Bioinspired fabrication of biosilica-based bone substitution materials [Elektronische Ressource] / Filipe André da Silva Raminhos Natálio

Filipe Andre Natalio

Sujets

• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 34
• Poids de l'ouvrage: 4 Mo

Extrait

Bioinspired fabrication of biosilica-based
bone substitution materials
Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften
im Promotionsfach Chemie
Am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg-Universität
in Mainz
Filipe André da Silva Raminhos Natálio
geb. in Caldas da Rainha (Portugal)
Mainz, 18.12.2009Tag der mündlichen Prüfung: 13.07.2010Index
1. Summary/Zusammenfassung………………………………………………………..8
2. Introduction
2.1. Biomineralization…………………………………………………………..………13
2.2. Sponges as Eumetazoa: A long pathway…………………………………………15
2.3. Biomineralization in sponges……………………………………………………...17
2.3.1. Geological background……………………………………………………17
2.3.2. Biosilification in spicules…………………………………………………18
2.3.2.1. Structural features………………………………………………18
2.3.2.2. Biochemical approach…………………………………………..19
2.3.2.3. Silicatein – Silica polymerizing protein…………………………20
2.3.2.4. Spiculogenesis…………………………………………………...22
2.4. Silicatein wide applications..……………………………………….……………...26
2.4.1. Bionanotechnological applications of silicatein…………………………..26
2.4.2.Biomedical approach………………………………………………………30
2.5. Calcium Phosphate biominerals………………………………………………….32
2.5.1. Bone………………………………………………………………………32
2.5.1.1. Bone has functional biomineral in animal evolution…………...32
2.5.1.2. Bone hierarchial composition…………………………………..33
2.5.1.2. Bone biomineralization…………………………………………36
2.6. Tissue engineering………………………………………………………………...39
2.6.1. Overview…………………………………………………………………39
2.6.2. Biological/medical approach……………………………………………..40
2.6.2.1. Bone grafts……………………………………………………..40
2.6.3. Synthetic and biomimetic materials……………………………………...42
2.6.3.1. Silica containing implant: Bioglass®………………………….46
3. Experimental Procedure
3.1. Component A: Microencapsulation of silicatein and respective
precursor into poly(D,L-lactide) biodegradable microspheres……………………50
3.1.1. Production of polymeric spheres containing both
silicatein and silica……………………………………………………………..50
3.1.2. Physical characterization of PLA microspheres………………………...51
3.1.2.1. Microscopic analysis of the PLA-silicatein
microspheres…………………………………………………………....51
3.1.2.2. Dynamic Light Scattering (DLS)……………………………...513.1.3. Immunodetection of encapsulated silicatein……………………………51
3.1.3.1. Immunoblotting……………………………………………….51
3.1.3.2. Immunochemistry……………………………………………..53
3.1.4. Elemental detection of encapsulated silica……………………………..54
3.1.5. Release of silicatein from polymeric
microspheres…………………………………………………………………..54
3.1.6. Enzyme linked Immunosorbent assay (ELISA)……............…………..55
3.1.7. Determination of enzymatic activity…………………………………...56
3.2. Component B: Production of plastic-like filler matrix
containing silicic acid (PMSA)……………………………………………………...57
3.2.1. Plastic-like filler matrix containing silicic acid (PMSA)……………....57
3.2.2. Microscopic analysis of the plastic-like filler matrix (PMSA)………...58
3.2.3. X-ray diffraction (XRD) of the plastic-like filler matrix (PMSA)……..58
3.3. Bifunctional 2-component implant…………………………………………….58
3.3.1. Physical properties of bifunctional 2-component implant……………..58
3.3.1.1. Hardness measurements……………………………………..58
3.3.1.2. Bifunctional 2-component implant behavior in
simulated body fluid (SBF)…………………………………………………59
3.3.1.3. Release/adhesion of silicatein to
bifunctional 2-component implant: Immunochemistry…………………59
3.3.1.4. Adhesion of silicatein to bifunctional 2-component
implant: Fourier Transform Infrared spectroscopy with
attenuated total reflection (FT-IR ATR) for
protein-surface interaction…………………………………………………60
3.3.1.5. Imaging properties on bifunctional
2-component implant……………………………………………….…60
3.3.2. Cell proliferation assay on bifunctional
2-component implant……………………………………………….60
3.4. Animal experiments…………………………………………………………….62
3.4.1. Surgery and implantation……………………………………………....62
3.4.2. Computed tomography (CT) and
micro-computed tomography (µ-CT) analysis……………………………….64
4. Results …………………………………………………………………………….65
4.1. Component A: Microencapsulation of silicatein and silica
precursor (sodium metasilicate) into poly(D,L-lactide)
biodegradable microspheres………………………………………………………..65
4.1.1. Production of polymeric spheres containing both
silicatein and silica……………………………………………………………65
4.1.2. Physical characterization of PLA microspheres……………………….664.1.3. Immunodetection of encapsulated silicatein…………………………..68
4.1.3.1. Dot-blot……………………………………………………...68
4.1.3.2. Thin cuts……………………………………………………..71
4.1.4. Detection of encapsulated silica source……………………………….75
4.1.5. Release of silicatein from polymeric microspheres…………………...76
4.1.6. Determination of enzymatic activity…………………………………..79
4.2. Component B: Production of plastic-like filler matrix containing
silicic acid (PMSA)………………………………………………………………….80
4.2.1. Preparation of a plastic-like filler matrix
containing silicic acid (PMSA)……………………………………………....81
4.3. Bifunctional 2-component implant……………………………………………83
4.3.1. Physical properties of bifunctional 2-component
implant: Hardness measurements……………………………………………83
4.3.2. Bifunctional 2-component implant in
simulated body fluid (SBF)………………………………………………….85
4.3.3. Release and adhesion of silicatein
onto the bifunctional 2-component implant………………………………….86
4.3.4. Imaging properties of bifunctional 2-component implant…………….90
4.3.5. Cytoxicity assay……………………………………………………….90
4.4. Animal experiments…………………………………………………………….92
4.4.1. Surgery and implantation..............................................................……..92
4.4.2. Computed tomography (CT) and
micro-computed tomography (µ-CT) analysis of
implant contrast within the bone......................................................................93
4.4.3. Analysis of bone regenerative capacity
after 9 weeks of implantation...........................................................................95
4.4.3.1. External Morphology……………………………………..…96
4.4.3.2. Computed tomography (CT)...................................................98
5. Discussion………………………………………………………………………..101
5.1. Microencapsulation of silicatein and respective
precursor (sodium metasilicate) into poly(D,L-lactide)
biodegradable microspheres………………………………………………...103
5.2. Release of silicatein from polymeric
microspheres (PLASSM)…………………………………………………...109
5.3. Component B: Production of plastic-like filler
matrix containing silicic acid (PMSA)……………………………………..113
5.4. Bifunctional 2-component implant…………………………………….117
5.4.1. Cytoxicity studies…………………………………………….123
5.5. Bifunctional 2-component implant: ex vivo and in vivo studies……….125
5.5.1. Ex vivo studies……………………………………………….125
5.5.2. In vivo preliminary studies…………………………………..1266. Conclusion……………………………………………………………………..128
7. Bibliography…………………………………………………………………...130
8. List of abbreviations…………………………………………………………..142Summary
1. Summary
Until today, autogenic bone grafts from various donor regions represent the gold standard
in the field of bone reconstruction, providing both osteoinductive and osteoconductive
characteristics. However, due to low availability and a disequilibrium between supply
and demand, the risk of disease transfer and morbidity, usually associated with
autogeneic bone grafts, the development of biomimic materials with structural and
chemical properties similar to those of natural bone have been extensively studied. So far,
only a few synthetic materials, so far, have met these criteria, displaying properties that
allow an optimal bone reconstitution. Biosilica is formed enzymatically under
physiological-relevant conditions (temperature and pH) via silicatein (silica protein), an
enzyme that was isolated from siliceous sponges, cloned, and prepared in a recombinant
way, retaining its catalytic activity. It is biocompatible, has some unique mechanical
characteristics, and comprises significant osteoinductive activity.
To explore the application of biosilica in the fields of regenerative medicine,
silicatein was encapsulated, together with its substrate sodium metasilicate, into
poly(D,L-lactide)/polyvinylpyrrolidone(PVP)-based microspheres, using w/o/w
methodology with solvent casting and termed Poly(D,L-lactide)-silicatein-silica-
containing-microspheres [PLASSM]. Both silicatein encapsulation efficiency (40%) and
catalytic activity retention upon polymer encapsulation were enhanced by addition of an
essential pre-emulsifying step using PVP. Furthermore, the metabolic stability, cytoxicity
as well as the kinetics of silicatein release from the PLASSM were studied under
biomimetic conditions, using simulated body fluid. As a solid support for PLASSM, a
polyvinylpyrrolidone/starch/Na HPO-based matrix (termed plastic-like filler matrix 2 4
8 Summary
containing silicic acid [PMSA]) was developed and its chemical and physical properties
determined. Moreover, due to the non-toxicity and bioinactivity of the PMSA, it is
suggested that PMSA acts as osteoconductive material.
Both components, PLASSM and PMSA, when added together, form a
bifunctional 2-component implant material, that is (i) non-toxic (biocompatible), (ii)
moldable, (iii) self-hardening at a controlled and clinically suitable