XXVIII cycle Doctoral School in Materials Science and Engineering Biomimetic and Bioinspired Biologically Active Materials Thi Duy Hanh Le Tutor Claudio Migliaresi, prof. Antonella Motta, prof. March 2016 BIOMIMETIC AND BIOINSPIRED BIOLOGICALLY ACTIVE MATERIALS THI DUY HANH LE E-mail: thiduyhanh.it Approved by: Ph. Claudio Migliaresi, Advisor Prof.
ssa Ilaria Cristofolini, Department of Industrial Engineering, Department of Industrial Engineering University of Trento, Italy. University of Trento, Italy. Antonella Motta, Co-advisor Prof. Paolo Antonio Netti, Department of Industrial Engineering, Dipartimento di Ingegneria Chimica, University of Trento, Italy.
dei Materiali, e della Produzione industriale. University of Naples Federico II, Italy. Maurizio Vedani, Dipartimento Meccanica Politecnico of Milano,Italy. University of Trento, Department of Industrial Engineering March 2016 University of Trento - Department of Industrial Engineering Doctoral Thesis Thi Duy Hanh Le - 2016 Published in Trento (Italy) – by University of Trento ISBN: - - - - - - - - - … To people who come into my life…by anyway.
Sometime, I forget to thank you… But I cannot express how much I appreciate… CONTENTS ABSTRACT. 1 Chapter 1: General Introduction.1 General introduction on diatoms .2 Organic component of diatom cell wall.1 Long chain polyamines .2 Cell wall proteins .3 The silica chemistry.4 The understanding of diatom cell wall formation .1 Silicic acid transport.2 Mechanism of diatom silica biogenesis .2 Morphogenesis of silica deposition valve (SDV) .5 Diatom cell cycle .6 Biomaterials, bio-inspired and biomimetic materials .1 Biomaterial definition and classification .2 Bioinspired and biomimetic materials from nature .3 Silk fibroin biopolymer .7 Biomineralization and biomaterials .2 Hard bone tissue and the role of silicon on bone maintenance .1 Hard skeletal tissue formation .2 The role of silicon on bone formation and maintenance .3 Bone healing and tissue engineering.8 Diatomite and strategies for biological applications.2 Diatomite and diatom strategies for biological applications 26 i 1.9 Objectives and outline. 27 Chapter 2: Processing and Characterization of Diatom Nanoparticles and Microparticles as Potential Source of Silicon for Bone Tissue Engineering .2 Materials and Methods .2 Raw diatomite purifications .1 Acid-purified raw diatomite powder .2 Acid-purified calcined diatomite powder .3 Diatom microparticles and nanoparticles from purified diatoms .4 Diatomite, purified diatomite and diatom particles characterization .5 Silicon release from diatom particles in DI water .3 Results and discussion .1 Purification of the raw diatomite powder .2 Characterization of raw diatomite and purified-diatomite .3 Diatomite nanoparticles preparation and morphology .4 Diatoms microparticles morphology.5 BET surface area of nanoparticles and microparticles .6 Silicon ion release from dissolution of diatom particles in DI water .7 Cytotoxicity of diatom particles. 49 Chapter 3: Enhancing Bioactive Properties of Silk Fibroin with Diatom Particles for Bone Tissue Engineering Applications .2 Materials and Methods .1 Cell proliferation and metabolic activity .2 Cells morphology and adhesion .3 Live and dead assay .5 Alkaline phosphatase quantification .3 Results and discussion .2 Evaluation of in vitro cells bioactivity in various scaffold formulations .1 Metabolic activity and proliferation .2 Cells viability and distribution .3 Cell morphology and adhesion .3 Bone formation markers .4 Alkaline phosphatase quantification.
73 Chapter 4: Osteoinductive Silk fibroin/ Diatom Particles Scaffold for Bone Tissue Regeneration .2 Materials and methods .3 In vitro experiment .3 Alkaline phosphatase quantification .3 Results and discussion .3 Alkaline phosphatase quantification. 85 iii List of figure Figure 1-1: a) Diversity of diatom morphology in different sources including fossil, freshwater and marine environment, b) Diatom skeleton structure and c) the patterned porous structure of the gird bands. 3 Figure 1-2: Chemical structures of some long-chain polyamines characteristic of three different diatom species. 5 Figure 1-3: A) the primary structure of peptide precursors sil1p presenting the signal peptide (italics), highly acidic peptide sequence attached regular peptides (108- 271) is shown silaffin sequences and B) The schematic chemical structure of Silaffin 1- A of C.fusiformis cell wall.
7 Figure 1-4: Schematic to show the range of functionalities possible for fundamental silica particles. 8 Figure 1-5: The proposal of different pathways of silicic acid uptake of transport in diatom intracellular, reproduced and adapted from [35]. 10 Figure 1-6: The drawing scheme of the mechanism of silicon oxide deposition in diatom cell wall by using phase separation model. Silicon oxide depicted at the position showed by the white grey colour.
13 Figure 1-7: The scheme of diatom cell cycle (asexual). The cross section of diatom was illustrated, reproduced from [4]. 14 Figure 1-8: The hierarchical organization of bone structure ranging from nano to marco length scale [89]. 21 Figure 2-1: Morphology (SEM micrographs) and mineral composition (X- ray diffraction) of raw diatomite (RD) and purified diatomite (AD and CAD) powders.
37 Figure 2-2: The whole XPS spectra of diatomite powders. 38 Figure 2-3: High energy resolution C1s, O1s and Si2p core lines obtained by XPS for raw diatomite powder (RD) and purified diatom powders (AD) and (CAD). 40 Figure 2-5: High magnification of SEM showed the porous structure of diatom cell wall encompassing patterned porous structure with different pores size. 41 Figure 2-6: TEM observation of diatom fragment structure and its chemical composition by EDS.
42 Figure 2-7: Size distribution of two different nanoparticles measured by dynamic light scattering (DLS) in DI water and PBS A) Size distribution of AD-NPs, B) Size distribution of CAD-NPs. 43 Figure 2-8: Morphology and elemental composition of nanoparticles obtained from acid-purified diatomite (AD-NPs) and acid-purified calcined diatomite (CAD-NPs). A) and B) TEM micrographs of AD-NPs and CAD-NPs, C) and D) elemental composition of AD-NPs and CAD-NPs determined by EDS. 44 Figure 2-9: SEM morphology of diatom microparticles.
A) Diatom microparticles produced from acid-purified raw diatomite (AD- MPs), B) Diatom microparticles from acid-purified calcined diatomite (CAD-MPs). 45 Figure 2-10: Nitrogen physisorption isotherms of (A) diatom microparticles AD-MPs and (B) nanoparticles AD-NPs prepared from acid-purified raw diatomite powders. 46 Figure 2-11: Silicon release profile from diatom nanoparticles and microparticles quantified by inductively couple plasma/optical emission spectroscopy (ICP/OES). 48 Figure 2-12: Percentage of cytotoxicity of the different groups of diatom particles on 3T3 cells determined with LDH assay performed by both elution and direct contact method.
49 Figure 3-1: Scanning electron microscopy (SEM) images presented three different scaffold architectures and high magnification of SEM to observe difference of their structures. 59 Figure 3-2: Diatom distribution of all groups scaffolds detected by using BSE of FE- SEM. Arrows presented diatom particles placed in scaffolds. 60 Figure 3-3: FTIR spectra of 3 different scaffolds including SF– silk fibroin, SF-(N+M)0.8 – composite comprising of 0.8% diatom particles mixed diatom nanoparticles (DNPs) and diatom v microparticles (DMPs) and SF-(N+M)3.2% of diatom particles mixture of DNPs and DMPs.
61 Figure 3-4: Compressive elastic moduli elastic modulus of composite scaffolds and b) the selected of stress-strain curve in the linear region of three different scaffolds. 62 Figure 3-5: Cell metabolic activity performed by Alamar Blue® at 3 two different concentration of cell seeded initially at a) 9.10 cells/ 2 3 2 mm and b) 4.10 cell/mm for all scaffold groups. Statistically significant difference compared with the control at the same time of culture was representative at * (p<0. 63 Figure 3-6: Cell proliferation quantified by PicoGreen Kit of two 3 3 different cell seeded initially at A)9.10 cells/ mm2 and b)4.10 2 cell/mm for all group scaffolds.
Statistically significant difference compared with the control at the same time of culture was representative at * (p<0. 64 Figure 3-7: Confocal scanning laser microscopy images of cell live/ dead stained with calcein AM/ PI after day 3 and 7 of culture 3 of two concentration of the cells initially seeded A) 9.5 10 cell/ mm in different scaffoldswith scale bar = 50µm. 66 Figure 3-8: SEM micrographs of cell morphology (after day 3) and attachment on different scaffolds after 7 day of culture of the high concentration of cell seeded. Red arrows is depicted the position where cell presented at day 3.
67 Figure 3-9: Confocal scanning laser microscopy images of samples stained with specific antibody for observation of the signal and organization of Osterix (red) after day 3, 7 and 14 of culture and DAPI for nuclei (blue) of all scaffolds (scale bar = 50 µm). 69 Figure 3-10: Confocal scanning laser microscopy images of samples stained with specific antibody for observation of the signal and organization of collagen type I (red) occurred after day 3, 7 and 14 of incubation and DAPI for nuclei (blue) of all scaffolds (scale bar = 50µm). 71 Figure 3-11: The effect of scaffold formulations on alkaline phosphatase (ALP) activity induced by MGG3 during 3, 7 and 14 days of culture. Significant difference was representative at * (p<0.005), compared with the control at the same time of culture.
72 vi Figure 4-1: Cell proliferation in expansion and differentiated medium up to 21 day of culture of two scaffold groups, pure silk fibroin (SF) and silk fibroin loading 3.2% of diatom particles mixed nanoparticles and microparticles. 78 Figure 4-2: Confocal scanning laser microscopy images of samples stained with specific antibody for observing fibronectin (green) synthesized after day 7, 14 and 21 of hMCSs incubation and DAPI for nuclei (blue) of two scaffold groups in two different medium (scale bar = 50µm). The arrows may show the region of bone lacunae. 79 Figure 4-3: Confocal scanning laser microscopy images of samples stained with specific antibody for observing collagen type I (red) synthesized after day 7, 14 and 21 of hMCSs incubation and DAPI for nuclei (blue) of two groups of scaffold in two different medium (scale bar = 50µm).
80 Figure 4-4: Quantification of alkaline phosphatase activity induced by hMSCs seeded into two different scaffolds; pure silk fibroin (SF) and silk fibroin loading 3.2% of diatom particles mixed nanoparticles and microparticles; up to 21 day in expansion and differentiated medium, respectively. 81 vii List of tables Table 1-1 Numerous silaffin’s maturation formed by precursor variants and different post- translational modification, adapted from [18]. 6 Table 1-2: Some examples of different biomaterial and their applications, modified [57]. 16 Table 2-1: Elemental composition of raw diatomite powder (RD) and purified diatomite powders (AD) and (CAD) as determined by X-ray photoelectron spectroscopy (XPS).
39 Table 2-2: Average size of diatom nanoparticles measured by dynamic light scattering (DLS) in DI water and in PBS. 43 Table 3-1: Composition of the silk fibroin/diatom particles scaffolds. 55 Table 3-2: Porosity of all scaffold groups was determined by the hexane replacement. 62 viii ABSTRACT Tissue engineering is an interdisciplinary field aimed to design and engineer an efficient system for tissue and organ regeneration, for instance, for bone healing, based on the combined use of scaffolds, cells, bioactive or signalling molecules.
An optimal tissue engineering procedure requires materials and scaffolds fulfilling several requirements, one of those being the ability to trigger and control the crosstalk with the biological environment both in vitro and in vivo, and to induce and control the extracellular matrix production and assembling. Diatomite is one of the most abundant natural sources of hydrated amorphous silica resulting from the accumulation of diatom skeletons. Diatoms possess particular features in structure, morphology as well as composition. Interestingly, it has been recognized that the formation process of diatom skeleton is possibly related to that of human bone.
In this study, we wanted to utilize diatoms as silicon donor additives in scaffolds for bone tissue engineering, having been demonstrated the important role of silicon in bone formation. In this first part of the project, we used several methods to eliminate impurities in the raw diatomite. Diatom microparticles (DMPs) and nanoparticles (DNPs) were successfully produced by fragmentation of purified diatoms under alkaline condition. Our result showed that both DMPs and DNPs were able to release silicon, as detected in- vitro by inductively coupled plasma optical emission spectrometry (ICP/OES).
In addition, diatom microparticles and nanoparticles - derived from diatom skeletons - showed minimal or non-cytotoxic effects in-vitro as determined by lactate dehydrogenase assays on cell cultures. These findings suggest that diatom particles derived from diatom skeleton as a silicon donor might have potential use for bone tissue engineering.