Study on synthesis of combination of silver nanoparticles and mesenchymal stem cell products for wound healing

The result from the burn model suggested the potential of using CM derived from hUCB MSCs for burn wound treatment besides MSCs-based therapy. Both AgNPs and CM exhibited healing [r]

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

NGUYEN THI THANH HOAI

STUDY ON SYNTHESIS OF COMBINATION OF SILVER

NANOPARTICLES AND

MESENCHYMAL STEM CELL PRODUCTS FOR WOUND HEALING

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

NGUYEN THI THANH HOAI

STUDY ON SYNTHESIS OF COMBINATION OF SILVER

NANOPARTICLES AND

MESENCHYMAL STEM CELL PRODUCTS FOR WOUND HEALING

MAJOR: NANOTECHNOLOGY CODE: 8440140.11QTD

RESEARCH SUPERVISORS: Prof Dr Sc NGUYEN HOANG LUONG Associate Prof HOANG THI MY NHUNG

i

ACKNOWLEDGMENTS

First of all, I would like to express my deepest gratitude to my supervisors Prof Dr Sc Nguyen Hoang Luong and Assoc Prof Hoang Thi My Nhung for their enthusiastic guidance and inspiration throughout the implementation of the thesis I also wish to thank Assoc Prof Nguyen Hoang Nam, Dr Luu Manh Quynh (Center for Materials Science, VNU University of Science), Dr Le Tra My, MSc Bui Thi Van Khanh (Department of Cell Biology, VNU University of Science) for the wholehearted instruction and useful suggestion Besides, I am extremely grateful to Dr Than Thi Trang Uyen (Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Health Care System) for all her support

My sincere thanks to lecturers in the Nanotechnology program for their helpful instruction when I have learned at Vietnam Japan University

I am truly thankful for all the encouragement from my family and my friends My thesis would not be done without their support

Finally, I would like to thank my classmates and my friends from Vietnam Japan University, VNU University of Science, Vinmec Research Institute of Stem Cell and Gene Technology who help me accomplish this thesis

Hanoi, July 2020 Student

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS i

TABLE OF CONTENTS ii

LIST OF FIGURES iv

LIST OF TABLES v

LIST OF ABBREVIATIONS vi

INTRODUCTION

CHAPTER 1: OVERVIEW

1.1 Cutaneous wound and wound healing process

1.1.1 Cutaneous wound

1.1.2 The normal wound healing process

1.1.3 The two therapeutic targets in wound treatment

1.2 AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase

1.2.1 AgNPs as a topical antimicrobial agent

1.2.2 AgNPs as an anti-inflammatory agent 10

1.2.3 Concerned factors for using AgNPs in wound treatment 11

1.2.3.1 Effect of particle size 12

1.2.3.2 Effect of capping agents 12

1.3 Products derived from MSC - cytokines and growth factors-modulated agent in wound healing 14

1.3.1 Stem cells and mesenchymal stem cells 14

1.3.1.1 What are stem cells (SCs)? 14

1.3.1.2 Mesenchymal stem cells (MSCs) 14

1.3.1.3 Products derived from MSCs 15

1.3.2 MSC-derived conditioned medium (CM) in wound healing 16

1.4 Combined using of silver nanoparticles and bio-factors for wound healing 17 CHAPTER 2: MATERIALS AND METHODS 19

2.1 Overview of experimental design 19

2.2 Preparation of AgNPs 20

2.2.1 Synthesis of AgNPs 20

2.2.2 Characterization of AgNPs 21

2.2.2.1 Physicochemical properties 21

2.2.2.2 Evaluation of the antimicrobial activity of AgNPs 22

2.2.2.3 Determination of the cytotoxic effect of AgNPs on NIH 3T3 cell 24

2.3 Preparation of CM and effect of CM on NIH 3T3 migration in vitro 26

2.3.1 Preparation of CM 26

2.3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro 26

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2.4.1 Deep partial-thickness burn wound model 29

2.4.2 Excisional wound model 31

2.4.3 Wound analysis 31

2.5 Statistical analysis 33

CHAPTER 3: RESULTS AND DISCUSSION 34

3.1 Characterization of AgNPs 34

3.1.1 Physicochemical properties 34

3.1.1.1 XRD pattern 34

3.1.1.2 TEM image 35

3.1.1.3 UV-Vis spectra 36

3.1.2 Evaluation of the antimicrobial activity of AgNPs 37

3.1.2.1 Sterility of AgNPs 37

3.1.2.2 Antimicrobial effect of AgNPs 38

3.1.3 Cytotoxic effect of AgNPs solution on NIH 3T3 cells in vitro 39

3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro 44

3.3 Skin wound model in vivo 48

3.3.1 Deep second-degree burn model 48

3.3.2 Excisional model 51

CONCLUSIONS AND PERSPECTIVES 59

CONCLUSIONS 59

PERSPECTIVES 59

iv

LIST OF FIGURES

Page

Figure 1.1 Phases in wound healing

Figure 1.2 The types of wound treatment applied for different wound categories

Figure 1.3 Mechanism of antimicrobial action of AgNPs

Figure 1.4 Factors impacted their cytotoxicity 11

Figure 1.5 MSC capacity of differentiation 15

Figure 2.1 Overall experimental design of the study 19

Figure 2.2 Schematic procedure of AgNPs synthesis 21

Figure 2.3 Examination of media on NIH 3T3 cells migration 28

Figure 2.4 Analysis of wound images by Image-J 29

Figure 2.5 Analysis of wound area by Image-J 32

Figure 2.6 Determination of wound area based on the stage of healing process 32

Figure 3.1 XRD pattern of synthesized AgNPs 35

Figure 3.2 TEM image shows the morphology of AgNPs and sizes of particles ranged from 10 to 45 nm 35

Figure 3.3 UV-Vis spectra of synthesized AgNPs 37

Figure 3.4 Agar plate without detection of microbial colony 38

Figure 3.5 AgNPs plates with less of microorganisms than the Control (-) plates 39

Figure 3.6 Morphology of NIH 3T3 cells 40

Figure 3.7 Image of 96-well plate after SRB staining 42

Figure 3.8 Cell viability measured by SRB assay on NIH 3T3 cells 43

Figure 3.9 Effect of media on the migration of fibroblast cells 45

Figure 3.10 The migration rate of fibroblast treated with media 46

Figure 3.11 The healing process of burn wounds in mice 48

Figure 3.12 Statistical analysis of healing rate of burn wounds at day 23 and day 30 after creating burns Values are represented as mean ± SD 49

Figure 3.13 Uneven healing rate in the MSC group 51

Figure 3.14 Statistical analysis of healing rate of excisional wounds with different treatments 52

v

LIST OF TABLES

vi

LIST OF ABBREVIATIONS

AgNPs Silver nanoparticles

CM Conditioned medium

DFUs Diabetic foot ulcers

DMEM Dulbecco’s modified Eagle’s medium

EGF Epidermal growth factor

ECM Extracellular matrix

EVs Extracellular vesicles

FBS Fetal bovine serum

fcc Face centered cubic

FDA Food and Drug Administration

hUCB CM Human umbilical cord blood-derived mesenchymal stem cell conditioned medium

hUCB MSCs Human umbilical cord blood-derived mesenchymal stem cells IL-1, IL-6, IL-8 Interleukin-1, Interleukin-6, Interleukin-8

IGF Insulin-like growth factor

KGF Keratinocyte growth factor

MSCs Mesenchymal stem cells

OD Optical density

PDGF Platelet-derived growth factor

ROS Reactive oxygen species

SDF Stromal cell-derived factor

SRB Sulforhodamine B

TEM Transmission electron microscopy

TGF-α, TGF-β Transforming growth factor α, transforming growth factor β

TSC Trisodium citrate

UV-Vis Ultraviolet visible spectroscopy

1

INTRODUCTION

Wounds are a silent burden on the healthcare system In 2018, Medicare beneficiaries analyzed that around 8.2 million people who have at least one type of wounds Wounds often classified into acute (traumatic, abrasions, surgical) and chronic wounds (diabetic foot ulcers (DFUs), leg ulcers, and pressure ulcers) based on healing time The challenges of healing wounds are the increase of infection, age and pathological background of the patient Hence, we need to come up with novel strategies to solve these problems Over the past few decades, silver nanoparticles (AgNPs) attract rapt attention in wound treatment due to various featured natures such as the history of using silver, simple and effective synthesized methods, and above all the outstanding antimicrobial activity These make AgNPs become one of the most widely used agents for preventing infection On the other hand, mesenchymal stem cells (MSCs) and products derived from MSCs, which appear as advanced therapies, have recently been studied and applied in the field of medicine In terms of wound healing, many studies suggest that paracrine signaling of MSCs rather than tissue differentiation and engraftment is a pivotal element for promoting wound healing That indicates the capacity to use conditioned medium (CM), which is one of the products derived from MSCs for wound treatment CM contains a variety of cytokines, growth factors, chemokines that modulate the healing process through induction of re-epithelialization, angiogenesis, and remodeling Therefore, we assume the synergistic effect of the combined use of AgNPs and CM, in which AgNPs with antibacterial, anti-inflammatory activities support CM to promote wound healing Our target is chronic wounds that require advanced therapies for treatment At the beginning of the research process, we aim to examine the healing effect of the combined use of AgNPs and CM on an acute wound, then perform it on a chronic wound model at a later stage This thesis is the first step of research, so in this study, three objectives need to be fulfilled

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(2) Evaluate the healing potential of conditioned medium (CM) by scratch assay in vitro;

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CHAPTER 1: OVERVIEW

1.1 Cutaneous wound and wound healing process

1.1.1 Cutaneous wound

Wounds are considered a growing challenge for the healthcare system There are variety of reasons that can lead to injury, from extrinsic factors, such as shear, thermal, pressure to underlying causes such as diabetes, stress [61] The injuries not only cause burden to patients, family, and healthcare system but also resulted in significant economic costs A retrospective analysis of Medicare beneficiaries (2018) reported that approximately 8.2 million people who suffered from at least one type of wounds with or without infection The cost of wound care ranged from $28.1 billion to $96.8 billion involving costs for chronic and acute wounds [52] Wound injuries are often classified into acute wounds including surgical wounds, traumatic, abrasions, or superficial burn, and chronic wounds, such as ulcers, diabetic foot ulcers (DFUs) Risk of chronic wounds is developed from an increase of age, the complication of diabetes, vascular diseases, obesity, etc The market for advanced wound care for chronic and surgical wounds is expected to $22 billion by 2024 [61]

On the other hand, acute wounds are at risk of wound infection, particularly in post-surgery [61] Another challenge for acute wounds is that prolonged healing can lead the wounds to enter a chronic state (non-healing) [16] Therefore, novel concepts to prevent infection and promote the healing process are vital to managing wounds

1.1.2 The normal wound healing process

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Figure 1.1 Phases in wound healing [43]

Hemostasis begins immediately after an injury created, platelets form a plug and release several mediators, for example, platelet-derived growth factor (PDGF), which subsequently recruit leukocytes to the wound site

5

concert of extracellular matrix (ECM) molecules and growth factors, PDGF, TGF-β induce fibroblasts around the wound to proliferate and migrate into the wound area The structural molecules of new ECM involving fibrin, fibronectin, hyaluronic, providing a scaffold for cell migration and the formation of granulation tissue The fibroblasts play an important role in synthesis, deposition, and remodeling of the ECM Besides, the formation of new blood vessels, called angiogenesis, initiates with the angiogenic molecules such as acidic or basic fibroblast growth factor (a-FGF, b-FGF), VEGF, TGF-β, angiogenin

After proliferation, the final phase is remodeling including the contraction and reorganization of ECM Fibroblast and macrophage release several proteolytic enzymes called matrix metalloproteinases that degrade collagen type III of the granulation tissues Collagen type I was replaced and aligned into paralleled fibrils, resulting in the formation of a scar

In summary, cytokines and growth factors are protein molecules that coordinate cellular processes These act to regulate a wide range of functions involving cell proliferation, cell differentiation, angiogenesis, wound healing, tissue modeling, immune cell activity through autocrine, paracrine, juxtacrine, or endocrine mechanisms [13] Hence, modulation of cytokines and growth factors can enhance the healing process

1.1.3 The two therapeutic targets in wound treatment

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Figure 1.2 The types of wound treatment applied for different wound categories (adopted from [54])

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in wound healing, but one of the major targets is to regulate the cytokines, growth factors, chemokines involved in wound healing

Therefore, two targets that may be intervened to promote the healing process are (1) prevent external infections, and (2) administration of internal cytokines, growth factors, chemokines In a recent study, the topical antimicrobial agent (AgNPs) was aimed to prevent the infiltration of microorganisms that enhance the inflammatory phase Simultaneously, a product derived from MSCs, herein, conditioned medium (CM) display healing ability through regulating mediators (cytokines, growth factors, chemokines) The combination of the two strategies, which use the basic wound care to support the active wound care, is expected to create a synergistic effect to accelerate the healing process

1.2 AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase

1.2.1 AgNPs as a topical antimicrobial agent

Preventing infection is the crucial target of effective wound management [56], [61] Recently, the use of topical antibiotics and antiseptics are markedly increased Topical antibiotic shows several benefits over the systemic use, such as the reduction in systemic toxicity, but can lead to rising bacterial resistance [1], [56] Antiseptics, on the other hand, prefer more useful in the reduction of bacteria but more toxic than antibiotics [58] Current representatives of topical antimicrobial agents, in which their benefits and drawbacks were listed in Table 1.1

Table 1.1: Topical antimicrobial agents for wound healing

Type Description Ref

Antiseptics Advantages: Broad spectrum of antimicrobial activity Disadvantages: toxic to host cells

[58]

Hydrogen peroxide

Use for wound irrigation and remove necrotic tissues H2O2 was detected in normal healing, rapidly

8

(H2O2) decomposed to water and oxygen High concentration

cause cell damage through corrosion, formation of oxygen gas, and lipid peroxidation

Povidone-iodine

A solution of 0.1 – 0.2% minimize cytotoxicity and rise the release of free iodine for antibacterial activity Disadvantages: irritation, allergy, cytotoxic to vulnerable people (pregnant women, newborns, etc.)

[56]

Chlohexidine Chlohexidine showed long-lasting residual ability, and active activity against positive and Gram-negative bacteria but poor activity against non-enveloped viruses and bacterial spores The mechanism of its action is disrupting the cytoplasmic membrane

[1]

Alcohol Bactericidal action of an aqueous solution of 70% - 92% alcohol is rapid, but short-time action, and can cause irritation and dryness

[56]

Nanoparticles: silver, gold, zinc (NPs)

NPs exhibit the bactericidal effect with wide-spectrum by the release of metal ions or generation of Reactive Oxygen Species (ROS) The toxicity can be governed by modulating several factors such as size, shape, concentration [49] Antibiotics: Bacitracin, Mupirocin, Bacitracin, Silver sulfadiazine

Advantages: the cytotoxic to host cells is less than antiseptic

Disadvantages: Antibacterial spectrum is narrower, and resistance to antibiotics is more frequent than that of antiseptics

[58]

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development of nanotechnology, decreasing the size of the material to the nanoscale, for example, increases its surface-to-volume ratios, leading numerous advantages for applications [42] The use of silver for preventing microorganisms and treating burns have seen for hundreds of years [63] In the 18th -20th century, silver nitrate was widely used for treating burns and then a commercial product of silver sulfadiazine has been commonly used as topical antibiotics [75]

Over the past few decades, silver nanoparticles (AgNPs) attract great interest due to their well-known antimicrobial activity The mechanism of this action is not fully understood yet, but it is suggested that this action is in relation with (1) AgNPs anchor to the cell wall of bacteria then penetrate it, altering the permeability of cell membrane, (2) AgNPs penetration damage the bacterial organelles including mitochondria, vacuoles, ribosomes, and denature protein, as well as DNA, (3) The formation of free radicals and generation of ROS, (4) AgNPs can modulate the signal transduction, dephosphorylate of peptides substrate on tyrosine residues, resulting in inhibition of cell growth [14], [57], [76] (Figure 1.3)

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Besides, AgNPs are easily incorporated into the cotton fabric and dressing and able to synthesize by simple and safe approaches [49] The anti-inflammatory activity is another advantage of AgNPs in wound healing, which then is discussed further

1.2.2 AgNPs as an anti-inflammatory agent

The inflammatory phase is a range of early immunological response against bacteria and other foreign particles by the production of pro-inflammatory cytokines In normal healing, both the pro-inflammatory and anti-inflammatory are present However, if these responses take place inappropriately, a prolonged inflammatory phase can lead to non-healing wounds [16] Scientific studies suggest that AgNPs possess anti-inflammatory activity besides the well-known antimicrobial activity Tian et al (2007), who first found evidence in the anti-inflammatory activity of AgNPs, compared the healing process between the mice treated with AgNPs and those treated with amoxicillin and metronidazole, two widely used antibiotics The result showed that the AgNPs-treated group healed faster than the antibiotic-treated group, suggesting other influence of AgNPs besides antimicrobial action The findings showed the expression level of IL-6 (pro-inflammatory cytokines) was lower in the AgNPs-treated group whereas IL-10 (anti-inflammatory cytokines), VEGF (angiogenic cytokine), TFN-γ (cytokines in remodeling phase) were stayed higher than the control group The overall reduction in inflammation might be predicted, and the decrease of neutrophils in the wound area confirms it [67] The efficiency of AgNPs in inflammatory reduction without toxic effects was found in a postoperative peritoneal adhesion model [77]

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Besides, AgNPs showed an influence on improving tensile properties of healed wounds, modulating collagen deposition in the wound healing process [36] Taken together, not only AgNPs display broad antimicrobial spectrum, but also exhibit anti-inflammatory properties through cytokine regulation at the inflammatory phase This reinforces the potential of using AgNPs for wound treatment

1.2.3 Concerned factors for using AgNPs in wound treatment

AgNPs differ from size, shape, surface electric charge, and other physicochemical properties AgNPs are aimed to use for wound repair, hence, the cytotoxicity (ability to destroy cells), genotoxicity (property that damage genomic information) of AgNPs are needed to take into account apart from antimicrobial activity Size, range of concentration, and agglomeration are vital factors impacting their cytotoxicity [3] (Figure 1.4)

12 1.2.3.1 Effect of particle size

Size is considered to be the most relevant factor to the cytotoxicity of AgNPs AgNPs revealed the effect on cell viability, ROS generation, lactate dehydrogenase (LDH) activity relying on sizes as well as testing cell lines (Table 1.2)

Table 1.2: Effect of AgNPs size on cytotoxicity

Synthesized method

Size (nm)

Cell type Findings Ref

Chemical

reduction method (PVP coated)

5, 20, 50

Human cells: A549,

HepG2, SGC-7901, MCF-7

Smaller particles were easier to enter cells than larger ones, causing higher toxicity

[39]

Unknown method (PVP-coated)

10, 50, 100

Human liver cells HepG2

Size-dependent toxicity through autophagy activation

[45]

Unknown method (Hydrocarbon-coated)

15, 30, 55

Alveolar macrophages

Increase of ROS level when cells treated with AgNPs 15 nm suggested cytotoxicity through oxidative stress

[11]

Unknown method (citrate-coated)

20, 110

Citrate-coated AgNPs 20 nm generates neutrophilic inflammation in lung compared to citrate-coated 110 nm

[73]

13 1.2.3.2 Effect of capping agents

To prevent aggregation, capping agents stabilize the nanoparticles by producing electrostatic repulsions between particles Because they act in preservation of the surface chemistry by upholding shape and reducing Ag+, they may affect the cytotoxicity of AgNPs The capping agents can be divided into organic (polysaccharides, polymer, citrates, protein, etc.) and inorganic (sulfide, chloride, carbonate)

In the study of K C Nguyen et al (2013), citrate- and polyvinylpyrrolidone (PVP)-coated AgNPs (10, 50, 75 nm) and un(PVP)-coated AgNPs (20, 40, 60, 80 nm) were examined their cytotoxicity to J774A.1 macrophage and HT29 epithelial cells The results showed that uncoated AgNPs suppressed inflammatory responses and promoted oxidative stress in testing cells, so were higher toxic than coated AgNPs Besides, PVP-coated AgNPs displayed higher toxic than citrate-coated ones The studies again confirmed the cytotoxic effect of AgNPs is size- and coating-dependent [50] Anda R Gilga et al (2014) investigated the cytotoxicity of citrate-coated AgNPs (10, 40, 75 nm), PVP-citrate-coated (10 nm), and uncitrate-coated AgNPs (50 nm) against BEAS-2B human lung cells They found that cytotoxicity was only presented in the 10 nm AgNPs groups and independent of the coating agent Moreover, although citrate- and PVP-coated AgNPs showed different agglomeration patterns, there was not confirmed the difference in the cellular uptake and intracellular localization between them [22] Xiaoquing Guo et al (2016) studied the impact of size and coating on cytotoxicity and genotoxicity of AgNPs types including PVP- and citrate-coated (20, 50, 100 nm) and silver nitrate in L5178Y and TK6 cells The results confirmed that AgNPs were less cytotoxic and genotoxic than ionic silvers The smallest sized of citrate-coated AgNPs (20 nm) had the highest mutagenic potency and micronucleus frequency than other AgNPs, then more cytotoxic and genotoxic than PVP-coated (20 nm) [25]

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by the FDA for use as a preservative in many foods, and drug for preventing gout or kidney stones [FDA data] Hence, sodium citrate was used as a capping agent in the recent study

1.3 Products derived from MSC - cytokines and growth factors-modulated agent in wound healing

1.3.1 Stem cells and mesenchymal stem cells

1.3.1.1 What are stem cells (SCs)?

Stem cells (SCs) have recently been receiving great interest in the field of medicine SCs are described as cells that can be self-renewal and the ability to differentiate More clearly, SCs can produce daughter cells which are identical to them and can generate several cell types relying on their types of stem cell [44] SCs are found both in embryos and adult cells Based on the differentiation potency, SCs are classified to be totipotent, pluripotent, multipotent, oligopotent, and unipotent Totipotent SCs can divide and generate cells of the whole organism Zygote, the structure formed after fertilization of a sperm and an egg, is representative of the totipotent cell Pluripotent SCs (PSCs) are capable of differentiation into all germ layers’ cells The examples are embryonic stem cells (ESCs) originated from the inner cell mass of preimplantation embryos, and induced pluripotent stem cells (iPSCs) which are derived from somatic tissues including skin and blood cells, then they have been reprogrammed back into an embryonic-like state Multipotent cells may be adult cells including hematopoietic stem cells, mesenchymal stem cells (MSCs), and neural SC

1.3.1.2 Mesenchymal stem cells (MSCs)

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Human MSCs first isolated from bone marrow (BM), then they have been isolated from other tissues such as adipose tissue, amniotic fluid and membrane, peripheral blood, umbilical cord (UC), umbilical cord blood (UCB), Wharton’s jelly, salivary gland, etc [68] (Figure 1.5)

Figure 1.5 MSC capacity of differentiation [44] 1.3.1.3 Products derived from MSCs

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MSCs are cytokines, growth factors, chemokines that can be divided into many families: angiogenesis (Ang 1, FGF, HGF, PDGF, VEGF, etc.), immune-modulation (TGF-β, HO-1, IL-6, IL-10, etc.), proliferation (FGF, HGF, IGF-1), anti-fibrosis (MMPs, TIMP-1, KGF, HGF) [47], [70] Besides, extracellular vesicles (EVs) are nano-sized and micro-sized particles classified as exosomes, microvesicles and apoptotic bodies EVs regulate a range of biological responses via transferring bioactive factors such as protein, lipids, nucleic acids (mRNA, miRNA) so EVs plays an important role as mediators in cell-cell communication [66], [71] MSC-derived secretome is contained in the conditioned medium (CM), which is spent medium harvesting after MSC cell culture in vitro [30-31] MSC-derived EVs are isolated and purified from CM [28 -29], [35]

Cell-free therapies, in this case, MSC-derived secretome display crucial advantages besides stem-cell therapies, (1) using secretome has more benefits on safety issues than MSC transplantation; (2) MSC-derived secretome can be stored for a long time without the addition of toxic cryopreservative agents also decrease in quantity; (3) using MSC-derived secretome (CM and EVs) is more practical for therapeutic application because the cell collection is not needed [70]

For these reasons, cell-free therapies (MSC-derived CM and EVs) are viable options along with cell-based therapy for treating many diseases such as lung disorders, bone defects, Alzheimer’s disease, wound healing, etc [47], [70]

1.3.2 MSC-derived conditioned medium (CM) in wound healing

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Liwen Chen et al (2008) used CM derived from BM MSC for wound model in vivo and obtained noticeable wound repair, indicating that paracrine factors of BM MSC could promote wound healing Their data showed that BM MSCs released differential levels of cytokines than dermal fibroblasts, including EGF, KGF, IGF-1, VEGF-a, PDGF-BB, EPO, and TPO, and significantly lower amounts of IL-6 and osteoprotegerin [12] Many other studies investigate the presence of cytokines, growth factors in CM that promote the migration of fibroblast cells in the scratch assay in vitro Walter et al (2010) found various cytokines IL-6, IL-8, TGF-β1, MCP-1, Rantes in BM CM that accelerate the migration and proliferation of L929 fibroblasts and HaCaT keratinocytes co-culture in vitro [72] Jiajia Zhao et al (2013) reported the synergistic actions of many cytokines in enhancing migration and proliferation of dermal fibroblast than single cytokine [82] Moreover, data from animal models in vivo showed the potential of using CM for wound repair The study of Sun et al., 2018 showed Wharton’s jelly-derived MSC CM promotes HUVEC proliferation, regeneration of sebaceous glands, angiogenesis in a radiation-induced wound in rats [64]

The release of cytokines and growth factors from MSCs is depended on the sources and culture conditions [70] Human umbilical cord blood-derived MSCs (hUCB MSCs) have biological advantages such as anti-inflammatory activity through reducing the expression level of pro-inflammatory cytokines (IL-1α, IL-6, IL-8) [33] On the other hand, using CM derived from hUCB MSC for wound repair has not investigated in Vietnam yet Therefore, it is essential to examine the potential of hUCB CM in the terms of healing injuries

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amoxicillin combined with AgNP-water showed an antagonistic effect against methicillin-resistant S aureus strain (MRSA) [15] Besides, a large number of studies are investigated the wound dressing fabricated from natural polymers (collagen, chitosan, etc.) incorporated with AgNPs [49], [76], [80]

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CHAPTER 2: MATERIALS AND METHODS

2.1 Overview of experimental design

In the present study, AgNPs and CM were prepared and evaluated before using for wound treatment in vivo In brief, synthesized AgNPs have characterized the physicochemical properties in terms of size, shape, crystal structure, antimicrobial activity, and cytotoxicity On the other hand, CM was harvested and evaluated the effect on the migration of fibroblast cells Finally, AgNPs and CM were aimed to treat wounds in mice (Figure 2.1)

20 2.2 Preparation of AgNPs

2.2.1 Synthesis of AgNPs

AgNPs were synthesized using a chemical reduction method where sodium borohydride (NaBH4) as a reductant agent and trisodium citrate (TSC) as a stabilizing agent

Materials

Chemical: Silver nitrate AgNO3 (Merck), sodium borohydride (NaBH4) (Scharlau), trisodium citrate (TSC) (Bio Basic), citric acid (Bio Basic), distilled water

Equipment: 200 ml glass beakers, measuring cylinder, magnetic stirrer hot plate

Procedure

The synthesis process was described in Figure 2.2

Step 1: 74 ml distilled water was prepared in 200 ml glass beakers

Step 2: The beaker was placed on a magnetic stirrer ml of AgNO3 0.1 mM was added to the beaker

Step 3: ml of TSC 0.05 mM was added to the beaker and stirred in minutes Step 4: 20 ml of NaBH4 0.01 mM was added to the beaker and continued stirring for

50 minutes The pH of the solution was measured after stirring 50 minutes Step 5: The last solution was measured by UV-Vis spectroscopy

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Figure 2.2 Schematic procedure of AgNPs synthesis

2.2.2 Characterization of AgNPs

2.2.2.1 Physicochemical properties

Synthesized AgNPs were quantified with the following techniques

Ultraviolet-visible spectroscopy (UV-Vis)

UV-Vis technique is based on the absorption of ultraviolet light or visible light of the matter, which undergoes excitation and de-excitation, leading to the formation of distinct spectra UV-Vis technique can give qualitative and quantitative data of given compound [20]

Synthesized AgNPs were measured using UV-Vis spectroscopy (Shimadzu) at Center of Materials Science, VNU University of Science

Transmission electron microscopy (TEM)

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elastically or inelastically scattered electrons when the electron beam interacts with the specimen

TEM analysis (TEM, JEOL-JEM 1010) was performed at National Institute of Hygiene and Epidemiology

X-ray diffraction (XRD)

XRD is a primary analytical technique for the identification of various compounds, determination of molecules and crystal structure of material, etc [18] When X-rays strike on crystal, resulting in the formation of many diffraction patterns that exhibit the physicochemical properties of the crystal structures

Synthesized AgNPs were measured by X-ray spectroscopy (Rigaku Miniflex 600) at VNU University of Science

2.2.2.2 Evaluation of the antimicrobial activity of AgNPs

Synthesized AgNPs solution was examined the sterility, which means a test of whether microorganisms present in the solution Then, the AgNPs solution was tested antimicrobial activity against microorganisms in the air through incubating it in agar plates and observing the presence of microorganisms

a) Sterility testing of AgNPs

Materials:

Chemicals: Soyabean Casein Digest Agar (Himedia), distilled water, AgNPs solution

Equipment: Biological Safety Cabinet Class I, Microbiological incubator, Autoclaving, Glass petri plates (20 dishes), ml Pipette and tips

Procedure:

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20 g Soyabean Casein Digest Agar was dissolved in 500 ml of distilled water This was sterilized by autoclaving at 121ºC for hours Subsequently, the sterile solution was poured into sterile glass petri plates, and waited until frozen This step was performed in the biological safety cabinet

Sterility test of AgNPs:

Stock AgNPs of 300 µg/ml was diluted with sterile distilled water to make AgNPs solutions with a concentration of 30 and 10 µg/ml

1 ml AgNPs solutions of 30 and 10 µg/ml were added alternately into agar plates for each solution Those plates were shaken well to make sure that the solution covers the surface of the plate evenly plates were not treated AgNPs solution and kept as the control

Finally, those plates were wrapped and placed in the microbiological incubator The plates were examined the presence of microorganism colonies and took photograph after days

b) Antimicrobial activity of AgNPs against microorganism in the air

Agar plate preparation: the step was followed the procedure in the previous section Test the bactericidal effect of AgNPs:

The agar plates were divided into groups: 10 µg/ml AgNPs-treated group, untreated control, agar plate control (ensure there were no errors in the process of plate preparation) groups were marked names: 10 µg/ml AgNPs, Control (-), and Control (+), respectively

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wrapped and placed in the microbiological incubator The plates were examined the presence of microorganism and took photograph after days

2.2.2.3 Determination of the cytotoxic effect of AgNPs on NIH 3T3 cell

Sulforhodamine B (SRB) assay is one of the most well-known methods for testing drug cytotoxicity SRB is a bright pink aminoxanthene dye having two sulfonic groups that can bind to basic amino acid residues of protein in cells fixed in trichloroacetic acid under mildly acidic conditions The amount of SRB extracted from stained cells under basic conditions is proportional to the cell mass [62]

Cell line

The NIH 3T3 mouse fibroblast cell line used in this experiment Cells were stored in liquid nitrogen, at Experimental Oncology Research Group, Department of Cell Biology, Faculty of Biology, VNU University of Science

Procedure

Cell viability was tested using SRB assay according to the instructions of manufacturer [69]

Cell preparation

NIH 3T3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (100 units/mL) Cells were maintained in a cell culture incubator at 37 ºC, 5% CO2 The medium

was changed every 2-3 days and cells were sub-cultured when they covered 70 - 80% confluence until reached the required number of cells

Cells were seeded in 96-well plate (1 x 103 cells/well) with 180 µl culture medium/well and incubated overnight at 37 ºC and 5% CO2 for cells adhered to the surface of the culture plate

25

20 µl AgNPs solution were added into each well with concentrations in the range of 30 µg/ml, 15 µg/ml, 7.5 µg/ml, 3.75 µg/ml, 1.875 µg/ml, and 0.9375 µg/ml Gently tapped the wall of the plate to ensure even distribution of the sample, and incubated in the incubator for 48h at 37 °C, 5% CO2

Cell morphology after incubation with AgNPs was observed under an inverted microscope at 10x magnification

After 48 h, the plate was taken out of the culture room Supernatants were aspirated out and cells were fixed by adding 50 µl of TCA 50% and incubated at °C for h SRB incubation and Optical density (OD) measurement

50 µl of SRB 0.4% was added in each well and then stained for 10 minutes at room temperature Anxcessive amount of SRB was removed by the cleaning solution of acetic acid 1%, repeated times Then dye was dissolved in Tris-based and absorbance at 540 nm wavelength was recorded using a Microplate reader

Analysis

Cell viability ( ) (%) was calculated from values of OD, thereby the potential toxicity of AgNPs against fibroblast cells was assessed Cell viability ( ) was written as

: The average value of OD of solvent-treated wells : The average value of OD of AgNPs-treated wells

26

2.3 Preparation of CM and effect of CM on NIH 3T3 migration in vitro 2.3.1 Preparation of CM

Human umbilical cord blood-derived MSCs (hUCB MSCs) were provided by Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Health Care System, Hanoi Condition medium (CM) was generated from MSCs culture

Step 1: hUCB MSCs were cultured in free-serum completed culture medium (StemMACS, Mitenyl, Germany) through passage II, at 37 ºC and a humidified atmosphere containing 5%CO2 Culture medium mentioned here was completed,

with supplement

Step 2: When cells covered 80% surface of the culture dish, cells were harvested and seeded on a 100 nm culture dish in the free-serum culture medium

Step 3: After days, the medium was collected, and then cellular debris in the medium were removed by 0.22 µm filter After filtration, the medium was considered as conditioned medium (CM)

Step 4: CM was stored at 4ºC and not be used after more than days of storage

2.3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro

Procedure

Cell culture

NIH 3T3 cells were prepared using the same process as described in 2.1.2.3 Briefly, NIH 3T3 were cultured in DMEM supplemented with 10% FBS and 1% penicillin (100 units/mL) and were maintained in the incubator at 37 ºC and 5% CO2.

When reached the required number of cells, cells were seeded in tissue culture dishes (diameter 40 mm), 5x104 cells/ dish, and incubated overnight at 37 ºC, 5% CO2 to permit cell adhesion to the dish surface

27 Scratch assay

NIH 3T3 cells were examined morphologically to ensure the adhesion and confluence of the monolayer

The monolayer was then scratched using a sterile pipette tip type 200 µl to create a gap of approximately 0.2 – 0.4 mm in width

28

Figure 2.3 Examination of media on NIH 3T3 cells migration Image capture and analysis

29

Wound closure (%) was calculated by the following formula

Wound closure (W0-Wt)

W0 x100 Where W0: average width of scratch at time point (pixel); Wt: average width of scratch at time point (pixel)

Figure 2.4 Analysis of wound images by Image-J (A-C) the gap was measured at points including the top, middle, and bottom along their verticals (D) results in

length column were calculated to average 2.4 Skin wound model in vivo

AgNPs, CM, and combined use of AgNPs and CM have investigated the healing properties in wound models: burn and excisional models

2.4.1 Deep partial-thickness burn wound model

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10 Swiss male mice (50 – 60 g, – 10 weeks old) were obtained from the National Institute of Hygiene And Epidemiology Animal experiments conducted in the laboratory following the regulation on animal use for scientific purpose Mice were supplied with food and water, in the condition of 12h light/dark cycle The mice were anesthetized with intraperitoneal ketamine (Ketamine Hydrochloride Injection, 50 mg/ml) A dosage of 140 mg/kg body-weight was used

The dorsum of mice was shaved using an electric shaver, then the hairs were removed completely with the hair removal cream An alcohol swab was used to disinfect the shaved skin Each mouse was inflicted on the right side of the body Creation of burn wounds and treatment

A metal plate (1.5 x 1.5 x 0.3 cm) (sterilized with 70% alcohol overnight) was heated on the fire lamp in minutes and placed on the skin for 10 s Mice were randomly divided into groups, treated immediately after creating the wounds Group (Control): non-treated mice

Group (AgNPs): mice were treated with a topical AgNPs solution (200 µl of AgNPs 10 µg/ml) applied to the wound bed daily and for the first days [74]

Group (CM): mice were injected intradermally with CM (200 µl) in the wound bed daily and for the first days [38]

Group (MSCs): mice were injected intradermally with MSCs (1x106 cells/200 µl PBS) into the wound bed daily and for the first days [31]

Group (Culture medium): mice were injected intradermally with culture medium (200 µl) in the wound bed daily and for the first days

Group (PBS): mice were injected intradermally with PBS - buffer solution (200 µl) in the wound bed daily and for the first days

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2.4.2 Excisional wound model

Preparation of mice: 30 mice (25-30 g, 4-6 weeks old) were prepared followed the process as described in the previous part

Creation of excisional wounds and treatment

Full-thickness excisional wounds (approximately x cm2) were made on shaved skin, on the right side of body The mice were randomly divided into groups and treated immediately after creating the wounds

Group (Control): non-treated mice

Group (AgNPs): mice were treated with a topical AgNPs solution (200 µl of AgNPs 10 µg/ml) applied to the wound bed daily and for the first days

Group (CM): mice were injected intradermally with CM (200 µl) in the wound bed daily and for the first days

Group (MSCs): mice were injected intradermally with MSCs (1x106 cells/200 µl PBS) in the wound bed daily and for the first days

Group (AgNPs + CM): mice were injected intradermally with CM (200 µl) in the wound bed, then treated with a topical AgNPs solution (200 µl of AgNPs 10 µg/ml) applied to the wound bed CM was used for the first days, AgNPs was used for the first days

2.4.3 Wound analysis

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Figure 2.5 Analysis of wound area by Image-J How to determine wound area:

In the early stage, when the eschar had not peeled off, the wound area included the eschar that covers the wound (Figure 2.6A) When the new tissues of the wound were revealed, the wound area was calculated according to the wound bed (Figure 2.6B)

33

Wound closure (%) was calculated by the following formula

Wound closure (S0-St)

S0 x100 Where S0: wound area at day (cm2);

St: wound area at day (cm2) 2.5 Statistical analysis

34

CHAPTER 3: RESULTS AND DISCUSSION

3.1 Characterization of AgNPs

AgNPs were synthesized by chemical reduction using NaBH4 as the reducing agent and the TSC as the stabilizing agent Subsequently, the physicochemical properties (UV-Vis, XRD, TEM), antimicrobial activity, and the cytotoxic effect of AgNPs have been characterized

3.1.1 Physicochemical properties

3.1.1.1 XRD pattern

35

Figure 3.1 XRD pattern of synthesized AgNPs

The crystallite size of AgNPs obtained from XRD pattern using Debye-Scherrer equation were 27.8 nm corresponding to (111) as the most intense plane and approximately 9.3 nm corresponding to (200) and (220) planes

3.1.1.2 TEM image

TEM is a powerful tool for determining the morphology and size of nanoparticles TEM images revealed mostly synthesized AgNPs have the spherical shapes with the size ranged from 17.5 to 42.5 nm, the average size is 27.5 nm (Figure 3.2)

Figure 3.2 TEM image shows the morphology of AgNPs and sizes of particles ranged from 10 to 45 nm

36

smallest specific surface energies, such as {111}, which might contributed to encase the nanocrystal to larger particles (red arrow) [65]

Besides, the blurred area surrounding particles might be citrate ions remaining after reaction (black arrows) This was confirmed the existence of residual TSC in the solution as seen in XRD pattern The capping agent acts to stabilize the shape of nanocrystals via binding to the surface of nanocrystals [79] However, the capping agent gradually degraded and released into the solution during long-time storage, which facilitated the dissolution of the small particles and the growth of larger particles, like Ostwald ripening [23] In this case, AgNPs particles had have fused into larger particles via coalescence during 3-month storage

3.1.1.3 UV-Vis spectra

37

Figure 3.3 UV-Vis spectra of synthesized AgNPs

3.1.2 Evaluation of the antimicrobial activity of AgNPs

3.1.2.1 Sterility of AgNPs

For further use of cytotoxicity assay, it is needed to test whether the AgNPs solution was sterile If the solution is not sterile, it can cause contamination in cell culture, and consequently lead to cell death In the recent study, the AgNPs solution was incubated with Soybean casein agar in the bacteriological incubator at 37ºC for days There was not detected any microbial colony on all the agar plates including control, AgNPs solution of 10 µg/ml, and AgNPs solution of 30 µg/ml This proved the AgNPs solution is sterile itself (Figure 3.4)

38

Figure 3.4 Agar plate without microbial colony indicated AgNPs sterility 3.1.2.2 Antimicrobial effect of AgNPs

For using in vivo wound model, AgNPs have evaluated the antimicrobial activity through testing on the agar plate The agar plates were opened the cover to allow microorganisms to enter the surface of the plates Table 3.1 showed there was only bacterial colony detected in AgNPs-treated plates, which is much less than the control (-) group, with colonies Otherwise, the size of the colony detected in AgNPs-treated plates was smaller than the control (-) group This indicated the antimicrobial activity of AgNPs (Figure 3.5)

Table 3.1 Number and size of microbial colonies in each group

Group Number of colonies Size

Control (+) -

Control (-) +++

10 µg/ml AgNPs +

39

Figure 3.5 AgNPs plates with less of microorganisms than the Control (-) plates, indicating the antimicrobial activity

3.1.3 Cytotoxic effect of AgNPs solution on NIH 3T3 cells in vitro

Cytotoxicity assay is a crucial type of test to evaluate the toxic potential of products that are aimed to use for biological objects including humans and animals In the recent study, the evaluation of the cytotoxic effect of AgNPs solution was performed on NIH 3T3 cells using the SRB assay

40

Figure 3.6 Morphology of NIH 3T3 cells (A) Unexposed cells (B) Solvent-exposed control (H2O) (C-F) AgNPs of 3.75, 7.5, 15, 30 µg/ml-exposed cells,

respectively (10x magnification lens, zoom 5.6x)

41

Besides, most cells exposed AgNPs of 15 µg/ml remained their shape and attached to the surface of the culture dish, whereas some others had shrunken and contracted in small clusters (Figure 3.6E) When cells exposed to AgNPs of 30 µg/ml, most cells were seen as less multipolar, shrunken, and coalesced into large clusters However, a few floating cells were seen under the microscope (Figure 3.6F) Similar changes were recorded by other researchers in other cell types treated with AgNPs Up to a concentration of 25 µg/ml, photo-reduction AgNPs with size – 20 nm did not lead to shape change compared to control primary fibroblast cells With increasing concentration from 50 to 100 µg/ml, cellular morphology was seen to be less polyhedric and shrunken [5] Other paper showed that when rat alveolar macrophages treated hydrocarbon-coated AgNPs of 25 µg/ml (15 nm), cells clustered with each other, with agglomerated AgNPs on the surfaces [11]

Dose-dependent cytotoxicity of AgNPs

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Figure 3.7 Image of 96-well plate after SRB staining The amount of pink dye was less than the remaining wells in the first column (AgNPs of 30 µg/ml-treated cells)

(red arrows) and uneven pink color in the second column (AgNPs of 15 µg/ml-treated cells) (black arrows) X was not counted for analysis

The results of the SRB assay showed that AgNPs exhibited toxicity to NIH 3T3 cells relying on the concentrations AgNPs of 0.9375 to 7.5 µg/ml displayed a slight impact on cell viability, by 80 to 100% When cell exposed AgNPs of 15 µg/ml, the cell viability was around 75% in both two trials The ratio decreased to around 30% at the first performance and 50% at the second performance when cells treated AgNPs of 30 µg/ml

According to calculation, the IC50 values, which are concentrations of AgNPs that

required for 50% inhibition of cell viability in vitro, were 26.02 µg/ml and approximately 30 µg/ml, respectively (Figure 3.8) The result was consistent with the qualitative comparison of cell morphology (Figure 3.6)

43

Figure 3.8 Cell viability measured by SRB assay on NIH 3T3 cells after treatment with AgNPs (0.9375 – 30 µg/ml) for 48 h OD values of solvent-exposed cells were

taken as 100% viability All values are expressed as the mean ± SD

For example, the cytotoxic potential of – 20 nm AgNPs to primary fibroblasts cells, in which IC50 value was found to be 61 μg/ml by using the XTT assay [5]

This was significantly higher than those values of same AgNPs exposed to secondary human cells, which were 10.6 μg/ml for HT1080 cells and 11.6 μg/mL for A431 cells, found in their previous study [4]

44

capped AgNPs with size distribution from - 50 nm on NIH 3T3 by MTT assay [59] This is relatively lower than IC50 values in this study, which means higher toxic than the synthesized AgNPs There was a slight difference among the results from SRB, MTT, and XTT assays, however, the comparison of IC50 values among

these assays was feasible [34]

In summary, citrate-capped AgNPs were synthesized, which possessed the spherical shape with the size ranged from 17.5 to 42.5 nm AgNPs was aimed to use for biological objects, so AgNPs was then examined their antimicrobial and cytotoxic effect Synthesized AgNPs with the concentration of 10 μg/ml exhibited sterile nature, bactericidal properties against microorganisms in the air, and mild cytotoxic on mouse fibroblast as well Therefore, AgNPs of 10 μg/ml was used for mouse wound model in vivo

3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro

Fibroblasts are primary active cells of connective tissue, which play important roles in synthesis, deposition, and remodeling of ECM in the proliferation phase In the healing process, fibroblasts were stimulated to proliferate and migrate into the wound site, thus was an extremely important event in wound healing [53] The evaluation of factors effected on the migration of fibroblasts thus can aid in finding therapeutic targets for wound repair

45

46

Figure 3.10 The migration rate of fibroblast treated with media

47

It was clear that CM induce the migration of fibroblasts Considering CM and CM (-) groups, MSCs might release trophic factors into both culture media with and without supplement CM showed a better influence on fibroblast migration than CM (-) Besides, using CM collected from MSCs cultured in the serum-free and xeno-free culture medium, which is the cell culture medium that did not contain elements derived from animals, is met the requirement of safety issue [30]

Coming up with Culture medium and CM groups, when MSCs cultured in Culture medium with supplement; nutrient, growth factors, etc inside medium had been supported for MSCs growth and expansion At the same time, MSCs secreted soluble compounds into the culture medium, which were key factors supporting the migration of NIH 3T3

To evaluate the potential of using CM for wound treatment, the scratch assay is used widely to assess their impact on migration of other cell types, herein, mouse fibroblast cells These results matched studies reported by other researchers The study on the influence of BM CM on L929 fibroblast and HaCaT keratinocytes co-culture found that there were various cytokines and growth factors inside CM involving IL-6, IL-8, TGF-β1, MCP-1, rantes, in which TGF-β1 and IL-6 might act to accelerate cell migration [72] The number of migrated human fibroblast cells (CCD-986sk) in the CM-exposed group was around times higher than the control group [31] In case of hUCB CM, diverse anti-aging cytokines and growth factors including EGF, bFGF, TGF-β, PDGF, HGF were found that promote migration of human dermal fibroblast (HDF) compared to CM derived from human bone marrow and human adipose tissue [34]

48 3.3 Skin wound model in vivo

3.3.1 Deep second-degree burn model

To investigate the healing effect of each treatment, the deep second-degree burn wound in mice was created Immediately after wound creation, wounds were treated with: (1) Control: no treatment, (2) AgNPs solution, (3) hUCB CM, (4) hUCB MSCs, (5) Culture medium, (6) PBS

49

Burn wounds were created by contact mouse skin with the hseated metal plate for 10s This was the same approach with the creation of deep second-degree burn in the other study [74] Burn injuries were also compared with burn classification based on the depth of wounds [32] Therefore, in this experiment, the wounds were supposed to be deep second-degree burn wounds

All groups exhibited higher healing rates compared with the control group Table 3.2 showed a descriptive qualitative assessment for epidermal change in different groups adopted from Lamiaa G Wasef’s study [74] The control group was observed moderate epidermal necrosis and losses, as well as a slow epidermal regeneration In the AgNPs-treated and CM-treated group, epidermal regeneration was notable as seen from Figure 3.11

Figure 3.12 Statistical analysis of healing rate of burn wounds at day 23 and day 30 after creating burns.Values are represented as mean ± SD

50

Table 3.2 Descriptive qualitative assessment for the healing process in the burn model

Group Time Epidermal necrosis Epidermal losses Epidermal regeneration

Control

Day + + -

Day 15 + + -

Day 23 + + -

Day 30 ++ ++ +

AgNPs

Day + + -

Day 15 ++ ++ -

Day 23 + + +

Day 30 - - +++

CM

Day + + -

Day 15 ++ ++ -

Day 23 + + +

Day 30 - - +++

MSC

Day + + -

Day 15 ++ ++ -

Day 23 ++ ++ +

Day 30 - - ++

Culture medium

Day + + -

Day 15 + + -

Day 23 + + +

Day 30 - - ++

PBS

Day + + -

Day 15 + + -

Day 23 - - +

Day 30 - - ++

51

Another noteworthy point is that the MSCs group presented an uneven speed of wound healing, in which one mouse healed faster, completely closed wound after 30 days with a normal scar Whereas the remaining one had a slow healing rate, the wound closure was only 50% after 30 days This might a limitation of the direct injection of stem cells because of low cell survivability in the host after injection [51] (Figure 3.13)

Figure 3.13 Uneven healing rate in the MSC group

3.3.2 Excisional model

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Figure 3.14 Statistical analysis of healing rate of excisional wounds with different treatments

53

Figure 3.15 The healing rate of excisional wounds All values are expressed as the mean ± SD p ≤ 0.05 compared with groups

On day 3, the wounds in the AgNPs-treated groups were significantly closed, with approximately 47% Followed by combined use group and control group, with around 41% and 40%, respectively On day 7, the healing rate of AgNPs and combined use groups were nearly equal, with approximately 80%, and the control group with 77% Meanwhile, CM and MSCs displayed similar healing ability and were slower than the other groups in early-stage and subsequently accelerated in the later stage of the healing process The wound closure rates in both groups were about 35% and 65% on day and day 7, whereas completed 96% wound area in day 10 (Figure 3.15)

Discussion

a) AgNPs

54

In many cases, the level of bacteria that suppressed the healing process but did not express the standard clinical sign of infection has been called “critical colonization” At this level, burn wounds have become infected and virtually hard to heal [46] On the other hand, removing skin in the excisional wounds (open injuries) facilitated bacteria to penetrate the wound The risk of wound infection after trauma ranges from to 17.5% [16], [19]

In this study, AgNPs displayed the most remarkable healing effect among groups in both wound models In the burn wound model, the presence of pus, redness in the wound site, which was the normal inflammatory response seen by the naked eyes, was not observed due to the covered burnt skin However, it could be seen the eschar of wounds in the AgNPs group had completely peeled off after 15 days This demonstrated that epidermal cells at the margin of the wound had proliferated and fibroblast cells around the wound site had migrated and proliferated into the wound area That means the inflammatory phase was promoted and the re-epithelialization phase had been started earlier in the AgNPs than other groups Similar observation was seen on the excisional wound model, re-epithelialization phase demonstrated the significant healing rate in the early stage (i.e day and day 7)

55

maximum from day to day 17, whereas that of the AgNPs-treated group was reached a peak on day 12 and then significantly decreased Surprisingly, E coli was still detected in the control and SSD-treated group and was not markedly found in the AgNPs-treated group throughout the healing process [74]

Also, the enhanced and excessive production of pro-inflammatory cytokines could tend to prolong the inflammatory phase [16] It is suggested that apart from preventing infection, AgNPs might display anti-inflammatory activity by regulating the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in the inflammatory phase Tian el at., 2007 reported that during the process of healing, level of cytokines including IL-6, TGF-β1, IL-10, VEGF, and IFN-γ in the AgNPs-treated group were significantly different compared to the control group They found that level of IL-6 was maintained lower during the process IL-6 was pro-inflammatory cytokine, which considered as a pioneer of inflammation events after injury IL-6 enhanced inflammatory response through the activation of monocyte and macrophage Decreased level of IL-6 might result in fewer immune cells (neutrophils and macrophages) recruited to the wound site, then lower paracrine signal for cellular migration and proliferation, and synthesis of ECM Therefore, their findings confirmed AgNPs induced IL-6 reduction, leading to an accelerating inflammatory phase and overall healing process [7], [67] Additionally, AgNPs regulated pro-inflammatory and anti-inflammatory cytokines in the early stage of the healing process AgNPs suppressed IL-6 expression significantly at day and day 3, resulting in the reduction of prolonged inflammation, hence promoting wound healing [81]

b) CM, MSC and Culture medium

56

the later stage of the process, by 35% and 65% on day and day 7, and completed 96% wound area in day 10

Fibroblasts located surround the wound migrate and proliferate to the wound area is one of the early events in the proliferation phase In this study, the effect of CM on the migration of fibroblasts has been proved in the scratch assay in vitro Therefore, it was likely that in this burn model, CM promoted the migration of fibroblasts, thus accelerated wound healing Besides, wounds of the CM groups nearly healed after 23 days, with approximately 90%, and completed after 30 days with normal scars, suggesting CM affected wound contraction and remodeling Several studies reported a variety of cytokines and growth factors found in CM that involved these stages Umbilical cord CM (UC CM) increased fibroblast migration and proliferation and changed the phenotypic characteristics of fibroblast, including a decreased ratio of TGF-β1/TGF-β3 and an increased ratio of MMP/TIMP [38] Human bone marrow-derived MSC CM (BM CM) induces re-epithelialization, tissue formation, angiogenesis in a deep second-degree burn model in rat [6] Meanwhile, many crucial anti-aging cytokines and growth factors in hUCB MSC CM such as EGF, bFGF, TGF-β, PDGF, HGF in which these cytokines mostly acted to induce fibroblast migration and proliferation, forming of new blood vessels and aligning ECM They also reported growth differentiation factor (GDF-11) induced growth and production of ECM protein involving Collagen type I, III, elastin, and fibronectin in human dermal fibroblast [34]

In the case of MSCs, the MSCs-treated groups showed almost the same healing rate as the CM-treated group This result coincided with recent studies on its healing mechanisms that paracrine signaling is the main mechanism for the healing effects of MSCs [10], [27], [41] That means CM acts relatively similar mechanisms as MSCs on wound healing, primarily through autocrine and paracrine effects of cytokines and growth factors in the healing process

57

scratch assay, the Culture medium group accelerated artificial “wound” closure faster than the CM group However, in the burn wound model, CM revealed better healing properties than the Culture medium group This could be explained by secretome, which means trophic factors presented in these media Culture medium was serum-free optimized for cell expansion in vitro, including manipulated recombinant factors Therefore, it was understandable that the Culture medium group healed faster than other groups in vitro On the other hand, CM involved in various factors released from MSC, these are key elements that contributed to wound repair This proved that trophic factors secreted from MSCs were more natural and exhibited better wound healing properties than recombinant factors in Culture medium

c) The combined use of AgNPs and CM

The result from the burn model suggested the potential of using CM derived from hUCB MSCs for burn wound treatment besides MSCs-based therapy Both AgNPs and CM exhibited healing capacity for burn treatment Therefore, it was expected the synergic effect of the combined use of AgNPs and CM for wound repair

Interestingly, in cases of combined use and CM only groups, the combined-use group was seen accelerated healing rate, by 41% compared with 35% of the CM group in day The difference between healing rate of the combined use and CM group was more noticeable with 80% and 65% in day 7, respectively Due to the noticeable difference in the early stage, it is suggested that AgNPs promoted the inflammatory phase of the combined group via antimicrobial and anti-inflammatory activity, thereby enhance the proliferation, re-epithelialization and contraction phases For this reason, it might be the first signs of the synergic effects of using both treatments, although the mechanism of action (i.e which cytokines, growth factor are contained in CM, and the action of AgNPs and CM in each phases) needs to be studied further

58

59

CONCLUSIONS AND PERSPECTIVES

CONCLUSIONS

1 Citrated-coated AgNPs were synthesized by the chemical reduction method AgNPs exhibited the spherical shape with the size ranged from 17.5 to 42.5 nm AgNPs possess sterility, antimicrobial activity and mild cytotoxicity on mouse fibroblasts with the values of IC50 from 26.02 – 30 µg/ml

2 CM derived from human umbilical cord blood MSCs exhibited the effect on the migration of NIH 3T3 fibroblast cells in vitro

3 Both AgNPs and CM displayed the healing nature for wound treatment, in which AgNPs showed the most prominent influence among treatments in both two models Besides, the combined use of AgNPs and CM revealed as the promising candidate for wound repair on the excisional wound model

PERSPECTIVES

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