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

LIST OF TABLESPageTable 1.1: Topical antimicrobial agents for wound healing...7Table 1.2: Effect of AgNPs size on cytotoxicity...12 Table 3.1.. Over the past few decades, silver nanopart

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

NGUYEN THI THANH HOAI

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

Hanoi, 2020

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 theirenthusiastic 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) forthe wholehearted instruction and useful suggestion Besides, I am extremely grateful

to Dr Than Thi Trang Uyen (Vinmec Research Institute of Stem Cell and GeneTechnology, Vinmec Health Care System) for all her support

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

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

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

Hanoi, July 2020Student

Nguyen Thi Thanh Hoai

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS i

TABLE OF CONTENTS ii

LIST OF FIGURES iv

LIST OF TABLES v

LIST OF ABBREVIATIONS vi

INTRODUCTION 1

CHAPTER 1: OVERVIEW 3

1.1 Cutaneous wound and wound healing process 3

1.1.1 Cutaneous wound 3

1.1.2 The normal wound healing process 3

1.1.3 The two therapeutic targets in wound treatment 5

1.2 AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase 7

1.2.1 AgNPs as a topical antimicrobial agent 7

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

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

REFERENCES 60

LIST OF FIGURES

Page

Figure 1.1 Phases in wound healing 4

Figure 1.2 The types of wound treatment applied for different wound categories 6

Figure 1.3 Mechanism of antimicrobial action of AgNPs 9

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 4 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 4 media on the migration of fibroblast cells 45

Figure 3.10 The migration rate of fibroblast treated with 4 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

Figure 3.15 The healing rate of excisional wounds 53

LIST OF TABLES

PageTable 1.1: Topical antimicrobial agents for wound healing 7Table 1.2: Effect of AgNPs size on cytotoxicity 12

Table 3.1 Number and size of microbial colonies in each group 38

Table 3.2 Descriptive qualitative assessment for the healing process in the burnmodel 50

Extracellular matrixExtracellular vesiclesFetal bovine serumFace centered cubicFood and Drug AdministrationHuman umbilical cord blood-derived mesenchymal stem cellconditioned medium

Human umbilical cord blood-derived mesenchymal stem cellsInterleukin-1, Interleukin-6, Interleukin-8

Insulin-like growth factorKeratinocyte growth factorMesenchymal stem cellsOptical density

Platelet-derived growth factorReactive oxygen speciesStromal cell-derived factorSulforhodamine B

Transmission electron microscopyTransforming growth factor α, transforming growth factor βTrisodium citrate

Ultraviolet visible spectroscopyX-ray diffraction

Wounds are a silent burden on the healthcare system In 2018, Medicarebeneficiaries analyzed that around 8.2 million people who have at least one type ofwounds Wounds often classified into acute (traumatic, abrasions, surgical) andchronic wounds (diabetic foot ulcers (DFUs), leg ulcers, and pressure ulcers) based

on healing time The challenges of healing wounds are the increase of infection, ageand pathological background of the patient Hence, we need to come up with novelstrategies to solve these problems Over the past few decades, silver nanoparticles(AgNPs) attract rapt attention in wound treatment due to various featured naturessuch as the history of using silver, simple and effective synthesized methods, andabove all the outstanding antimicrobial activity These make AgNPs become one ofthe most widely used agents for preventing infection On the other hand,mesenchymal stem cells (MSCs) and products derived from MSCs, which appear asadvanced therapies, have recently been studied and applied in the field of medicine

In terms of wound healing, many studies suggest that paracrine signaling of MSCsrather than tissue differentiation and engraftment is a pivotal element for promotingwound healing That indicates the capacity to use conditioned medium (CM), which

is one of the products derived from MSCs for wound treatment CM contains avariety of cytokines, growth factors, chemokines that modulate the healing processthrough induction of re-epithelialization, angiogenesis, and remodeling Therefore,

we assume the synergistic effect of the combined use of AgNPs and CM, in whichAgNPs with antibacterial, anti-inflammatory activities support CM to promotewound healing Our target is chronic wounds that require advanced therapies fortreatment At the beginning of the research process, we aim to examine the healingeffect 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 inthis study, three objectives need to be fulfilled

(1) Synthesize and characterize properties of silver nanoparticles (AgNPs) includingphysicochemical properties, sterility, antimicrobial activity and cytotoxicity;

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

vitro;

(3) Initially evaluate the therapeutic effect of each treatment: AgNPs and CM and

the combined use of AgNPs and CM on the wound models in vivo.

$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 foradvanced 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 surgery [61] Another challenge for acute wounds is that prolonged healing can leadthe wounds to enter a chronic state (non-healing) [16] Therefore, novel concepts toprevent infection and promote the healing process are vital to managing wounds

post-1.1.2 The normal wound healing process

Wound healing is a dynamic process involving 4 phases – hemostasis, inflammation,proliferation, and remodeling, that overlap in time This process is regulated by acomplex system of mediators, which are responsible for cell-cell communication,

involving various cytokine, growth factors, and chemokines [9], [13], [53] (Figure 1.1).

Figure 1.1 Phases in wound healing [43].

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

In the inflammatory phase, neutrophils start to cleanse the injury area frommicroorganisms and foreign contaminants, and then phagocytosed by macrophages

or formed the eschars Chemokines, transforming growth factor β (TGF-β), andmonocyte chemoattractant protein 1 (MCP-1) are released that lead to theinfiltration of monocytes to the injury site that later transformed to macrophages.The monocyte and macrophages play a crucial role in inflammatory phase byreleasing various cytokine such as vascular endothelial growth factor (VEGF),colony-stimulating factor 1 (CSF-1), PDGF, transforming growth factor α (TGF-α),TGF-β, interleukin-1 (IL-1), etc that initiate the formation of granulation tissue.The proliferation phase started with re-epithelialization, in which several growthfactors including TGF-α, epidermal growth factor (EGF) and keratinocyte growthfactor (KGF) were released to stimulate the proliferation of epidermal cells at the

concert of extracellular matrix (ECM) molecules and growth factors, PDGF, TGF-βinduce 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 Thefibroblasts play an important role in synthesis, deposition, and remodeling of theECM Besides, the formation of new blood vessels, called angiogenesis, initiateswith 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 andreorganization of ECM Fibroblast and macrophage release several proteolyticenzymes called matrix metalloproteinases that degrade collagen type III of thegranulation 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 coordinatecellular processes These act to regulate a wide range of functions involving cellproliferation, cell differentiation, angiogenesis, wound healing, tissue modeling,immune cell activity through autocrine, paracrine, juxtacrine, or endocrinemechanisms [13] Hence, modulation of cytokines and growth factors can enhancethe healing process

1.1.3 The two therapeutic targets in wound treatment

Wound care depends on the wound type, along with the purpose of treatment.Superficial burns are normally needed primary care, which is cleaning the woundand applying antibiotics For deep partial-thickness and full-thickness burn,

common treatment are topical antimicrobial agents, and skin grafts in case of a

large-area wound For non-healing wounds, the purpose of care is more complex

including wound debridement, preventing injection, and avoiding the pressure at

the wound site The treatment approach for improving wound healing, was listed in

Figure 1.2 [54].

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

(adopted from [54])

For active wound care, several therapeutic strategies have been studying includingusing growth factor, chemokine, cell therapies, microRNA, and non-endogenousmolecules such as silver-based agents, synthetic chemical pirfenidone (PFD), etc.[54] For instance, a gel containing recombinant PDGF, which is a vital growthfactor throughout the healing process, called Becaplerim showed to enhance woundhealing in DFUs, was approved by Food and Drug Administration (FDA) for non-healingwound treatment [55] These approaches target different cellular processes

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 theinflammatory 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 tocreate 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 thereduction 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 butmore 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

Antiseptics Advantages: Broad spectrum of antimicrobial activity [58]

Disadvantages: toxic to host cellsHydrogen Use for wound irrigation and remove necrotic tissues [21]peroxide H2O2 was detected in normal healing, rapidly

(H2O2) decomposed to water and oxygen High concentration

cause cell damage through corrosion, formation ofoxygen gas, and lipid peroxidation

Povidone- A solution of 0.1 – 0.2% minimize cytotoxicity and rise [56]iodine the release of free iodine for antibacterial activity

Disadvantages: irritation, allergy, cytotoxic tovulnerable people (pregnant women, newborns, etc.)Chlohexidine Chlohexidine showed long-lasting residual ability, and [1]

active activity against positive and negative bacteria but poor activity against non-enveloped viruses and bacterial spores The mechanism

Gram-of its action is disrupting the cytoplasmic membrane

Alcohol Bactericidal action of an aqueous solution of 70% - [56]

92% alcohol is rapid, but short-time action, and cancause irritation and dryness

Nanoparticles: NPs exhibit the bactericidal effect with wide-spectrum [49]silver, gold, by the release of metal ions or generation of Reactive

zinc (NPs) Oxygen Species (ROS) The toxicity can be governed

by modulating several factors such as size, shape,concentration

Antibiotics: Advantages: the cytotoxic to host cells is less than [58]Bacitracin, antiseptic

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

Over the past few decades, silver nanoparticles (AgNPs) attract great interest due totheir well-known antimicrobial activity The mechanism of this action is not fullyunderstood yet, but it is suggested that this action is in relation with (1) AgNPsanchor to the cell wall of bacteria then penetrate it, altering the permeability of cellmembrane, (2) AgNPs penetration damage the bacterial organelles includingmitochondria, vacuoles, ribosomes, and denature protein, as well as DNA, (3) Theformation of free radicals and generation of ROS, (4) AgNPs can modulate thesignal transduction, dephosphorylate of peptides substrate on tyrosine residues,

resulting in inhibition of cell growth [14], [57], [76] (Figure 1.3).

Figure 1.3 Mechanism of antimicrobial action of AgNPs [14]

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 isanother 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 bacteriaand other foreign particles by the production of pro-inflammatory cytokines Innormal healing, both the pro-inflammatory and anti-inflammatory are present.However, if these responses take place inappropriately, a prolonged inflammatoryphase can lead to non-healing wounds [16] Scientific studies suggest that AgNPspossess anti-inflammatory activity besides the well-known antimicrobial activity.Tian et al (2007), who first found evidence in the anti-inflammatory activity ofAgNPs, compared the healing process between the mice treated with AgNPs andthose treated with amoxicillin and metronidazole, two widely used antibiotics Theresult showed that the AgNPs-treated group healed faster than the antibiotic-treatedgroup, suggesting other influence of AgNPs besides antimicrobial action Thefindings showed the expression level of IL-6 (pro-inflammatory cytokines) waslower in the AgNPs-treated group whereas IL-10 (anti-inflammatory cytokines),VEGF (angiogenic cytokine), TFN-γ (cytokines in remodeling phase) were stayedhigher than the control group The overall reduction in inflammation might bepredicted, and the decrease of neutrophils in the wound area confirms it [67] Theefficiency of AgNPs in inflammatory reduction without toxic effects was found in apostoperative peritoneal adhesion model [77]

On another paper, this team reported AgNPs accelerated the rate of wound closurethrough the migration and proliferation of keratinocytes in the stage of re-epithelialization AgNPs, at the same time, inhibited the fibroblast proliferation, notdue to their toxic effect The enhanced expression of α-SMA (myofibroblast’smarker), suggested AgNPs stimulate the differentiation of fibroblast intomyofibroblast in the healing process [40] Other finding showed nanocrystallinesilver solution exhibited the anti-inflammatory activity with a pH of 9 [48]

Besides, AgNPs showed an influence on improving tensile properties of healedwounds, modulating collagen deposition in the wound healing process [36] Takentogether, not only do AgNPs display broad antimicrobial spectrum, but also exhibitanti-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 physicochemicalproperties AgNPs are aimed to use for wound repair, hence, the cytotoxicity (ability

to destroy cells), genotoxicity (property that damage genomic information) ofAgNPs 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).

Figure 1.4 Factors impacted their cytotoxicity

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 Size Cell type Findings Ref method (nm)

Chemical 5, 20, Human cells: Smallerparticles were [39]reduction method 50 A549, easier to enter cells than

(PVP coated) HepG2, SGC- larger ones, causing higher

7901, MCF-7 toxicityUnknown method 10, 50, Human liver Size-dependent toxicity [45]

activationUnknown method 15, 30, Alveolar Increase of ROS level when [11](Hydrocarbon- 55 macrophages cells treated with AgNPs 15

through oxidative stress

1.2.3.2 Effect of capping agents

To prevent aggregation, capping agents stabilize the nanoparticles by producingelectrostatic repulsions between particles Because they act in preservation of thesurface chemistry by upholding shape and reducing Ag+, they may affect thecytotoxicity 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 coated AgNPs (10, 50, 75 nm) and uncoated AgNPs (20, 40, 60, 80 nm) wereexamined their cytotoxicity to J774A.1 macrophage and HT29 epithelial cells Theresults showed that uncoated AgNPs suppressed inflammatory responses andpromoted oxidative stress in testing cells, so were higher toxic than coated AgNPs.Besides, PVP-coated AgNPs displayed higher toxic than citrate-coated ones Thestudies 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-coated (10 nm), and uncoated AgNPs (50 nm)against BEAS-2B human lung cells They found that cytotoxicity was onlypresented in the 10 nm AgNPs groups and independent of the coating agent.Moreover, although citrate- and PVP-coated AgNPs showed differentagglomeration patterns, there was not confirmed the difference in the cellular uptakeand intracellular localization between them [22] Xiaoquing Guo et al (2016)studied the impact of size and coating on cytotoxicity and genotoxicity of 6 AgNPstypes including PVP- and citrate-coated (20, 50, 100 nm) and silver nitrate inL5178Y and TK6 cells The results confirmed that AgNPs were less cytotoxic andgenotoxic 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]

(PVP)-Therefore, it is hard to confirm PVP-coated AgNPs have greater cytotoxicity thancitrate-coated counterparts due to a variety of factors such as size, synthesizedmethod, agglomeration, etc However, sodium citrate and citric acid were allowed

by the FDA for use as a preservative in many foods, and drug for preventing gout orkidney stones [FDA data] Hence, sodium citrate was used as a capping agent in therecent 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 cangenerate several cell types relying on their types of stem cell [44] SCs are foundboth in embryos and adult cells Based on the differentiation potency, SCs areclassified to be totipotent, pluripotent, multipotent, oligopotent, and unipotent.Totipotent SCs can divide and generate cells of the whole organism Zygote, thestructure formed after fertilization of a sperm and an egg, is representative of thetotipotent cell Pluripotent SCs (PSCs) are capable of differentiation into all germlayers’ cells The examples are embryonic stem cells (ESCs) originated from theinner cell mass of preimplantation embryos, and induced pluripotent stem cells(iPSCs) which are derived from somatic tissues including skin and blood cells, thenthey have been reprogrammed back into an embryonic-like state Multipotent cellsmay be adult cells including hematopoietic stem cells, mesenchymal stem cells(MSCs), and neural SC

1.3.1.2 Mesenchymal stem cells (MSCs)

Human MSCs are multipotent, adult stem cells International Society for CellularTherapy proposed the minimum criteria for MSCs identification, in which MSCsare stem cells exhibiting 3 properties (1) adherence to plastic, (2) specific surfacemarkers expression including CD73, CD90, CD105 and lack the expression ofCD14, CD34, CD45 and human leucocyte antigen-DR (HLA-DR), (3) ability to

differentiate to osteoblast, adipocyte, chondrocyte in vitro [17].

Human MSCs first isolated from bone marrow (BM), then they have been isolatedfrom other tissues such as adipose tissue, amniotic fluid and membrane, peripheralblood, 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

The initial idea for investigating the healing potential of MSCs stemmed from theability to differentiate to new tissues and replace injured cells However, otherresearch suggested MSCs have enabled to repair damaged cells, improve theirfunction without engraftment or differentiation The main benefits of MSCs havecome from their secretome through autocrine and paracrine signals [8], [10], [41].The secretome is a product derived from MSCs, described as the variety ofmolecules secreted to the extracellular space including soluble proteins, free nucleicacid, lipids, and extracellular vesicles (EVs) [70] Bioactive factors secreted by

MSCs are cytokines, growth factors, chemokines that can be divided into manyfamilies: 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 viatransferring 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 advantagesbesides stem-cell therapies, (1) using secretome has more benefits on safety issuesthan MSC transplantation; (2) MSC-derived secretome can be stored for a long timewithout the addition of toxic cryopreservative agents also decrease in quantity; (3)using MSC-derived secretome (CM and EVs) is more practical for therapeuticapplication because the cell collection is not needed [70]

For these reasons, cell-free therapies (MSC-derived CM and EVs) are viable optionsalong 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

Scientific evidences indicate that paracrine signaling of MSCs rather than tissuedifferentiation may be a key factor to accelerate wound healing That opens up thepotential to use CM for healing wounds In 2007, Liwen Chen et al firstinvestigated the benefit of BM MSCs in wound treatment They found the BMMSCs-treated group showed accelerated wound closure, re-epithelialization, andangiogenesis The high levels of VEGF, angiopoietin-1 presented in BM CM,suggesting BM MSC enhanced wound healing through differentiation and release ofpro-angiogenesis compound [78] To examine the effects of soluble factors in CM,

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 MSCcould promote wound healing Their data showed that BM MSCs releaseddifferential 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 andosteoprotegerin [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 migrationand 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 promotesHUVEC proliferation, regeneration of sebaceous glands, angiogenesis in aradiation-induced wound in rats [64]

The release of cytokines and growth factors from MSCs is depended on the sourcesand culture conditions [70] Human umbilical cord blood-derived MSCs (hUCBMSCs) have biological advantages such as anti-inflammatory activity throughreducing 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 hasnot investigated in Vietnam yet Therefore, it is essential to examine the potential ofhUCB CM in the terms of healing injuries

1.4 Combined using of silver nanoparticles and bio-factors for wound healing

AgNPs have been used in combination with other materials for wound treatment.The common strategy is to use the combination of AgNPs-antibiotic against drug-resistant bacteria De’ Souza et al studied the combination of 19 antibiotics and thesolution of silver-water, in which AgNPs size was 15 nm In this study, thecombination of amoxicillin or clindamycin with silver–water dispersion exhibited

an additive effect on B subtilis, S aureus, S flexneri, and S typhi, however,

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]

Herein, we suggest another strategy for a combination of AgNPs with active woundcare (CM) AgNPs play the role of a topical antimicrobial agent, preventing theinfection of microorganisms At the same time, CM derived from MSCs modulatesmediators (cytokines, growth factors, chemokines) involved in wound healing Thecombination of the two treatments, which use the AgNPs (basic wound care) tosupport the CM (active wound care), is expected to create a synergistic effect asdouble therapies to enhance the healing process

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, antimicrobialactivity, and cytotoxicity On the other hand, CM was harvested and evaluated theeffect on the migration of fibroblast cells Finally, AgNPs and CM were aimed to

treat wounds in mice (Figure 2.1).

Figure 2.1 Overall experimental design of the study

2.2 Preparation of AgNPs

2.2.1 Synthesis of AgNPs

AgNPs were synthesized using a chemical reduction method where sodiumborohydride (NaBH4) as a reductant agent and trisodium citrate (TSC) as astabilizing agent

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 1 ml of AgNO3 0.1 mM wasadded to the beaker

Step 3: 1 ml of TSC 0.05 mM was added to the beaker and stirred in 5 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

Step 6: Synthesized AgNPs solution was stored at 4 ºC for future use

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 ofthe matter, which undergoes excitation and de-excitation, leading to the formation

of distinct spectra UV-Vis technique can give qualitative and quantitative data ofgiven compound [20]

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

Transmission electron microscopy (TEM)

TEM provides data of the chemical information and morphology of nanomaterials.The incident light is transmitted via a thin foil specimen is transformed into

elastically or inelastically scattered electrons when the electron beam interacts withthe specimen.

TEM analysis (TEM, JEOL-JEM 1010) was performed at National Institute ofHygiene 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-raysstrike on crystal, resulting in the formation of many diffraction patterns that exhibitthe physicochemical properties of the crystal structures

Synthesized AgNPs were measured by X-ray spectroscopy (Rigaku Miniflex 600) atVNU 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 ofwhether microorganisms present in the solution Then, the AgNPs solution wastested 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), 1 ml Pipette and tips

Procedure:

Agar plate preparation:

20 g Soyabean Casein Digest Agar was dissolved in 500 ml of distilled water Thiswas sterilized by autoclaving at 121ºC for 2 hours Subsequently, the sterile solutionwas poured into sterile glass petri plates, and waited until frozen This step wasperformed in the biological safety cabinet.

Sterility test of AgNPs:

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

1 ml AgNPs solutions of 30 and 10 µg/ml were added alternately into 3 agar platesfor each solution Those plates were shaken well to make sure that the solutioncovers the surface of the plate evenly 3 plates were not treated AgNPs solution andkept as the control

Finally, those plates were wrapped and placed in the microbiological incubator Theplates were examined the presence of microorganism colonies and took photographafter 5 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 3 groups: 10 µg/ml AgNPs-treated group,untreated control, agar plate control (ensure there were no errors in the process ofplate preparation) 3 groups were marked names: 10 µg/ml AgNPs, Control (-), andControl (+), respectively

6 plates of 2 groups (Control (-) and 10 µg/ml AgNPs) were opened the cover for 5minutes to allow bacteria in the air fall on agar plates Group of 10 µg/ml AgNPswere added 1 ml of 10 µg/ml AgNPs into 3 agar plates Those plates were shakenwell to make sure that the solution covers the surface of the plate evenly 3 plateswere not treated AgNPs solution and kept as control (+) Finally, those plates were

wrapped and placed in the microbiological incubator The plates were examined thepresence of microorganism and took photograph after 5 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 testingdrug cytotoxicity SRB is a bright pink aminoxanthene dye having two sulfonicgroups that can bind to basic amino acid residues of protein in cells fixed intrichloroacetic acid under mildly acidic conditions The amount of SRB extractedfrom 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 CellBiology, Faculty of Biology, VNU University of Science

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

Incubation with AgNPs

20 µl AgNPs solution were added into each well with 6 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, andincubated in the incubator for 48h at 37 °C, 5% CO2

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

After 48 h, the plate was taken out of the culture room Supernatants were aspiratedout and cells were fixed by adding 50 µl of TCA 50% and incubated at 4 °C for 2 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 roomtemperature Anxcessive amount of SRB was removed by the cleaning solution ofacetic acid 1%, repeated 5 times Then dye was dissolved in Tris-based andabsorbance at 540 nm wavelength was recorded using a Microplate reader

Analysis

Cell viability ( ) (%) was calculated from values of OD, thereby the potentialtoxicity of AgNPs against fibroblast cells was assessed Cell viability ( ) was writtenas

: The average value of OD of solvent-treated wells

: The average value of OD of AgNPs-treated wells

When = 50%, AgNPs exhibited the toxic effect causing 50% of cell death Theconcentration of the sample at which the value was 50%, called Inhibitoryconcentration IC50 This value is used to evaluate the toxicity of AgNPs to NIH 3T3

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 byVinmec Research Institute of Stem Cell and Gene Technology, Vinmec Health CareSystem, 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 humidifiedatmosphere containing 5% CO2 Culture medium mentioned here was completed,with supplement

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

Step 3: After 2 days, the medium was collected, and then cellular debris in themedium were removed by 0.22 µm filter After filtration, the medium wasconsidered as conditioned medium (CM)

Step 4: CM was stored at 4ºC and not be used after more than 2 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 4 tissue culturedishes (diameter 40 mm), 5x104 cells/ dish, and incubated overnight at 37 ºC, 5%

CO2 to permit cell adhesion to the dish surface

At the same time, CM was generated from MSC culture with and without

supplement as described in 2.2.1.

Figure 2.3 Examination of 4 media on NIH 3T3 cells migration.

Image capture and analysis

The changes of wounds were captured each hour with an inverted microscope(ZEISS Axio) The images were transferred into equal size of 2048x1536 pixels andanalyzed using Image-J software by measuring the width of scratch in the pixel at 3points including the top, middle, and bottom along their verticals, and then averaged

Wound closure (%) was calculated by the following formula.

Wound closure (W0 -Wt) x100

W0

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 3points 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 2 wound models: burn and excisional models

2.4.1 Deep partial-thickness burn wound model

Preparation of mice

10 Swiss male mice (50 – 60 g, 8 – 10 weeks old) were obtained from the NationalInstitute of Hygiene And Epidemiology Animal experiments conducted in thelaboratory following the regulation on animal use for scientific purpose Mice weresupplied with food and water, in the condition of 12h light/dark cycle The micewere 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 wereremoved completely with the hair removal cream An alcohol swab was used todisinfect 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) washeated on the fire lamp in 5 minutes and placed on the skin for 10 s Mice wererandomly divided into 5 groups, treated immediately after creating the wounds

Group 1 (Control): non-treated mice

Group 2 (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 5 days [74]

Group 3 (CM): mice were injected intradermally with CM (200 µl) in the wound

bed daily and for the first 3 days [38]

Group 4 (MSCs): mice were injected intradermally with MSCs (1x106 cells/200 µl

PBS) into the wound bed daily and for the first 3 days [31]

Group 5 (Culture medium): mice were injected intradermally with culture

medium (200 µl) in the wound bed daily and for the first 3 days

Group 6 (PBS): mice were injected intradermally with PBS - buffer solution (200

µl) in the wound bed daily and for the first 3 days

Wounds were observed and photographed daily