone tissue, two-dimensional images often lack detail on the interfacial morphological complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar length scale. Electron tomography (ET) can meet these challenges by enabling high-resolution three-dimensional imaging of biomaterial interfaces. In this article, we review the fundamentals of ET and highlight its recent applications in probing the three-dimensional structure of bioceramics and their interfaces, with particular focus on the hydroxyapatite-bone interface, titanium dioxide-bone interface and a mesoporous titania coating for controlled drug release.

Keywords
electron tomography, interface, hydroxyapatite, bone, titanium dioxide, implant interface
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-171539 (URN)10.1098/rsta.2011.0253 (DOI)000300631900004 ()
Available from: 2012-03-21 Created: 2012-03-21 Last updated: 2018-02-08Bibliographically approved
4. Resolving the CaP-bone interface: A review of discoveries with light and electron microscopy
Open this publication in new window or tab >>Resolving the CaP-bone interface: A review of discoveries with light and electron microscopy
2012 (English)In: Biomatter, ISSN 2159-2527, Vol. 2, no 1, p. 15-23Article in journal (Refereed) Published
National Category
Materials Engineering
Research subject
Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-179313 (URN)10.4161/biom.20062 (DOI)
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2018-02-08Bibliographically approved
5. Bone-titanium oxide interface in humans revealed by transmission electron microscopy and electron tomography
Open this publication in new window or tab >>Bone-titanium oxide interface in humans revealed by transmission electron microscopy and electron tomography
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2012 (English)In: Journal of the Royal Society Interface, ISSN 1742-5689, E-ISSN 1742-5662, Vol. 9, no 67, p. 396-400Article in journal (Refereed) Published
Abstract [en]

Osseointegration, the direct contact between an implant surface and bone tissue, plays a critical role in interfacial stability and implant success. Analysis of interfacial zones at the micro- and nano-levels is essential to determine the extent of osseointegration. In this paper, a series of state-of-the-art microscopy techniques are used on laser-modified implants retrieved from humans. Partially laser-modified implants were retrieved after two and a half months' healing and processed for light and electron microscopy. Light microscopy showed osseointegration, with bone tissue growing both towards and away from the implant surface. Transmission electron microscopy revealed an intimate contact between mineralized bone and the laser-modified surface, including bone growth into the nano-structured oxide. This novel observation was verified by three-dimensional Z-contrast electron tomography, enabling visualization of an apatite layer, with different crystal direction compared with the apatite in the bone tissue, encompassing the nano-structured oxide. In conclusion, the present study demonstrates the nano-scale osseointegration and bonding between apatite and surface-textured titanium oxide. These observations provide novel data in human specimens on the ultrastructure of the titanium–bone interface.

Keywords
dental implant, laser, focused ion beam, transmission electron microscopy, electron tomography, bone
National Category
Medical Materials Engineering and Technology
Research subject
Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-160563 (URN)10.1098/rsif.2011.0420 (DOI)000298380100018 ()
Available from: 2011-10-25 Created: 2011-10-25 Last updated: 2017-12-08Bibliographically approved
6. Three-dimensional structure of laser-modified Ti6Al4V and bone interface revealed with STEM tomography
Open this publication in new window or tab >>Three-dimensional structure of laser-modified Ti6Al4V and bone interface revealed with STEM tomography
2013 (English)In: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 127, p. 48-52Article in journal (Refereed) Published
Abstract [en]

The early interaction between an implant's surface and bone is a leading factor for implant success, where multiple surface properties contribute to improved bone anchorage. An important parameter is surface topography, both on the micron and nanoscale. Laser-modification has been performed in the thread valleys of Ti6Al4V screws to alter their surface chemistry and topography to form a nanostructured surface titanium-dioxide. Implants were placed in the rabbit tibia, removed with surrounding bone after 8 weeks, fixated, dried and resin embedded. Focused ion beam milling (FIB) was used to prepare specimens from the resin blocks for transmission electron microscopy (TEM). Z-contrast electron tomography offered the possibility to explore the interfacial structure with high-resolution in three-dimensions. With this technique, collagen fibers of the surrounding bone appear to have been laid down parallel to the implant surface. Accordingly, visualization of the laser-modified interface with nanoscale three-dimensional resolution, as offered by Z-contrast electron tomography, gives new insights into bone bonding mechanisms between roughened titanium-dioxide surfaces and bone.

National Category
Materials Engineering
Research subject
Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-179312 (URN)10.1016/j.ultramic.2012.07.007 (DOI)000316659100008 ()
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2018-02-08Bibliographically approved
7. Mesoporous titanium dioxide coating for metallic implants
Open this publication in new window or tab >>Mesoporous titanium dioxide coating for metallic implants
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2012 (English)In: Journal of Biomedical Materials Research. Part B - Applied biomaterials, ISSN 1552-4973, E-ISSN 1552-4981, Vol. 100B, no 1, p. 82-93Article in journal (Refereed) Published
Abstract [en]

A bioactive mesoporous titanium dioxide (MT) coating for surface drug delivery has been investigated to develop a multifunctional implant coating, offering quick bone bonding and biological stability. An evaporation induced self-assembly (EISA) method was used to prepare a mesoporous titanium dioxide coating of the anatase phase with BET surface area of 172 m2/g and average pore diameter of 4.3 nm. Adhesion tests using the scratch method and an in situ screw-in/screw-out technique confirm that the MT coating bonds tightly with the metallic substrate, even after removal from bone. Because of its high surface area, the bioactivity of the MT coating is much better than that of a dense TiO2 coating of the same composition. Quick formation of hydroxyapatite (HA) in vitro can be related to enhance bonding with bone. The uptake of antibiotics by the MT coating reached 13.4 mg/cm3 within a 24 h loading process. A sustained release behavior has been obtained with a weak initial burst. By using Cephalothin as a model drug, drug loaded MT coating exhibits a sufficient antibacterial effect on the material surface, and within millimeters from material surface, against E.coli. Additionally, the coated and drug loaded surfaces showed no cytotoxic effect on cell cultures of the osteoblastic cell line MG-63. In conclusion, this study describes a novel, biocompatiblemesoporous implant coating, which has the ability to induce HA formation and could be used as a surface drug-delivery system.

Keywords
mesoporous materials, titanium oxide, drug delivery, bioactivity, implant coating
National Category
Biomaterials Science Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials; Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-159881 (URN)10.1002/jbm.b.31925 (DOI)000297949800010 ()
Available from: 2011-10-11 Created: 2011-10-11 Last updated: 2018-02-08Bibliographically approved

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