Luận văn thạc sĩ synthesis characterization and biological activity of nano sio2 material

TABLE OF CONTENTS CHAPTER 1 OVERVIEW OF RESEARC 1.1 Overview of bone tissuc forming techniques, 1.1.1 Structure and role of bones 1.1.2 Treatment of bone damage 1.1.3 Bone Tissuz Forming

HANOI UNIVERSITY OF SCIENCE & TECHNOLOGY HUST

Instructor: PGS TS, Nguyén Kim Nga

Instructor’s signature

School: Chemical Engineering

HANOL 10/2021

TIANOI UNIVERSITY OF SCIENCE & TECIINOLOGY IUST

SOCIALIST REPUBLIC OF VIETXAM

Independence — Freedom - Happiness

GRADUATION APPROVAL

Full name of thesis author: Yudy Paola Moreno Ganzalez

Research Title: Synthesis

, Characterization and Bioactivity of SiOz nanoparticles

Specialization: Inorganic Chemistry

Student Identification: CA19027

Author, Scientific Instructor and Dissertation Tudging Commuillee accept the

author who has corrected and supplemented the thesis seeerding to the minutes of

the meeting of the board of directors on the day sees With the

following contents:

Dey month year

CHAIRMAN OF THE ASSOCIATION

SUBJECT TO THESIS

Research Title: Synthesis, Characterization and Bioactivity of SiOz nanoparticles School: Chemistry

Specialization: Inorganic Chemistry

Instructor: PGS TS Nguyễn Kin Nga

Instructor

Sign and full name

Acknpwledgement

‘Lhe completion of this study could not have been possible without the expertise of Assoc Prof Dr Nguyễn Kim Nga, my thesis supervisor Thank you for pointing me towards the corrcel path and supporting with the guides for my work

1 want to express my deepest thanks to Hanai University of Science and ‘Tecnhology, for

affording me the opportunity to advance my studies and have reached one more step up

in my carrer devclopmenl, My gratitude for having the chance to moot lecturers and teachers who with their careful guidance were valuable source of knowledge for my study both theoretically and practically To my colleagues, for all the patience, quidance and understading

Finally, and most importanlty, to my caring, loving and supportive family -Claudia, Laura and Ruud-, my heartfelt thanks, You were always avaliable whenever I need a helping hand and a word of encouragement Without you none of this would be possible

Student

Yudy Paola Morsna Gonvatcy

Project Summary

In this work, the successful synthesis of SiOz nanoparticles by the hydrothermal technique with addition of optital conditions of cetyllrimethylammonium bromide (CTAB) is slucidaicd and defined The determination in the composition and characterization of the material is supported with 4 specific analysis methods (FE-SEM,

XRD, FI-IR, EDX) Additionally, the biomineralization capability of the SiO: nanorods

is confirmed through in viiro tests in simulated body fluid The main objective of this roscarch is achicved by showing that highly bioactive SiO: nanoparticles with suilable rod-like shapes can be easily prepared by the hydrothermal method, highlighting the roll

of silica Oxide as a potential nanomaterial for bone regeneration

Student

Yudy Paola Moreno Gonzalez

TABLE OF CONTENTS

CHAPTER 1 OVERVIEW OF RESEARC

1.1 Overview of bone tissuc forming techniques,

1.1.1 Structure and role of bones

1.1.2 Treatment of bone damage

1.1.3 Bone Tissuz Forming Techniques

1.2 Materials used in Bone Tissue Enginecring +

1.2.1 organic Biomaterials

1.2.2 Biodegradable polymers

1.2.3 Composite materials (1.2.3)

1.3 Overview of Silicon dioxide

1.3.1 Structure of Silica Oxide

1.3.2 Properties of SiOz nanoparticles

1.33 Applications of Silica Oxide

1.4 Methods to synthesis Silica Oxide

1.4.1 Sol-gel method

142 High temperature method -

1.43 Conventional chemical precipitation

1.4.4 Raverse microemulsion method

1.4.5 Hydrothermal method

CHAPTER 2 METHODOLOGY OR EXPERIMENTAL METHODS

2.1 Equipment and tools

2.2 Reagents and method

2.3.3 Infrared spectroscopy (FT-IR) -

2.3.4 Energy Dispersive X-Ray Specestopy EDN

2.3.5 Bioactivity of nano-SiO2 materials

CHAPTER 3 RESULTS AND DISCUSSIO:

3.4 Results of characterization of nano-SiO2 material:

3.1.1 E-SLM analysis result

3.1.2 XRD analysis result

3.1.3 FT-IR analysis result

3.1.4 EDX analysis result

3.1.5 Mechanism of SiO» particles formation

LIST OF SIGNS AND ABBREVIATIONS

BPMs: Bone Morphogenetic proteins

MIIC: Major Histocompatibility Complex

TCP: Tricalcium phosphate

BTE: Bone Tissne Engineering

CS: Chitosan

ALP: Alkaline phosphatase

MSCs: Mesenchymal Stem Cells

TCP: Tricalcium Phosphate

ECM: Extraczfldlar Malrix

SBF: Simulated Body Fluid

HAp: Hydroxyapatite

'TEOS: 'Tetraethyl orthosilieate

FE-SEM: Fiel Emission Scanning Electron Microscope

XRD: X-ray diffiuction

EDX: Energy Dispersive X-ray Spectroscopy

FT-IR: Fowier-Transform Infrared Spectroscopy

PDLLA: Poly(D, L-Lactic Acid)

CTAB: Cetyltrimethylammonium Bromide

LIST OF TABLES

Table 3.) Particle sive and CTAB concentration uscd for the synthesis of SiOz naro-

‘Table 3.2 Characteristic oscillations and corresponding wavermmbers in nano-S101, 44

Table 3.3 Results of pI measurements of Si02/PDLLA membrane mineralization culture

Figure 1.4 Image of 31 Chitosan/Ilydroxyapatite (Cs/Ilap) scaffold Image adopted fiom

Figure 1.9 Hydroxyl carbonate apatite (HCA) growth on a GCs surface after immersion

Figure 1.10 Representation of two tetrahedral (4-sided pyramid) shape connected at the

Figue 1.1] Representation of amorphous SiO> and different polymorphs crystalline

Figure 1.12 Diagram of the phase transition paths between silica polymorphs conrad

by tomperature

Figure 1.13 Process fo the production of pyrogenic SAS from precursors (SiCH), Thạc

igure 1.14 Extemal and intemal surfaces with silanols fonned reversibily by Trảng and condensation of sitoxancs, Image adaplad from Ref [50]

Figure 1.15 Types of silanol groups and siloxane bridges on the surface of maghoe silica with internal OH groups

Figure 1.16 Schematic ilustration of the

formation on the surface of silica-based malarials Image arlopted from Ref [45] 27 Figure 1.17 Flow chart of sol-gel proccss to synthesize silica nanoparticles Image

Figure 1.18 Mechanism of particle growth and structural finmework of nanosilica

Figure 1.19 High-lemporalure lame pyrolysis of fied silica forms weakly soluble, salid spherical nanoparticles, Image adopted from Ref [50] - „3i Figure 1.20 Schematic representation of the inicroemilsion method for synthesis of silica

Figure 1.21 General purpose autoclave popularly used for hydrothermal synthesis 34 Figuc 1.22 Representation of SiO» nanoparticles formation under hydrothermal

Figure 2.3 Pxpsrimental procedure for biicaclivity of nano-SiO2 particles 40

Tigurc 3.LFE-SEM images of sample M2 (0.02M CTAB) al differcnt magnifications a) Magnification 100k: b) Magnification 50K scscsestmenieneinentninieinreneneneind

Figure 3.2 X-Ray diffiaction pattem of SiO2 samyflc (M2 - 0.02M CTAB) 43 Figure 3.3 FT-IR spectrum of SiO2 sample (M2 —0.02M CTAB) sample

+igure 3.4 Representative EDX spectrum of SiO powder sample

Higure 3.5 Possible mechanism of SiO, nanoparticle imaging process: a) In presence of CTAR, b) Growth of ri pjected to hydrothermal conditions

Figure 3.6 SEM image of s: ple M M2 - SiOYPDLLA A ae 02M CT TAR) afier culture in SRF

INTRODUCTION

Bone is a dynamic tissue that experiences renewal throughout life in a process whereby osieoclasis Tasorh worn bone and osteoblasis synthesize new bone Being one of the most important systems in the human body and in which infinitics of responsibilitics fall, the care and optimization of fonctions require some attention Inequalities in bone tumover lead to bone loss and development of osteoporosis and eventually fracture; a debilitating condition that today is more present in society and that represents a high morbidity rate For some considerable time, a great deal of tescarch offorl has boon dedicated lo enhancing the bioactivity of the bone-implanted materials and ensuzing, the continuous progress of techniques that can adapt to normal bone fimctionality and that in tum represent a decrease in the high costs and exhaustive searches for donors and human transplants Bioactive biomatzriats (such as bioglass and calcium sificate-based biomaterials) have drawn significant attention due to its excellent bioactivity and their ability to promote the development of bene tissue when in eantact with physiological Thúds

Silica oxide, most commonly found in nature and in various living organisms, is one of the materials that has shown its great skills and abilities in the creation of biomaterials that can easily be adapted and help in the prompt creation of new bone, !t is known that different effects on the reaction conditions such as the amount of KOS, pl values and reaction time have a different influcnce on the synthesis of particles that rescmble those found in bone, There are ditierent methods for preparation of amorphous and crystalline silicate material in the form of powder (ditferent chemical methods) or bulk materials (as glass fusion) All these methods have some advantages and disadvantages as energy consuming and their higher cost precursors, Hydrothermal method is one teclmique thal has been placed in the sights of many researchers calling their attention to the synthesis

of nanomaterials overall It permits preparation of materials having high parity and different shapes However, the studies that have been reported on the creation of SiO nanopartiotss using this technique aso successful in their results, Inl, unfortunately the lack of information to support such studics affzcts the litcraturc ercating the nccd for in- depth exploration in this field,

Moreover, silica oxide materials have attracted significant attention due to their structural characteristics including their uniform pore size distribution, high spocific surface arca, high pore volume and tunable pore size, encompassing the main characteristics required for application as bioactive materials The in vitre bioactivity (apatitz formation on the material surface) of 2 biomaterial has been shown to depend not only on its chsmical composition but also on its susfave morphology and microstructure For instance, silica materials with high surface area have been reporled to be cllective in inducing apatils fonnation, with the micropores on the material surtice acting as apatite nucleation sites,

shortening the time to reach the super saturation required for apatite precipitation proving

that amorphous structure of silica contributes in improving the biodegradability since ili more reactive, Sitica-hasct! materials are promising candidate bioactive matcriats thal have becn investigated for bone repair

The present sludy is porformed with the aim of sytihetizing SiO; wanoparticles with adcquate characteristics and controlled sizes, The hydrothermal method was applied for particle synthesis, using an optimal concentration af CYAB and hydrothermal duration based on previous studies, ta produce rod-like SiO: nanoparticles with narrow size distribution for development of bone scaffold materials due to particles with sive sinnilar

lo thase of bone mincrals arc orucial for biamnineralization, colt adhosion, proliferation, and alkaline phosphate production, contibuting and resulting in rapid repair of hard tissue injury More importantly, different aspects of the siHca-based biomaterials such as composition, synthesis techniques, and surface characterization are discussed in detail Morcover, T fave allampled to highlight tho tole that silica has within ths banc mineralization process and present a possible model in the formation of silica nanorods and inspire broader interests of this material across various disciplines

CHAPTER 1 OVERVIEW OF RESEARCH

1.1 Overview of bone tissuc forming techniques

1.1.1 Structure and role of bones

Bone has been calcd the ultimate biomaterial, with a structure optimized so that it is strong but relatively lightweight, capable of adapting to diverse functional demands, and

a unique capacity of self-regenerating or self-remodeling to a certain extent throughout the lifetime Bone parferms soveral integral functions in the maiutenanee of body systems, such as protection of vital organs fiom possible harmful outside effects, providing support and silz of muscle atlachmen! for locomotion, goncration of red and white blood cells for immmnoprotection, axygenation of other tissues [2] and acting as a storshouse for minerals where 99% of the calcium, 85% of the phosphate and 50% of the magnesium are stored in bones [3]

The bones of the adult skeleton are complex composites constituted by an organic phase and a minzral phase [5] The organic malrix contains collagonons proteins (90%) mainly type 1 collagen, noncollagsnous proteins including osteocalcin, osteonectin, and fibronectin, bone morphogenetic proteins (BMPs), and growth factors [6] Tho rimcrat phase, also known as the inorganic phase, consists predominantly of phosphate and calcium ions comprising 50%-60% of the bone, however, significant amounts of sodium, polassinm, magnesium, fluorite, vane [5][6] and on the ullra-tirace level, Sificon [7], have also besn found in this phase Calcium and phosphate ions :ucleatz to originate the Tiydroxyapatite (HA) in the bone malrix, represented by the chemical formula Caro(PO4)s(OH) The strength, resistance, and flexibility of bone tissue are the result of the combination of collagen Gbers as a structural framework, noncollagcnous protcins, and crystalized Hydroxyapatite [9|

In order to enable bone ta perform fimelions previously described, specific hierarchicat levels have ‘bosn widely shuficd [4] Thess tevels involve from macroscopic via microscopic to molccular structurcs (Error! Reference source not found.) The macroscale displays the typical constitution of bane, Two main supporting structures of bone tissne are: cortical or compact bone (80%) forms the hard and outer layer of bone, and trabecular ot cancellous bone (20%) the sponge-looking center [2| The pores in trabecular bone are filled with bone marrow, a site where stem cells are contained ‘the stem cells mature into the red blood cells that carry oxygen through your body, white calls thal fight infections, and (he platelets thal help with blood ctolling, a process know

as Hemalopoicsis Those proportions may vary accordingly to the lype and location of the bones in the skeleton The basic wnit of cortical or compact bone is the ostzon It is composed and surrounded by uniform, and conecninic rings of matrix knewn as tamucllic Cells called osteocytes are disttibuted within the concentric lamellae Osteocytes form a complex network that 1s thought to be important in maintaining the viability and structural integrity of bone [3] ‘he Haversian canal is located af the center of the osteon,

which contains blood vessels and nerves [1] In contrast, trabecular bones consist of a

network of plates called Trabeculae dominating the elastic properties of the bone [3]

Following the hierarchical structure, at the nanostructural level, the bone is comprised

predominantly of biological plate-like Hydroxyapatite crystals with particular orientation

to the c-axis, where in combination with type-1 collagen form the collagen fibril [5]

Figure 1.1 Hierarchical structure of bone from Nano to Macro scales Image adopted from Ref [1]

But the aforementioned activities and functions could not be carried out in the same way

without the existence of cellular components at the biological level that would allow bone

as a dynamic organ to be reabsorbed and maintained continuously This process is known

as remodeling, a firmly regulated combination of bone formation and resorption [2] It

follows a set sequence of phases from the activation of different cell types to the

production of new bone (Figure 1.2) A layer of flat-lining cells (bone housekeepers)

located on a thin collagen membrane on the bone surface set in motion a cycle of

remodeling In the first place, the Osteoclasts, multi-nucleated cells, are formed and

differentiated when the recruitment of osteoclast precursors has been completed Their

cellular membranes consist of numerous folds that face the bone surface allowing the

rapid segregation of organic collagen and the inorganic calcium and phosphorous mediate

by the protease cathepsin K (CaK) The osteoblasts break down the damaged bone

creating a sunken place or hollow on the surface of bone known as Lacunae or pit

Minerals are released into the bloodstream whereas un-mineralized bone (osteoid) is

protected against osteoclastic resorption [3] [11]

Subsequently, macrophages and lining cells prepare the surface of the lacunae by the

removal of the debris left after resorption Meanwhile, bone builders cells or Osteoblasts,

derived cells from mesenchymal stem cells, are attracted to the clean lacunae for the

synthesis of new bone matrix, secreting mainly Type I collagen but also osteocalcin and

BMPs On the other hand, Osteoblasts play an important role in the mineralization process of bone: by the secretion of enzymes that release calcium ions from

proteoglycans and together with the phosphates ions released after the breakdown of the

phosphate-containing compounds by alkaline phosphatase, results in the formation of

hydroxyapatite crystals [6]

Figure 1.2 Stages of bone remodeling Image adopted from Ref [3]

At the final phase, as osteoblasts form new bone tissue, many of them become embedded within the newly formed matrix and differentiate into osteocytes As osteocytes are located within the lacunae and surrounded by a mineralized bone matrix wherein they show a dendritic morphology [3][15], forming an interconnected network with lining

cells on the surface of the bone and the marrow By that, osteocytes seem to act as

mechanosensors that help in the adaptation process of bone to daily mechanical pressures

I6l[12]

Dus to various faclors such as genctic, enviromental, and mecharical, the bone can suffer altcrations that may dirceily affect its function, mass, and strength that lcad to malformations, injuries, and diseases, However, because of its high regenerative capacity, the majority of frachwes or cracks can heal well without the exigency of a major and invasive intervention Conversely, in cases of bone defects and malformations, bone replacment is indispensable [7]

1.1.2 Treatment of bone damage

Bones cm be replaced within the hunian body by grafls or substilutes thal assinnilate its structure, eamposition, cnsurc continuity in iis operation and facilitate the urion of the fractures as well as the reconstruction of diseased tissue,

‘As the bone performs a fracture healing process where various cells, factors, and biochemical mediators are involved, the ideal grafts and substitutes must ensure compliance of three important biological propertios The first property slates tha ability to support the attachment of osteoblast and osteogenic cells and allow the migration and ingrowth of these cells within the architecture of the graft This process is known as Osteoconduction [18], ‘The stimulation of undifferentiated and pluripotent cells to develop into the bonc-forming ccll Linage arc attributed to the Oxteoindaction process, by which osteogenesis is prompted |20| The third property is Ostewintegration, the stable anchorage ability of an implant when there is direct bone-to-implant contact to form new bone tissue around it [19]

1.1.3 Boue Tissue Forming Techniques

Diseased or damaged bone tissue curently places an enotmous demand on bene substiinles for trausplanlation, being the second most transplanted tissue anual including not only costs for hospital admissions for traumatic fractures but from post- surgical teatment to the limited number of donors to cope with clinical demand ]23| Since many of the methods used to ensure bone regeneration and maintain its properties and functions within the hiram body intact, complications can oocur and the succass of the transplant would be highly affected Disadvantagcs and limitations that arc linked to these procedures, such as donor site morbidity, incompatibility, and high risk of carrying, infectious diseases caused by transplants, have interested scientists nowadays To overcome those limitations, alternative approaches lo providing effective and reliable bone grafts are being actively pursucd

‘Tissue engineering is a rapidly developing interdisciplinary field that seeks to repair, restore, Teplace, or crhanec biotogival fissie and organs Bone tissue engineering (BTR) strategics arc showing promisc to replace lost or damaged bone tissuc, over morc traditional bone grafting methods, such as autografts or allografts [28] Within those strategies, the scaffold fabrication techniques are very important in dictating the final structural, mechanical propertias, and biologicel responsc of the implanted biematcriats Seaffolds, as a key purl of regcnerative medicine, ean be designed and developed to

recruit and guide cells in the body to heal tissues that would no heal by any other means

In addition, biomolecules such as proteins or growth factors can be integrated into

scaffolds to control osteogenesis, bone tissue regeneration, and extracellular matrix formation [26] On the other hand, scaffolds have played an important role in drug delivery systems They can be injected into the body and they can hunt out diverse cells,

for instance, cancer cells (Error! Reference source not found.) In conclusion, tissue

engineering uses a combination of cells, signaling molecules, and biocompatible

materials to design bioactive scaffolds and achieve the successful restore of a variety of

‘The 3D scaffold should preserve mechanical properties close to that of the adjacent tissue

(27).

By facilitating and contnbuting to the function of the tissue to be replaced, the scaffold

performs the constructive remodeling and favorable clinical outcomes avoiding the activation of the innate immune system, including dendritic cells, neutrophils, and

macrophages, among others

B Biocompatibility

Describe its ability to perform with the appropriate host response in the tissue The scaffold should allow the bone cells to adhere, proliferate, and form an extracellular

matrix on its surface and pore In addition, it needs to support biological response such as

anormal cellular activity including molecular signaling systems without any toxic effects

as well as induce new bone formation by recruiting progenitor cells through biomolecular

signaling Further goals suggest the capacity of forming blood vessels in or around the

implant [26]

C Mechanical properties

Success in selecting an ideal scaffold is driven by the proximity of the mechanical properties of the host bone The large variation in mechanical properties within which are compressive strength, resilience, and stiffness makes it difficult the design an ideal scaffold Nevertheless, thermoplastic polymers with a higher stiffness range and porous bioceramics are often selected for BTE, mimicking the load-bearing nature required for bone tissue regeneration [28].

D Porosity and pore size

Tntcreormected porosity has heen corsiderzd an indispensable property whan it comes lo scaffold design To guarantee the successful diffusion of essential nutrients and oxygen for cell survivability, the pore size should be at least 100 mm in diameter Yet, it has been

determined that sizes in the range of 200-350 mm contribute to optimum bone tissue n-

growth [26]

Suficient porosity and interconnections belwcen the pores as znust have parameters, offer the right environment to promote vasculatization, circulation of nutrients as well as oxygen, and waste disposal, At the same time, the distribution of the pores in the scaffold structure provides a high capacity for cell penetration to result, iw their respecti differentiation, migralion, and inlegration On the other hand, the rate of scaffold degradation has bccn sclated to its porosity, The scaffold degradation rate needs to well match with the maturation and regeneration of new tissue [30]

E Biodegradability

Being another crucial requirement, an ideal scaffold should not only meet similar mechanical properties that of the host tissne, but also be able to degrade with time in vivo, rather at a controlled resorption rate to guarantee the necessary mechanical support until the regeneration process is completed, The higher the degradation rates, the greater the probability of early weakening in the mechanical strength of the scaffold ‘To prevent this, coating biomaterials seem to be necessary to improve their degradation resistance

[26]

1.2 Materials used in Bone Tissue Engineering

The usc of materials for a spcsific approach +0 medical enhancement must mimic properties and characteristics that are specific to the host tissue to be replaced

Nowadays, materials that demonstrate their tissue regenerative capacity, easy degradation

simultaneously with the growth of native tissue while invoking a non-immunogenic responsc, adcqualc cnviromment as well as ticchanical forces lo support the ecll attachment and proliferation, are highly qualified to be implemented in BTE The closer

to the mechanical properties of native bons tissue the better and successful the material is considered [31]

In this scetion, the most promising materials used for bonc tissue scaffold fabrication are described as follows:

used metals nowadays concerning orthopedic and dental implants given their non-toxicity

and corrosion resistance Unfortunately, these materials do not tend to degrade because of

their inability to incorporate biomolecules into their design after being implanted mm the

human body, so it is crucially important to remove the metal implant, especially in the casé of bones subjected to natural growth and development as in children [33]

Titanium scaffolds with an average pore size of 800 jm allow strong osteoblast cell

attachment and proliferation However, because of its poor biodegradation rate, surface

modification techniques are implemented to improve the functionality of Titanium scaffolds [26]

that they can reduce significantly the possibility of deformations or fractures throughout

the implantation process High compressive strength, biodegradation by corrosion, and

biocompatibility are some of the main advantages however in high-temperature rates for

processing have been reported as a drawback of these materials [34]

Figure 1.6 Porous Mg scaffold fabricated by laser- assisted mechanical perforation method

To overcome the big challenges encountered in BTE when implementing metals,

scientists due to their biocompatibility, corrosion resistance, and biological activity have considered innovative materials the object of deep analysis and study Unlike metals, bioceramics scaffolds in BTE gradually degrade by solution-driven and cell-mediated

processes after implantation in the body and finally replaced by new bone tissue

In general, bioceramics are strong, bioactive, and bioresorbable materials, designed to stimulate a specific biological reaction on their surface, resulting in the connection between living tissue and the material through the formation of biochemical bonds [33]

Its biochemical and mechanical properties play an important role in the reduced or almost

null effect on the immune system after implantation

‘These materials are categorized as (crystalline) ceramics, (amorphous) glasses or (partly crystalline) glass ceramics [34]

At moment, calcium phosphate (CaPs) based bioceramics and bioactive glasses have

been used in BTE due to their resemblance to the hierarchical nanostructure of native

bone Some limitations in mechanical strength for load-bearing applications can be

controlled when polymers are added Moreover, this type of ceramics can easily

incorporate a wide range of bioactive ions (including Ca“*, Mg”*, Si‘*, Li* and Ag*), mimics the inorganic phase of bone knows as carbonated hydroxyapatite, and signaling molecules and cells [28] Among different CaPs, Hydroxyapatite Cayo(POs)OH) and

tricalcium phosphate Cas(POs)2, are compounds known for their excessive bioresorption

capacity [26]

Hydroxyapatite Cais(PO;)OH):, with abbreviation HAp, is characterized by high mechanical stiffness, very low elasticity, brittleness, and thermodynamic stability Either alone or filled in polymeric composite, HAp has been widely studied in BTE field due its chemical similarity to native bone, strong affinity for host hard tissues, facilitates good differentiation and proliferation of osteoblasts, and encourages alkaline phosphatase (ALP) in mesenchymal stem cells (MSCs) [33], Nowadays, advanced technology and continuous studies of these scaffolds have focused on the creation of nano-sized crystals

of HAp with less than 10 jum in size that are capable of breaking down over time but ina

slow rate [35] On the other hand, their brittleness can cause difficulties with mechanical loading but also slow biogradation during the repair of bone defects [33]

Figure 1.7 FE-SEM image of HAp sample Image adopted from Ref [35]

Tricalcium phosphate (TCP), Cas(PO.)>, with two crystalline forms, a-TCP, and B-TCP, has recently received considerable attention as a bone graft substitute because of its biocompatibility, supports in vivo osteogenic differentiation of MSCs, and low setting temperature B-TCP is favored by researchers because it differs from HAp by its accelerated and high degradation rate which is beneficial to the growth of new bone around the implanted scaffold [29] Nevertheless, TCP-based scaffolds have lower mechanical strength than HAp To overcome this, injectable 3D scaffolds of beta-TCP (- TCP) in combination with other materials such as collagen, sphingosine 1-phosphate (S1P) and metal ions, can enhance its biomechanical properties and display osteogenic

GC is its ability for developing a biologically active amorphous hydroxyl carbonate

apatite (HCA) layers throughout its surface that bond to bone upon implantation The

HCA phase that forms on bioactive GCs is chemically and structurally equivalent to the mineral phase of bone [28]

Figure 1.9 Hydhoxyl carbonate apatite (HAC) growth on a GCs surface after immersion in

simulated body fluid (SBF) Furthermore, the incorporation and release of silica ions stimulate osteogenesis and neovascularization/angiogenesis, and enzymatic activity [28] At the same time, these

ions are necessary for activating gene-transduction pathways When in contact with body fluids, these materials undergo degradation over time, being replaced by new and healthy

bone tissue That explains why bioactive glasses as the first artificial materials with a

demonstrated mentioned abilities, has encouraged much interest to scientists and

clinicians [36]

Another factor to consider is the necessity to overcome some unsuitable characteristics of

this type of inorganic material such as brittleness and low strength that difficult the use of

it for load-bearing applications [33] For this reason, bioinorganic materials such as silica

(SiO›) and calcium carbonate (CaCOs) have been researched as an alternative in BTE

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1.2.2 Biodegradable polymers

Polymers are organic materials constituted of long chains of atoms joined by covalent bonds that have been extensively used in industrial applications such as farming, food

sectors, pharmaceutical, and biomedical fields specifically in bone-tissue engineering

For the latter, these materials renovate the traumatized tissue due to their unique

properties such as biocompatibility, reproducible mechanical, physical properties, workability, and low price [27]

Polymers involved in tissue engineering are either of synthetic or natural origin and miscellaneous structures can be obtained from their processing Flexibility in processing, the potential to tailor the chemistry of polymers, and the ability of producing monomers after degradation which are readily removed by the natural physiological pathway are

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added advantages However, dogradation of cortain polymers forms a local acidic cnvironment that can also have unfavorable tissue responses [26]

The principal motivation in using biodegradable synthetic polymers is related to their very high srongth and stiffness [28] Compared with nalural polymers, synthetic polymers offer more possibilitics for chcmical modifications and molccular altcrations in its structure that can be prepared by mamally controlling the design and synthesis parameters [34] In addition, these matenals have been proven to have @ controlled degradation rate as the resuili of technological manipulation of their respective molecular weight and crystallinity One concer of this type of polymers is the abruplly release af acidic degradation products that although they arc dischargcd through the natural metabolic “pathway, they could change the local pH value and trigger a strong inflammatory response at the local transplantation site thus, which in tum accelerates the implant dogradalion tals causing promaturs failure of seaffolds [28] Al the same time, synthetic polymers tend lo be oslcoconductive rather than osteoinductive, as they lack cell-recognition signals that can direct cell behavior [34] In recent years, synthetic polymers that have been studied in BI'E include: polylactic acid (PLA), polyglycolic acid (PGA), poly s-caprolaclone (PCI), poly (ethylene glycol) (PEG) and poly (lactic-co- glycolide) (PLGA) capalymers,

Alternatively, natural polymers are particularly advantageous in comparison to synthetic polymers, owing their similarity with the native FCM, biccompatibitity, and biodegradability [28] Natural polymer scaffolds usually demonstmic a lack of imme response and better cell interactions [27] Biopalymers can be broadly catcgorized as proteins (¢.g., collagens, and silk fibroin) and polysaccharides-based (¢.g., chitosan, and ILA), Proteins are composed of amino acid sequences that are typically associated with cdllular attachment via intogrin-binding domains, a faature nol Cound in polysaccharides Therefore, chemical surfacc modifications in polysaccharidic scaffolds by mixing with osteoconductive materials or by the incorporation of integrin-binding sequences are primarily necessary to improve osteoconductivity and cell adhesion respectively Regardless of whether they are proteins or polysaccharides, the control of their bioactivity as well as the madidation of porosily, charge and mechanical resistance ean be controlled through the addition of chemicals, proteins and cells ar by the introduction of finctional groups, polymerization conditions and concentration correspondingly [37]

The most commonty studied polymers of natural origin for BTE are collagen, alginate, chitosan, silk fibroin, hyaluronic acid, slastin, glycosamninoglycans (GAGs), poptides, and others

- Collagen

As a major constituent! of the FCM in various connective lissuss, callagens are the mosh plonfifid protein in the huraan body (making up the 25 to 35% of the whole-body protein content)(38], in which its structure contains amino-acid sequences (specifically, the adhesion ligand arginine-glycine-aspartic acid (RGD)} to which cells readily attach to the extracellular matrix [34] The collagen molecule architecturally consists of a triple helix of clongated fibrils, known as a collagen liclix, As a main component in bone, collagen fibrils serve as a template for mineralization Biocompatible, bioactive, and rich

in surface-binding sites for cells, collagen stinmlates cell adhesion, proliferation, and

14

differentiation, roprosenling therofare an idzal candidate for 31) scaffold design [37] Over more than 29 types of collagen arc presont in several tissues and have been extensively used actoss biomedical applications In tendon, bone, and skin, collagen type [ig the most prevalent beside the less frequent types II and V while collagen type Il is mostly found in cartilage [28]

- Chitosan

In bone tissue engineering, chitosan (CS) can be used alone or with other polymers or ceramics in the design of sponge-like struclures with variqus porms amangements aller advanced preparalion processes, such as 31D printing and nanotechmology Tl has the sarc lincar strachuc as a non-collagcn cxganic component of ECM, glycosaminoglycan (GAG), which has a widespread fimotions whitin the body Chitosan can be obtamed by deacetylation of chitin, which is an exoskeleton component of crastacsans By virtue of its biodogradability, mom-sliergemeoily, antibacterial and biocampalitility, and hydrophilic surfacc, CS might erihance cell adhesion, differentiation, migration and mineralization of matrices Conceming its application as a dental, bone or cartilage xmplant or as artificial skin, chitosan exihibts good flexibility, softness and intercomscled pores as a bensfitial characteristics however, given ils poor mechanical strength, ils very oflen mixcd with other natural polymers or biocsramies lo overeome difficulties [33]

Ba

of the materials by themselves Diverse surface modifications can be applied to approach the conizol of scaffold stiffness and structure, Additionally, the adoption of crosslinking strategy also has direct effect on the degradation behavior of the material, as same crosslinks are cleaved by enzymatic or hydrolytic mechartisms resulting in the simple and desirable construct degradation [28]

1.2.3 Composite materials (1.2.3)

As the nature of bone is largely composed of colfagen, inorganic mineral, cells and diverse factors and molecules, most of the difficulties and drawbacks faced in BYE are associated to the deficicncics for a single material to mimic the composition of natural bone, The development of composite materials has allowed researchers to come up with multiple solutions in the manufacture of structures that could be adapted to a greater

degree to the requirements that are nesded when considering replacement in BY

Composils materials include a polymer phase wilh toughness and compressive shrongiht and an inorganic phase with bioactivity, which improves the mechanical proparties and degradation rate An ideal ratio between inorganic and polymeric materials is critical to induce bone tissue For adjusting and keeping the porosity and mechanical strength of the composites, variations on ths lovels of precision using various techniques can and up in the formation of intreonncctivity between the fibers or by the integration of porogens A wide range of different combinations gives rise to composites with vary good performance for BTE, Frequently, bioceramics or bioglasses are added as a coating or filler to a polymer matrix [28] [31]

A cortain sludy indicated thal 3D scaffolds of HAp/chitosan-gelatin proscnt sirmilarities in its structure to human bone The presence of HAp improves mechanical properties, and together with natural polymers, ceramic material and cells exhibits the effect of biomineralizaiion after three weeks Chitosan-based composite biomaterials seem to show promising results nol only far being a very good option for cartilages and intervertebral dises, but also for gone therapy in arthopedies [33]

Calabrese et al also demonstrated the osteoinduotive potential of a type | collagen (30%) -HA (70%) scaffold, The scaffold in this instance was in addition of magnesium to create bioactive Mp-doped HA (MHA) nano-crystals Human MSCs isolated from adipose tissue were seeded onto the scaifold and cultured in vitro in the absence of specific

osteogenic inducing factors Quantitative PCR and immunchistochemistry at up to 8

weeks demonstraicd osteogenic differentiation of MSCs This sludy therefore showed that the scaffold materials alone could trigger osteogenic differentiation of MSCs, with extracellular matrix production, gene expression and mineralization analysis all demonstating the osteoinductive potential of the scaffold [31]

Several examples of successful bone and cartilage construcls with ctinical applications have beon developed with ccrami¢ and polymer composites perhaps having the greatest success as a result For future times, the continuing fabrication of composite scatfolds that include a combination of materials with desirable mechanical and cell-triendly properties will be preferable in RTE,

1.3 Overview of Silicon dioxide

New and innovative technological approaches holding greal potential for the fature of bone lissuc enginccring have demonstrated thal the addition of clstrents found in large portions in the carth to scaffolds contribute to the improvement and rapid rcgcncration of bone providing the mechanical support and promoting ostegenic differentiation on its surface Sinmifteanously, more specific fields such as gene therapy strategies and erowth

16

factor delivery have implemented materials that provide greater reliability and less

toxicity in the human body In the design of an ideal scaffold that are able to address the

aforementioned challenges, silicon dioxide has been associated with calcium in bone

metabolism resulting in a new and excellent strategy to develop bone scaffolds with

positive patient outcome

1.3.1 Structure of Silica Oxide

Silicon dioxide, SiO2, is commonly known with the name of silica and is a group IV

metal oxide Because of its interesting electronic, geophysical and chemical properties,

silica has attracted much attention in a variety of scientific disciplines The elemental

compositional unit of silica minerals is the silicon-oxygen tetrahedron (Si-O tetrahedron)

It consists of one silicon cation (Sỉ?) surrounded by 4 oxygen anions (O°) in a 4-sided

pyramid arrangement known as a tetrahedron (Figure 1.10), Each of the oxygen can form

two bonds, so they also bond to another silicon atom on the other side The Si-O-Si bond

can vary between 100 and 170 degrees and the O-Si-O bond is 109 degrees approximately [47] On the other hand, each silicate forms four bonds Chemically, the

silicon-oxygen tetrahedron has a net electrical charge of -4

(sio.)*

Since minerals are chemical compounds, which cannot have an electrical charge, this

charge is balanced by the addition of metals, which are positively charged Different

arrangements of metals and tetrahedrons within a silicate mineral’s crystal structure

create the major subclasses of silicate minerals The chemical bonds that connect the metals to the tetrahedral are very strong covalent bonds SiO; simplifies because each

oxygen atom is shared by two tretrahedra in silica making up a 1:2 ratio When absolutely pure, this creates one of the most abundant minerals in the earth’s crust, quartz

(Si02)[47]

Figure 1.10 Representation of two tetrahedral (4-sided pyramid) shape connected at

the corner Image adopted from Ref [37]

Differences in the orientation and position of the tetrahedral shape create the variances in symmetry and cell parameters that give rise to the various polymorphs, Polymorphs refer

to any of the crystalline forms of a polymorphic substance All silica forms are identical

in chemical composition, but have different atom arrangements SiO» can take two separate forms, crystalline (or c-silica) and amorphous silica (a-siliea or non-crystalline silica), When referring to c-Silica compounds, these are characterized by having structures with repeating patterns ofssilicon and oxygen, high melting point, hard and chemically inert Conversely, a-Silica chemical structures are more randomly linked and for the absence of long-range order

Although the basic crystalline structures are uncomplicated and under normal pressure, many different polymorhic conformations have been reported among them: quartz, tridymite and cristobalite Each has its own structural motif, a pattem of atomic level

symmetry and structure, that is unique At the same time, each of these owns a low

temperature form (a) and a high temperature form () The structures differ only in the connectivity of the basic tetrahedral units and hence show only minimal structural energy differences (Figure 1.11)[46]

a-quartz a- tridymite a- cristobalite Fused silica

Bequartz < 5 ftridymite 2 5 f-cristobalite 2 Silica melt

Slow coaling

me Figure 1.12 Diagram of the phase transition paths between silica polymorphs controlled by

temperature

18

At temperatures above 573°C, the quartz structure is hexagonal and is known as the stable form, B-quartz or high-quartz Below the transition temperature, B-quartz readily

converts to a-quartz (or low quariz), 30 all quartz relevant to human exposure is o-quartz,

a-quarts is the most widespread polymorph, and is found in large quantities in any rooks and sails worldwide By gradually inercasing the temperature, two of the most common forms of tridymite at standard pressure, a- and B-, can be observed The a-

tridymite is favored at temperatures above 870°C and it converts to B-cristobalite at

1470°C Tridymite can also be formed from pure f-quartz but only in presence of certain trace amounts of alkstianctals (Li, Na, K) The high tomporature phase of silica, stable between 1470 and 1710°C is known as [-cristobalite, which persists metastably down to

about 275°C and inverts to the low temperature phase o-cristobalite ‘he phase

transitions are all reversible, except for the transition directly from B-quartz to B- cristobalile at 1050°C wilhout the occurrence of the bridymits phase

Covering the broad classification of synthetic amorphous silica, two main groups are found ant explained, wel silica and thermal silica As the only subdivision of thermal, pyrogenic silica is produced in closed reactors by the hydrolysis af (alkyl) chiorosilancs (eg, SiCl.,) in an oxygen / hydrogen flame at temperatures between 1200°C and 1600°C

A precursor is subjected to 3 important processes, starting with nucleation, which then

follows condensation and finally coagulation of said precursors to generate proto- partictss of SiOz which combine to prittary particles Within diffrent conditions in the Teaction zone (hot and cool zone), SiO: aggregates particles, which later foun agglomerates, The result product has low water content

reas | prin | mm | Ae@vante | Am ọ

| tan | wo-emomn | 1am

Figure 1.13 Process to the production of pyrogenic SAS from precursors (SiCl4) Image

adopted from Ref [49]

Precipitated silica belongs to the wet silica It consists of randomly linked spherical

polymerized primary particles that can be produced from various raw materials It

characteristic properties are a result of the size and state of aggregation of the primary

particles and their surface chemistry Primary particles have a diameter of 5-100 nm with

an average pore size greater than 30nm The most relevant process in industry is to produce a gelatinous precipitate by the reaction of a neutral silicate (sodium silicate) with

a mineral acid (sulfuric acid) under acidic conditions The resulting precipitate is filtered, washed, dehydrated in the manufacturing process [49]

Silica gel is an amorphous form included in the category of wet silica Analogously to precipitated silica, silica gels are produced by addition of sodium silicate to sulfuric acid, process that is considered low cost, non-toxic species and easy to control It has been

reported the production of silica gels with generally more narrow microporous or

mesoporous structure with average pore diameters between 2 and 50 nm By controlling the washing, ageing, and drying conditions, the important physical parameters such as porosity, pore size, and surface area can be adjusted to produce a range of different silica gel types with well-defined particle size distributions

Another form encountered within the wet clasfication is colloidal silica This is stable

dispersions of particles in a liquid medium Its production is usually based on a multi-step process that begins with the partial neutralization of an alkali — silicate solution by

acidification, electrodialysis, or ion exchange, leading to the formation of silica nuclei,

typically in the size range of 1-5 nm The pH plays a very important role in the desired silica result When obtaining silica gel, this can be achieved by the reduction of the pH below 7 or by the addition of any salts into the mixture If however the pH is kept under basic conditions (pH 7-10), the subunits remain seaparetley and gradually grow to

colloidal silica (silica sol) With the addition of KOH, NaOH, NHs or HCl in amounts of

up to 10% by weight the resulting colloidal suspension is stabilized Spherical colloidal silica particles in suspension can also be obtained through the Stéber method by which uniform size and porosity is achieved by the hydrolysis of alkylsilicates and subsequent

condensation of silicic acid in an ethanolic solution with catalytic amounts of ammonia thus ensuring the controlled growth of the particles within the solution,

Silica powders van be oblained fort multiples silica proparalions, Most applications arc addresscd to the use of porous structurcs obtained by cvaporative drying of wet gcls (xerogel) powder or granules ‘These can be prepared quickly by crusting the gel and Hinsing it with water or organic solvents to remove residual reagents before drying in an oven Silica powders overall possess unique properties such as low densily, large surface arca and good dispersibilily, Thy arc used in the preparation of thermal insulation materials, and employed as catalyst supports, adsorbents, additives as well as thickeners

[50]

1.3.2 Propertics of SiOz nanopartickes

13.2.1 Physical properties of nano-8iO:

All forms of silica are odorless transparent to gray podwer ‘he solubility of the vations phases of siliea is very carnplox and depends upon sevzral factors Solubility inorcases with temperature and pH and is affected by the presence of trace metals, Particle size influences the rate of solubility Silica is rather poorly soluble inwater and organic solvents although solubility is higher for the amorphous than for the crystalline morphologics (amorphous silica is quite stable in organic media and its solubility in watcr strongly inercases by increasing the pH above 8) It is insoluble in most of the acids with the exception of hydrogen fluoride, In general, it has good abrasion resistance and it

is an electrical insulator and it count with the ability to refract and absorb light at high lenyperatrss The densily of silica varies depending on its form of exislence, for instance, the density of a-quarts is 2.648 g.om? white dense amorphous silica is 2.196 gon? [47] SiO2 has a melting point of 1600°C and a boiling point of 2230°C, The amorphous silica form can be obtained with lage swface area with pH where the surface is electrically neutral (Point of Zero Charge or PZC) of about 2.5-3 and therefore the surface will be negatively charged in aqueous solutions having a pH above the PAC [AQ] Silicu-based membranes prepared through sol-gel methods must have a tailored pore size and quite narrow pore size distribution, be thin enough to achieve high permeation fluxes, be stable

in the physical and chemical operating environment of the application and show lack of defects affecting the separation selectivity

13.2.2 Surface characterization of nano-SiOa

Silicon dioxide nanolrydrates (SiO2 xThO), hetler known as silica nanoparticles, are

charaoleri⁄cd by an inorganic uctwork of (ctrhedral SiOz unils bonded via siloxane

linkages (=Si-O-Si=) and bearing surface silanol groups (=Si-OH) Because of this, silica

nanoparticles are hygroscopic and easily agglomerate at room temperature [39] Given its

robust and high surface silanot concentration, silica displays open spaces interconnected

with cach other and structures that facilitate the introduction of fimetional groups, such as

aminos, mercaptos and carboxyls for the further binding of biomolecules [50] Studies

back in 1950 by Yaroslavsky proved for the first time the existence of hydroxyl groups

on the silica surface (porous glass) Since then, many researchers have focused their studies on the investigation of the silica surface to support and study a little more in depth

and in detail its composition and structure and in turn the role within the ion exchange

HM

ng

Figure 1.14 External and internal surfaces with silanols formed reversibly by hydrolysis and

condensations of siloxanes Image adopted from Ref [50]

Condensation polymerization and rehydroxylation are two main processes that have been involved in the formation of silanol groups on the silica surface During the condensation

polymerization of Si(OH)s, silanol groups are formed as silica synthesis occurs A polymeric form is obtained thanks to the supersaturation of the acid, to result in spherical

shape colloidal particles composed of silanol groups on its surface Upon drying, the hydrogel is transformed to xerogel as the final product, which retains the majority if the

silanol groups on its surface On the second process, as a result of rehydroxylation of

dehydroxylated the surface OH groups can be formed when it is treated with water or aqueous solutions The surface silicon atoms tend to have a complete tetrahedral

configuration

The silica surface is characterized for the configuration of three types of silanol (Figure 1.15) where the Q” terminology in NMR spectroscopy indicates the number of briding

‘bonds (-O-Si) tied to the central atom Jsolated silanols or free silanols expresses surface

silicon atom with three bonds into the bulk structure and one more bond attached to a

single OH group (£SiOH) Geminal silanols or silanediols consist of two hydroxyl groups

that are attached to one silicon atom (=Si(OH):) The third type of silanol is vicinal or

bridged where two-isolated silanol groups bound through the hydrogen bond to different

silicon atoms Additionally, siloxanes groups or =Si-O-Si= bridges with oxygen also cover

the SiO› surface [39]

Figure 1.15 Types of silanol groups and siloxane bridges on the surface of

amorphous silica with internal OH groups

Many of the surface propertios in amorphopus sifica arc altributed m tmmy cases to the prescnee of sillancl groups The surface is considered hydrophilic when a sufficient consentration of these groups are found Because of this, the high amount of silanol (Si- OU) groups on their surface atlows ths attachment of a large variety of polar molecules These molecules are covalenily bound to the silanol group including carboxylale, amine, aminefphosphonatc, polycihylene glycol), ocladecyl, and caboxylalcoctadscyl groups The higher the concentration of the silanol groups found on the nanoparticle the higher is its affinity for polar molecules [42] Bioactivity of silica particles and the concentration

of silanol groups has caused great interest in researchers, Due to the presence of silanol groups al jis surface, siliva has boon involved in biomincralivation process favoring tha formation of hydroxyapatite in in vivo conditions According to numerous studies focused on the phenomenan of apatite formation on siliea, adsorption of calcium ions, which was stronger than adsorption of phosphate ions, is the initial step of apatite nucleation, This phenomenan strongly depends on the pI of the solution as well as on zeta potential of the silica particles [51

Madhưmalhi, el al reported in their study thal Chitinfnanositica composite scaffolds revealed a crystalline Hap layer formed onfin the sealfolds after 7 and 14 days of biomineralization, The apatite forming ability comes especially ftom Si OH but also

from Ti-OH or Z:-OH groups in less propartion concluding that by the incorporation of

silica into the chitin structure the bioactivity of the structural complex can be reached by showing thal cormpounds thal include silica in their siruoturo Gan be uscd for tissuc engineering applications |52|

Carlisle carried out a study involving specimens of normal tibia from young mice and rats

in which the activity of silicon found in active calcification sites was evaluated In the earliest stages of calcification in these Tegions, both silicon and calcium contents vise congruently when the calcium content is vary ow In more advanced stages as calcium approaches the proportions present in hydroxyapatite, silicon is present only at the detection limit Carlisle concluded that the amount of silicon present in specific regions within the active areas appears to be uniquely related to maturity of the bone mineral

[53]

Wiens and coworkers investigated the effzct of biosilica on the expression of one of the

most crucial signaling systems that mediate and modulate bone resorption, OPG/RANKL

using the osteoblast-like $a0S-2 cell model ‘Their data showed that biosilica is a selective inducer of OPG-cxpression resulting in an augmented synthesis and release of this cytokine in the extracellular space, There, the cytokine OPG might bind to RANKL and eliminates its fiunction to trigger osteoclast differentiation |54|

Kokubo proposed that hydrated silica formed on the surface of glass ceramics provides siles for favorabie apalile mucloation They found thal silanol groups (Si-O) abundant

on the surface of silica arc responsible for apatite formation and this plays the csscntial roll in formmng the chemical bond of the glass-ceramic to the bone |55]

Rhee, et al in 2005 developed a chitosan membrane modified with silanol group and calcium ions and cvalualed ils potcntial application øs a bioseliveguided bong regeneration membranz The membrane showed apalite-formning abilily in SBF within 1 day [56], At the same time, Eun-Jung Lee, et al, found and confizmed that menibranes of hybrid chitosan-siliea xerogel showed high bioactivity m vitro compared to chitosan

23