Study on the co-operation ability of mesenchymal stem cells and nanobiomembrane for skin wound-healing

Key words: Electrospinning, nanobiomembrane, gelatin, Human Mesenchymal Stem Cells (MSCs), wound healing, fibers... The process of wound healing on the skin.[r]

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

VU THI NHUNG

STUDY ON THE CO-OPERATION ABILITY OF MESENCHYMAL STEM

CELLS AND NANOBIOMEMBRANE FOR SKIN WOUND-HEALING

MASTER’S THESIS

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY

VU THI NHUNG

STUDY ON THE CO-OPERATION ABILITY OF MESENCHYMAL STEM

CELLS AND NANOBIOMEMBRANE FOR SKIN WOUND-HEALING

MAJOR: NANOTECHNOLOGY CODE: 8440140.11QTD

RESEARCH SUPERVISORS:

Assoc Prof NGUYEN HOANG NAM

Assoc Prof HOANG THI MY NHUNG

ACKNOWLEDGEMENT

First of all, I would like to express my sincere appreciation to my supervisor, Assoc Prof Nguyen Hoang Nam and Assoc Prof Hoang Thi My Nhung who has guided and created favorable conditions and regularly encouraged me to complete this thesis Thank you for all your thorough and supportive instructions, your courtesy and your enthusiasm Without your dedicated guidance, I absolutely have not conducted this research well

Secondly, I would like to express my great thankfulness to Master’s Nanotechnology for their wonderful supports, especially Prof Yoji Shibutani, Prof.Dr.Sci Nguyen Hoang Luong, Dr Dinh Van An, Dr Nguyen Tien Thanh, Dr Bui Nguyen Quoc Trinh and Ms Nguyen Thi Huong Their encouragement and assistance has facilitated me a lot during years studying in the Vietnam-Japan University I also want to give my special thanks to all lecturers and staffs at the Osaka University for their warmly welcome and supports during in my internship in Japan

II

CONTENTS

ACKNOWLEDGEMENT I LIST OF TABLES III LIST OF FIGURES IV NOMENCLATURES AND ABBREVIATIONS IV ABSTRACT VII

CHAPTER 1: INTRODUCTION

1.1.Overview

1.1.1 The process of wound healing on the skin

1.1.2 Membrane fabrication methods for wound healing

1.1.3 Materials used for fabricating the membrane

1.1.4 Mesenchymal stem cells 12

1.1.5 The combination between gelatin bionanomembrane and MSCs in the introduction of wound healing 14

1.2.Research objectives 15

CHAPTER 2: MATERIALS AND METHODS 16

2.1 Materials 16

2.1.1 Chemical reagents 16

2.1.2 Equipment 17

2.1.3 Tools and consumable supplies 18

2.2 Experiment section 18

2.2.1 Fabrication of gelatin nanofibers 18

2.2.2 Human Mesenchymal Stem Cell (hMSC) studies 23

2.2.3. The combination ability of bionanomembrane and cells 28

CHAPTER 3: RESULTS AND DISCUSSION 33

3.1 Results of the fabrication nanofiber studies 33

3.1.1 FTIR results 33

3.1.2 SEM Images 36

3.2 The results of hMSCs studies 38

3.2.1 hMSCs culture 38

3.2.2 The determination of proteins in hMSCs 41

3.3 Results of the combination between bionanomembranes and cells 44

3.3.1 Nanobiomembrane sterilization 44

3.3.2 The effect of nanobiomembrane on the cell growth 47

3.3.3 The effect of nanobiomembrane on cell viability 49

CHAPTER 4: CONCLUSION 52

III

LIST OF TABLES

Table 1.1 Polymers used for nanofibers to support stem cells 12

Table 2.1 Chemicals used in the laboratory 16

Table 2.2 Equipment used in the laboratory 17

Table 2.3 Tools and consumable supplies used in the laboratory 18

Table 2.4 Parameters relate to the membrane fabrication 20

Table 2.5 Setting the data of electrospinning for fabricated nanobiomembrane 22

Table 3.1 FTIR spectra characteristics of gelatin and acid acetic 35

Table 3.2 The calculated proportion of two kind of fibers 37

Table 3.3 The Calculation of cell density average at t=24h 41

Table 3.4 Adhesion ratio (α), specific growth rate (µ) and doubling time (td) 41

Table 3.5 OD540 of standard BSA at varied concentration 42

Table 3.6 Calculation of total protein concentration in sample base on standard curve of BSA protein 43

IV

LIST OF FIGURES

Figure 1.1 Images for a process of wound healing on the skin [13]

Figure 1.2 Wound healing diagram by ECM Synthesis [3]

Figure 1.3 Electrospinning method creates structure similar to ECM [26]

Figure 1.4 The phenomena of electrospraying and electrospinning occur when the electrostatic repulsive forces overcome the surface tension of the liquid [1]

Figure 1.5 A schematic view of the electrospinning (a) downward electrospinning setup; (b) Upward electrospinning setup; (c) Horizontal electrospinning setup [1]

Figure 1.6 Materials used for fabricating the membrane [18] 10

Figure 1.7 Organic materials for manufacturing the membrane [11] 10

Figure 1.8 The basic characteristics for the application potential of MSCs [24] 13

Figure 2.1 Fabrication of nanobiomembrane by electrospinning method 19

Figure 2.2 Image for the important parts of the electrospinning equipment 19

Figure 2.3 The behavior of the electrospun jet divided into three main phases: Taylor cone formation, straight jet ejection, and whipping jet formation 20

Figure 2.4 The diagram of gelatin nanobiomembrane fabrication 22

Figure 2.5 Observed five positions in each well of 8-well rectangular dish; Culture area: 10.5 cm2 (3.76 cm × 2.79 cm); Captured image area: 0.021962 cm2 24

Figure 2.6 Overall the experimental process for Western blotting 27

Figure 2.7 Experimental procedure in-vitro research 28

Figure 2.8 In-vitro experiment model 31

Figure 3.1 FTIR spectra for acid acetic, and gelatin solution with 10%, 15%, 20%, and 25% concentration 33

Figure 3.2 FTIR spectra for powder gelatin, acid acetic, gelatin solution 25%, and gelatin fibers 25% 34

Figure 3.4 The percentage of large fiber, small fiber and ratio two kind of fiber 37

Figure 3.5 Nanobiomembrane with 25 % gelatin were fabricated 38

Figure 3.6 Representative images of hMSC cells at t= 24, 72 and 120 after cell seeding in different five positions Scale bar: 200 µm 40

Figure 3.7 Growth curves for hMSC cells in the experiment process 41

Figure 3.8 Standard curve of BSA protein concentration 43

Figure 3.9 The images of COL I and β-actin band using ChemiDoc MP imaging system (a) β-actin; (b) Col-1; (c) Maker 44

Figure 3.10 Results of band ratio between β-actin, and COL I 44

Figure 3.11 Examination the membrane sterilization by immerse in ethanol 70%, and UV lamp after 24 h incubation (a) untreated sample; (b) immerse in ethanol 70% within min; UV light at 135 45

V

VI

NOMENCLATURES AND ABBREVIATIONS

MSC Mesenchymal Stem Cells

ISCT International Society for Cellular Therapy

PBS Phosphate - buffered saline

FBS Fetal Bovine Serum

ECM Extracellular matrix

PDGF Platelet-derived growth factor

TGF-β Transforming growth factor-β

FGF Fibroblast growth factor

EGF Endothelial growth factor

DC Direct current

DMSO Dimethyl Sulfoxide

DF Dilution factor

USSCs Unrestricted somatic stem cells

PMSCs Plasma derived mesenchymal cells

ASCs Adipose-derived mesenchymal cells

NSCs Neural stem cells

PCL Poly(caprolactone)

PLGA Poly(lactic-co-glycolic acid)

PEG-PCL Poly(ethyleneglycol)- poly(caprolactone)

PLLA Poly-L-lactic acid

Ep Epidermis

D Dermis

VII ABSTRACT

The main purpose of this research is fabricating gelatin nanofibers and investigating the application of nanofibers for skin regeneration First, the gelatin nanofibers were fabricated by using the electrospinning method Then, the morphology and size of nanofibers were examined by the scanning electron microscope (SEM) SEM images results show that the connection between large and small size fibers varies significantly according to concentration of gelatin, which help to choose the best to apply for in-vitro research Second, study of hMSCs culture show that it could be used for this study while determining COLI is a take advantage for wound healing Finally, gelatin nanofibers were applied as scaffolding for three different cell types Hacat, Fibroblast, hMSCs The initial investigation demonstrated that the fabricated gelatin samples did not killed cells In particular, the survival rate was greater than 90%, and the total number of cells after 48 hours of culture was approximately greater than 50% for all samples

1

CHAPTER 1: INTRODUCTION

1.1. Overview

1.1.1. The process of wound healing on the skin

The largest organ in the human body is the skin, which has direct contact with the external environment The skin plays integral important role in the human body due to many reasons First, it has the main function that ensures homeostasis and protects the human body from aggressors and pathogens in the outside environment Second, it has taken part in many main processes in the body such as water balance and temperature regulation, signal perception, hormones, neuropeptides and cytokine production and activation, etc [25] Thus, it is easy to be damaged than any other parts In particular, burn injuries are extremely severe and difficult to treat because the area of damaged skin is very large, and the treatment time may be so long Three main parts make skin structure including the epidermis, the dermis, and the hypodermis [22] A large number of cells such as epidermal, stromal, endothelial, and neuronal cells and the complex structure of extracellular matrix (ECM) lie on below epidermis This system is an extremely important factor in tissue regenerating for wound healing on the skin after the burn injury The percentage of successful skin healing for burn wound has increased significantly when using various skin substitutes

2

Figure 1.1 Images for a process of wound healing on the skin [13]  Stage 1: Hemostatic

After injuring the hemostatic occurs immediately First, thrombin is formed and activates platelets, when platelets are activated which leads to the changes following - Active the coagulation layers As results, the forming of a fibrin clots, above of that protects the wound as well as the bottom acts like a scaffold for the attached cells

- the complement system began to worked

- Platelet aggregation and degranulation: The cells release a variety of growth factors and substances to start for wound healing on the human skin such as cytokines, growth factors, and vasoactive substances from the platelet α-granules, such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), endothelial growth factor (EGF), derived angiogenesis factor, serotonin, bradykinin, platelet-activating factor, thromboxane A2, platelet factor IV, prostaglandins, and histamine [32]

 Stage 2: Inflammation phase

3

neutral protease and collagenase as well as support to eliminate the damaged components of the extracellular matrix (ECM) [30] Finally, activated mononuclear cells have a macrophage phenotype and support host protection

 Stage 3: Proliferation

In this phase, the presence of two basic processes: (1) The formation of the ECM

(2) The beginning of angiogenesis

The main cells that proliferate and develop at this stage are fibroblasts and endothelial cells Therefore, a large amount of growth factors and cytokines are released from macrophages, platelets and mesenchymal cells or have been stored in fibrin clots Moreover, growth factors also induce activation and proliferation of fibroblasts [34] In the first 2-3 days after injury, fibroblast activity is primarily associated with migration and proliferation After this time, fibroblasts release collagen and compounds in response to the growth factors released by macrophages The combination of collagen and fibronectin forms the new ECM, which plays an integral important role in the growth of granulation tissue and eventually the wound will heal [31] The next stage is the beginning of angiogenesis In this period is accompanied by proliferation of fibroblasts and allows nutrients and healing elements to enter the wound space It is also necessary for the development of granulation tissue The FGF main growth factor controls angiogenesis; it is released by endothelial cells and damaged macrophages Besides, vascular endothelial growth factors (VEGF) are released by keratinocytes and macrophage cells [31]

This stage usually extends weeks after injury and up to years to complete tissue engineering [10] The newly formed collagen fibers in the wound are randomly arranged and disorganized The Remodeling of collagen fibers into the ECM structure increases the tensile strength of scar tissue, although many studies show that this never exceeds 80% of the strength of intact skin

4 Stage 4: Remodeling

This process takes place the balance between collagen synthesis and degradation of ECM [21] There are two types of injuries are chronic and acute Wounds are considered as chronic if the time lasts longer than months With acute wounds, there is a balance between production and degradation of molecules like collagen, while in chronic wounds, this balance is lost as well as the process of degradation plays a major role In addition, protease concentrations were high in chronic, and a number of growth factors and cytokines were lower than acute [8] As a result, prolonged protein degradation at high concentrations can lead to a significant reduction of growth factors, resulting in wounds during inflammation for too long [15]

1.1.2. Membrane fabrication methods for wound healing

5

Figure 1.2 Wound healing diagram by ECM Synthesis [3]

6

Figure 1.3 Electrospinning method creates structure similar to ECM [26] Fabrication of nanofibers by electrospinning technique

 History and development of electrospinning

The electrospinning was first patented by Morton and Cooley in 1902, which including electrospraying and electrospinning In that, electrospinning is called a direct extension of electrospraying Both these inventions used the electrostatic force for disperse liquids [1] Formhals invented the electrostatic apparatus in 1934, and he received more than 30 patent for this research [19]

7

Consequently, more than 200 polymers have been applied, in that, less than 20 pure biomolecules have been successfully used for biomedical applications, over 100 years after recommending electrospinning Many fields have been put on to apply electrospun materials such as filtration, solar systems, tissue engineering, drug delivery, wound dressing, electronics, etc [19]

 Basic theory of the electrospinning

It is remarkable that fibers formation in the electrospinning is continuous stretching, while the production of electrospraying was small droplets The basic principle was understood that there are two different forces effect on the spherical droplet of a liquid, which including the electrostatic force and the surface tension, as shown in Fig 1.4 The first one is a repulsive force in order to deform the droplet shape, while other force tries to keep the spherical droplet At equilibrium, the below equation expresses two forces balance each other

R R Q s   8 2  (Eq 1.1) where

Q is the charge located on the droplet surface R is the droplet radius,

0 is the vacuum permeability

and σs is the surface tension coefficient

8

Figure 1.4 The phenomena of electrospraying and electrospinning occur when the electrostatic repulsive forces overcome the surface tension of the liquid [1]

9

Also, Alghoraibi and Alomari in 2018 claimed that there were three different ways to set up a schematic of electrospinning: downward electrospinning setup; upward electrospinning setup; horizontal electrospinning setup Furthermore, it is noticeable that the orientation of fibers and the number of beads was given the basic differences between the upward and the downward electrospinning setups While the fibers in the upward electrospinning setup will collect a remarkable uniform, the downward method orients randomly In addition, the beads in the downward setup consider as higher than the upward process [16] As a consequence, while the upward setup is an optimistic option for the large scale as the industrial production because it is easy to produce massively the homogeneous nanofibers By contrast, the downward electrospinning is the most suitable for a small scale as laboratory or center research, owing to easily monitoring and simply optimizing

 The application potential of electrospinning method in wound healing The electrospinning method has allowed for design and synthesis of the new membrane fibers with properties that have advantages for tissue engineering applications For example, the structural membrane as well as their hydrophilic is easier to change, same as electrical conductivity, and antibacterial activity, etc Moreover, many authors have applied nanofiber scaffolds by using high voltage to make fibers into many fields such as engineer bone, vascular, neural, and cartilage tissue [1, 2] Meanwhile, thanks to the feature characteristic is simple, fast, cost-effective, besides its easy adaptability and versatility in spinning a wide variety of polymeric fibers, as well as its consistency in producing and its consistency in producing multifunctional nanofibers from various polymers However, the main disadvantage of this process is considered as the low productivity

1.1.3. Materials used for fabricating the membrane

10

properties including origin, structural characteristics, chemical and physical properties These properties are considered carefully when selecting materials for applications like the selectivity and permeability of the material; chemical resistivity; mechanical strength; thermal resistance; economic and technical feasibility [18]

Figure 1.6 Materials used for fabricating the membrane [18]

Figure 1.7 Organic materials for manufacturing the membrane [11]

11

nanobiostructures that allow cell binding, movement and proliferation, to promote tissue regeneration and wound healing

Furthermore, the properties of conventional, synthetic polymer fibers, fibers made from natural polymers, especially proteins with biocompatibility, biological activity and biodegradability For example, keratin, collagen, silk, elastin, zein and soy are most commonly used in fiber fabrication as shown in Fig 1.7 In particular, there are two types of noteworthy are collagen and gelatin

First, collagen is a fibrous protein that plays an integral important role for a major component of ECM A great deal of collagen found in the body can be classified as Type I, II or III, while up to 29 different types of collagen have been identified Among them, type I collagen is the most common using in the development biomaterials [7] The collagen fibers have allowed the creation of biological tissue engineering scaffolds similar to natural ECM As a result, mesh fibers have been applied to many parts of the body such as bones, cartilage, skin vessels, muscles, and nerves Collagen fibers allow cell binding, penetration and proliferation due to collagen capacity, interacting with cell surface receptors, such as integrations of α2β1, α1β1, α10β1 and α11β1 [11]

 Gelatin used for fabricating the membrane

12

gelatin is quite reusable It is also easy to found in nature and has the potential to be applied on a large scale

In addition, there are many studies combining natural and synthetic materials applied in the field of tissue engineering, as can be seen in the Table 1.1

Table 1.1 Polymers used for nanofibers to support stem cells

Polymer Stem cells

PEG-PCL PMSCs

PLGA ASCs

Collagen NSCs

PCL/Chitosan USSCs

PCL/ Gelatin MSCs

PCL MCSs

PLLA NSCs

1.1.4. Mesenchymal stem cells

Mesenchymal stem cells, first described by Friedenstein et al, they exist primarily in the bone marrow In addition, subsequent studies have shown that these bone marrow cells have the ability to differentiate into cartilage or muscle cells Due to the ability to differentiate into different cell lines of these cells, the name "Mesenchymal stem cells" was proposed by Caplan in 1991 [6] Later, the name of the MSC was officially standardized by the International Society for Cellular Therapy

 The basic criteria for determining the mesenchymal stem cells: There are three basic criterias for evaluating mesenchymal stem cells:

i Able to adhere on the surface ii Antigen expression

13

- Negative expression (≤ 2%) such as CD45, CD34, CD14/11b, CD79α/CD19, HLA-DR

iii The ability of multi-line differentiation into types of cells like bone cells, fat, cartilage cells s

14

matrix production and adjustment of angiogenesis Many mechanisms have been demonstrated that MSCs promotes wound healing by providing the necessary cytokines and growth factors and differentiating into different types of cells in the wound including endothelial cells, monocyte [29].It has been claimed that applying MSCs to wounds just increases wound healing as well as promote the formation of new appendages of the skin such as sweat glands [17] If this can be proven, the application potential of MSCs will be huge, not only in wound healing but also in other fields of tissue

1.1.5 The combination between gelatin bionanomembrane and MSCs in the introduction of wound healing

Until now, there are limitation studies combining hMSCs and products of electrospinning for wound healing treatment For instance, Yuna Qian et al (2017) claimed that PCL (Polycaprolactone) nanofiber could support the adhesion and proliferation of hMSCs In the permanent, three different kinds of human MSCs culture on PCL nanofiber maintain the viability as well as accelerated the proliferation In particularly, the osteogenic differentiation ability of hMSCs was considerably rose by culturing on PCL nanofiber scaffold [5, 6]

15

significantly technical advances in the electrospinning allow the design and synthesis of new polymeric materials with desirable properties which include the structural variation of nanofibers and the ability to modify their hydrophilicity, conductivity Especially, the small pore size prevents from the growth of bacteria and fungi, so that nanofibers can be easy to protect cells which is damaging [26] The great potential of electrospinning for the fabrication of nanofibers to be used as scaffolds in tissue engineering applications [26]

Based on these above results, in this research, we focus on the electrospinning method for the fabrication of nanofibrous scaffolds by using the gelatin material and the in-vitro cytotoxicity of this gelatin biomembrane

1.2. Research objectives

The objectives of the study include the main contents are:

 Fabricating nanobiomembrane by the electrospinning method, using DC high voltage spray and create nanoscale fibers Also, investigating some physical characteristics and morphology of membrane to choose the appropriate concentration for apply to the in-vitro studies

 Evaluate the effect of culture on hMSCs as well as determine the protein by the western blotting technique

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CHAPTER 2: MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemical reagents

Table 2.1 Chemicals used in the laboratory

Name Manufacturer Country

Gelatin Biotech Canada

Glacial Acetic Acid Sci-tech China

Soybean casein Himedia India

Blue Trypan Gibco USA

DMSO (Dimethyl Sulfoxide) Prolabo USA

FBS (Fetal Bovine Serum) Invitrogen USA

PBS (Phosphate Buffered

Saline) Gibco USA

Peniciline/Streptomicin Gibco USA

Trypsin – EDTA Gibco USA

DMEM Gibco USA

StemMACS Mitenyl Germany

CelLytic MT cell lysis reagent Merck Germany

Protease Inhibitor Cocktail Merck Germany

Pierce BCA protein assay kit Thermo USA

Pierce BCA protein assay kit Bio-rad USA

4x Laemmli sample buffer Bio-rad USA

SuperSep Ace precast gel Wako Japan

Immun-Blot® low

fluorescence PVDF Bio-rad USA

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Rabbit anti-β-actin CST USA

Rabbit anti-Colagen I antibody Abcam USA

Anti-Rabbit IgG, horseradish

peroxidase Healthcare Life Sience USA

2.1.2 Equipment

Table 2.2 Equipment used in the laboratory

Name Manufacturer Country

Nanospinner Inovenso Japan

Magnetic stirrer Ika Germany

Induction cooker Ika Germany

Electronic scales Sartorius Germany

Incubator Thermo USA

Incubator 5% CO2 Thermo USA

Phase contrast microscope Olympus Japan

Centrifuge Eppendorf Germany

Pipetman Eppendorf Germany

Refrigerator Panasonic Japan

ChemiDoc MP Bio- rad USA

Blotting and Vertical Electrophoresis System

18 2.1.3 Tools and consumable supplies

Table 2.3 Tools and consumable supplies used in the laboratory

2.2 Experiment section

2.2.1 Fabrication of gelatin nanofibers

Name Manufacturer Country

T-flask 25, 75 Corning USA

well plate Corning USA

Lamen Sail Brand China

Cryo tube Corning USA

Centrifugal tube 2, 15ml Corning USA

Sterile clamps - Pakistan

Measuring cup Duran Germany

Syringe Terumo Japan

19

Figure 2.1 Fabrication of nanobiomembrane by electrospinning method

Figure 2.2 Image for the important parts of the electrospinning equipment  The basic principle

To fabricated gelatin nanofibers, the electrospinning technique is applied in this research Important parts of the device including a DC high voltage power supply, a polymer solution, a conductive collector, and a nozzle There are numerous potential differences occurs on two electrodes as a positive electrode and a negative electrode One positive electrode on the bottom is connected to the syringe tip, while a negative electrode attaches to the conductive collector In addition, a viscous fluid was significantly changed for the properties by using a high voltage electrostatic field, as a results stretch of solution and formation of fibers into the conductive collector After forming fibers, the solvent evaporates into the outside environment, while fibers retain, and attach to the cylindrical collector This process is controlled by an automatic control panel device below the main system On the other hand, there are four different regions within electrospinning process [1]

20

(2) A jet region in which the solution path is straight

(3) A splay region in which the jets stretch and split forming nanofibers (4) A collector region in which nanofibers are positioned

In addition, the electrospun jet region can be divided into three main phases, as shown Fig 2.3

i The formation of the Taylor cone ii The ejection of the straight jet iii The unstable whipping jet region

Figure 2.3 The behavior of the electrospun jet divided into three main phases: Taylor cone formation, straight jet ejection, and whipping jet formation

 Parameters relate to the membrane fabrication of electrospinning Table 2.4 Parameters relate to the membrane fabrication

Parameters Effect on fiber morphology

i. Solution Properties

Concentration Increase in fiber diameter with increase of molecular

21

Viscosity Low-beads generations, increase in fiber diameter, disappearance of beads

Surface tension No conclusive link with fiber morphology, high surface tension results in instability of jets

Conductivity Decrease in fiber diameter with increase in conductivity, reduction in fiber diameter and beads with adding Salt (KH2PO4, NaH2PO4, NaCl)

ii. Operation Parameters

Applied voltage Decrease in fiber diameter with increase in voltage Feed rate/ Flow

rate

Decrease in fiber diameter with decrease in flow rate, generations of beads with too high flow rate

Types of collectors

Generations of beads with too small and too large distance, minimum distance required for uniform fibers

Tip to collector distance

iii. Environmental Conditions

Humidity High humidity results in circular pores on the fibers

22  Experimental procedures

Preparation sample

Figure 2.4 The diagram of gelatin nanobiomembrane fabrication Table 2.5 Setting the data of electrospinning for fabricated nanobiomembrane.

Gelatin concentration Water: Acid acetic Applied voltage (kV) Temperature (oC)

Distance (cm) 10%, 15%,

20%, 25%

1:1 25 30 10

Gelatin samples were prepared at the four different concentrations according to Table 2.5 Firstly, these samples concentrations were completely dissolved with acid acetic and water following ratio 1: and stirring 180rpm/ at 80oC Secondly, under the pressure of a pump, a polymer solution is injected from a syringe into a plastic tube with 15ml/h flow rate This tube is connected to a nozzle, with the activation of a high voltage, electrospinning carried out through the creation of an electric field due to the contact of the solution and the high voltage This process has occurred if the electric field exists between the nozzle and conductive collector The existence of distance between a nozzle and the collector was 10 cm and the rotation of drum was set up 250 rpm The membranes are collected on a plastic film placed on the collector Finally, to evaluate several characteristic and morphology of nanofibers were taken by SEM images Moreover, the application Image-J software in the computer calculates a number of fibers

1 hour

Electrospinning

45

Dry SEM, FTIR

23

 FTIR method tests the fabricated nanobiomembrane

Both the chemical structure of the pre-fabricated sample and the structure fiber were analyzed by Fourier transform infrared spectroscopy (FTIR) Then, origin lab 8.0 software was used to analyze the achieved results The FTIR (Fourier Transformation Infrared) method base on the absorption of infrared radiation of the material This method records the characteristic fluctuations of chemical bonds between atoms The advantage of this method is the analysis with very low samples; able to analyze structure, quality, and quantity with high sensitivity

 SEM method tests the fabricated nanobiomembrane

The gelatin nanofibers are produced by the electrospinning method, and see the size and shape on the surface of fibers were taken via SEM (Scanning Electron Microscope), after sputtering with gold Using the Image-J software on the computer calculate the total number of fibers, large and small fibers SEM is an electron microscope that can produce images with high magnification on the sample surface by using a narrow electrons beam, which scanned over the sample surface

2.2.2 Human Mesenchymal Stem Cell (hMSC) studies a) hMSCs culture

Preculture of hMSCs cells which have the origin from Umbilical cord-derived prepared at approximately 80% confluence were enzymatically passaged then counted by using hemocytometer, seeded with fresh growth medium at a density of 5.0 × 103 cells/cm2 in 8-well rectangular dish, and cultured at 37°C in a humidified atmosphere of 5% CO2 After seeding for 72 h, the medium was changed At 24, 72

24

Figure 2.5 Observed five positions in each well of 8-well rectangular dish; Culture area: 10.5 cm2 (3.76 cm × 2.79 cm); Captured image area: 0.021962 cm2

 Calculation of attachment efficiency ( ) [28]

The attachment efficiency (α) will be calculated using X24 (cell density at t = 24 h)

according to Eq 2.1

0 24

X X 

 (Eq 2.1)

Where (Eq 2.1)

 : Cell attachment efficiency X24: Cell density at 24 h (cells/cm2)

X0: Seeding density (5.0 x 103 cells/cm2)

 Specific growth rate ( ) [28]

After adhered to the culture surface, cells would grow, proliferate, and reach the exponential growth phase An increase in cell population during this phase could be mathematically described using the following equation:

X t

X 

d

25

Where (Eq 2.2)

X : Cell density (cells/cm2) t: Time (h)

 : Specific growth rate (h-1)

To define specific growth rate, Eq 2.2 could be rearranged and solved as shown in Eq 2.3 In this training µ was estimated from fitting the scatter plot

 X  t

X X t

X 0d d  ( 24) 24 t t

X X e  (Eq 2.3)

Where (Eq 2.3)

X 0: Seeding density (5.0x103 cells/cm2)

X: Final cell density (cells/cm2)  : Specific growth rate (h-1)

 Calculation of doubling time (td) [28]

The doubling time can be calculated directly from the following way using the growth rate

ln 0.693

d

t

 

  (Eq 2.4)

Where (Eq 2.4)

td : Doubling time ( h)

: Specific growth rate (h-1)

b) Protein determination in hMSCs by the western blot technique  Protein extraction and concentration measurement

26

suspended in buffer including CelLytic MT cell lysis reagent supplemented with protease and phosphatase inhibitor cocktails according to a certain ratio The next step was sonication on ice that help broken cell to protein leaks out of the cell membrane After that, sample conducted centrifugation at 40C, 12 0000 rpm and

minutes The solution was putted out another tube, in the case, not use immediately after protein extraction, it would be stored at -70ºC On the other hand, the protein extraction whole-cell protein extraction would be measured by the BCA method It was mean that using BCA protein assay kit which including BCA reagent A mixture with reagent B according to a definitely ratio Additionally, to determined protein extraction concentration, a standard straight line was founded by protein BSA standard II

 SDS-PAGE

With ꞵ- actin, protein extraction would be mixed with 4x Laemmli sample buffer containing 2-mercaptoethanol, and boiled at 95°C for min, while COL I only added 4x Laemmli sample buffer as well as boiled at 95oC for min, and 37°C

for 10 They were separated base on different molecular weight by SDS-polyacrylamide gel electrophoresis with 15% gel SDS-polyacrylamide This step was carried out 40 mA, 250 V, 75 minutes for gels

Protein extraction

1 2 SDS-PAGE 3 Transfer to membrane

_ + Protein quantification (BCA assay) Protein extract

using whole-cell or nuclear extraction reagent

Target protein

immobilized to

PVDF membrane

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Figure 2.6 Overall the experimental process for Western blotting  Western blotting and detection

Firstly, after processing SDS-PAGE, protein extraction was transferred on PVDF membrane by semi-dry transfer system at 60 minutes This step conducted at room temperature for h with 10V voltage and less than 360mA Secondly, before incubating with primary antibody at 1h in the room temperature, the membrane was experienced blocking with 40ml ECL 5% at 4oC overnight Then, washing membrane with TBS containing 0.1% Tween-20 (TBS-T), after incubating with secondary antibody at room temperature for h washed with TBS-T Finally, COL I and β-actin were detected using ChemiDoc MP imaging system

Meanwhile, the requirement of standard curve with R2 ≥ 0.95 would be accepted,

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2.2.3 The combination ability of bionanomembrane and cells

Figure 2.7 Experimental procedure in-vitro research a) Preparation bionanomembrane

 Sterilization methods  UV Light

29

microorganisms due to mutations The sterilization effect of lamps depends on the wavelength of the lamps, temperature, microorganisms, especially the ultraviolet intensity between the distance and ducts

 Ethanol 70%

Sample will be immersed in alcohol concentration 70% within from to 10 minutes for sterilization The mechanism is the coagulation of proteins by microorganisms Intergradient water in ethanol 70% plays an important role in the sterilization process, so never using pure alcohol The advantage of sterilize by ethanol 70% is low cost, no exist on toxic materials, so this disinfection method is widely applied in research laboratory as well as in the industry This method can kill bacteria, viruses, fungi

 Agar jelly method to the sterilization test

First of all, preparing a sterilization Soybean Casein Digest Agar by weight gram per 100ml of distilled water, then pasteurize at 121oC within 15 minutes When the temperature is reduced to approximately 50oC, pour agar into the peptri dish

Secondly, using a sterilized knife to cut up the jelly and place it on membrane samples at different locations The agar specimens were leaved in an incubator at 370C and monitored for 3, 5, and days to check for the sterility

b) Preparation cells  Cell lines

(1) Hacat provided by Faculty of Biotechnology, Hanoi University of Science (2) Fibroblast provided by Faculty of Biotechnology, Hanoi University of Science (3) Umbilical cord-derived hMSC provided by Vinmec Research Institute of Stem Cell and Gene Technology

 Prepare of culture medium

30

Changing color from red to yellow owing to cells consume the nutrition to take part in the metabolism of cells When the color of the culture medium changes, it is necessary to replace a new medium in order to ensure the smoothly biological process

 Cell activation

Firstly, cells were taken from -80oC Then next step is defrosting cells in a sterile

water-bath at 37°C for minute The cells sample was rapidly transferred into 15 mL centrifuge tube containing pre-warm culture medium for centrifugation Secondly, centrifuging at 1000 rpm/min within minutes was carried out, and then he supernatant was removed After adding a new medium and mix with cells, the mixture was transferred into a culture dish, and put on an incubator under optimal conditions as 37oC, 5% CO2 for adherent cells Finally, via phase-contrast microscope

observes the growth of cells as well as change the medium or days per time  Cell maintenance

To maintain and increase the growth of cells, it is necessary to create an incubation condition similar to the factors in the human body such as the temperature at 37oC,

5% CO2 throughout the controlled chamber The culture medium was replaced with

a new medium to provide nutrients to living cells throughout the phenol red indicator changes color Also, exchange CO2 occurs via the filters to make sure the healthy

cells Finally, checking the cell status was carried out once a day to ensure that the cell density reaches approximately 80%, and conduct the trypsin to cells seeding

 Cell counting

31 c) Experimental procedures

 The experiment was conducted on the well dish

 The three different types of cells were carried out including HaCat, human fibroblast, and MSC cells

M

Figure 2.8 In-vitro experiment model

Using a sterile tweezer to pick up and place each membrane sample into each well, repeat times to ensure the accuracy of the method The membrane area is 4cm2 used for each well The number of seeding cells Hacat, Fibroblast was 1.5 x105 per well

and 3x104 with hMSC, add 2ml of culture medium including DMEM and 10% FBS

to each well Then, 6-well plate was incubated at 37oC, 5% CO2 After incubating

two days, using trypsin- EDTA to detached cells and taking hemocytometer to calculate the total number of cells At the time t= 0h, 24h, and 48h observe and take photos via phase contrast microscope

d) Haversting

- After 24 h, and 48 h incubate, samples will be taken photo at both membrane area and no fibers position for observing the growth and morphology of the cell by a contrast microscope

- At t = 48 h, apply ml of trypsin to each well at 37°C, 5% CO2 within

minutes for cells separate from the peptri dish and examine under a microscope

- Then, stop trypsin-EDTA with 1ml the medium, centrifuge at 1000rpm within minutes, and remove the supernatant

Gelatin nanobiomembrane

32

- Finally, add 1ml medium of PBS and carried out cells counting, using hemocytometer as well as Cell: Trypan blue with the proportion 1:

 Total cells counting

Calculate the number of total cells was calculated by the formula below:

C= a x 104 x DF (Eq 2.5)

C: Total cells counting

a: Average number of cells counted on the hemocytometer DF: Dilution factor

Same as the cell preparation section, the hemocytometer (Neubauer chamber) was used to count the total number of cells in the sample via the phase- contrast microscope First, 10ul of the prepared sample was taken after adding Trypan blue to a small slot between the counting chamber and the slip cover Second, only those cells places on the squares were counted, while touching the right and bottom line If a number of cells lower than 100 cells, the average is given by calculating the x 16 chamber Finally, total cell counting is multiplied by 104 and the dilution factor (DF) In most of the cases, the value of DF is due to the ratio between cells and Trypan blue 1:1 Furthermore, the total number of cells were calculated using trypan blue when counting on 16 chambers The value a in (Eq 2.5) is calculated by plus a number of living cells (white color) and dead cells counting (blue color) when trypan blue

 Cell viability

At t = 0, and 48h, number of live cells nl, t (cells) and number of dead cells nd, t (cells)

based on trypan blue exclusive test were counted The cell viability R (-) can be

calculated from nl, t and nd, t using this following equation

t d t l t l n n n R , , , 

33

CHAPTER 3: RESULTS AND DISCUSSION

3.1 Results of the fabrication nanofiber studies 3.1.1 FTIR results

34

Figure 3.2 FTIR spectra for powder gelatin, acid acetic, gelatin solution 25%, and gelatin fibers 25%

Fig 3.1 showed the FTIR spectrum of gelatin solutions prepared with different gelatin concentrations and pure acetic acid The IR spectrum of four prepared samples dissolved in acetic acid and water solutions with different gelatin concentrations of 10%, 15%, 20%, 25% and gelatin powder have both the spectral bands characteristic of both gelatin and acetic acid In particular, there are IR bands of Amides I, II, and III In particular, Amide I (1650 cm-1) is related to C= O stretching vibration coupled

with the C-N stretch, while Amide II (1540 cm-1) derived from the out-of-phase

combination of C-N stretch and N-H extended strain modes in the plane Finally, Amide III (1234 cm-1) demonstrated the combined peaks between C-N stretching

35

On the other hand, acetic acid has IR band such as 1706 cm-1 (C=O stretching), 1271

cm-1 (C─O─H stretching), 1388 cm-1 (CH

2 scissors deformation), and finally, 3020

cm-1 and 2645 cm-1 (C─H asymmetric stretching) Regarding Fig 3.2, there is no typical band of acetic acid were detected in the FTIR spectra of the electrospun fiber gelatin 25% by the electrospinning method From the results above, it is showed that the membrane sample after fabricating by electrospinning method is quite purity

Table 3.1 FTIR spectra characteristics of gelatin and acid acetic

Gelatin Acid acetic

Region Band in cm-1 Band

assignment

Band in cm-1 Band

assignment

Amide A 3380 N─H,C─H 1706 C=O

Amide B 3065 N─H 1271 C─ O

Amide I 1652 C=O, C─N 1388 H─C─H

deformation

Amide II 1540 N─H,C─N 3020 C─H

Asymetric

36 3.1.2 SEM Images

Figure 3.3 SEM images of electrospinning gelatin fibers as the different gelatin percentage as: (a) 10%; (b) 15%; (c) 20%; (d) 25%

It can be seen that the SEM image of the fabricated gelatin concentration was analyzed for the morphology of electrospun fibers There are significant differences SEM Images nanofibers with changing the gelatin concentration according to Fig 3.3 For instance, the formation fibers are very spare in the gelatin 10 % as well as create so many beads, while with 15% and 25%, there is no bead appears in SEM images and fibers are thicker Furthermore, there are two types of fibers including large fibers and small fibers in the gelatin 15% and 20%, but it is seen that the connection with them was broken Besides, in the gelatin 25%, the connection between large and small fibers quite uniform The size of the big membrane is approximately 150nm, while 50nm is the size of small fibers

(a) (b)

(c) (d)

500µm 500µm

37

Table 3.2 The calculated proportion of two kind of fibers

Concentration 15% 20% 25%

35.69 37.41 34.45

% Large fibers 64.30 62.79 65.55

%Large/small fibers 55.5 59.60 52.55

38

Figure 3.5 Nanobiomembrane with 25 % gelatin were fabricated

From the above results, it can be seen that the sample with gelatin concentration of 10% appear a great deal of beads on the fibers For concentrations of 15% and 20%, large and small fibers appear connected together are broken This predicts that these cases have low elasticity and not high durability In contrast, with a concentration of 25%, the fibers are fairly uniform and not broken lead to high strength, and good elasticity Thus, this is a suitable concentration for the next research application Fig 3.5

3.2 The results of hMSCs studies 3.2.1 hMSCs culture

39

determined to form the cell density As a result, the hMSC cells adhesion ratio and doubling time of experiment group was recorded as higher than positivecontrol group, while the specific growth rate less than another group as shown Table 3.3

40

Figure 3.6 Representative images of hMSC cells at t= 24, 72 and 120 after cell seeding in different five positions Scale bar: 200 µm

Center Upper

right

Upper left

Lower left

Lower right

41

Figure 3.7 Growth curves for hMSC cells in the experiment process Table 3.3 The Calculation of cell density average at t=24h.

Time

Cell density average, X24 (Cells/cm2)

Experiment Positive control 24 h 4703.56±361.84 4681.82 ± 479.57

Table 3.4 Adhesion ratio (α), specific growth rate (µ) and doubling time (td)

Group Adhesion

ratio (α)

Specific growth rate (µ, 10-2 h-1)

Doubling time (td)

Experiment 0.94 ± 0.07 1.18 58.74

Positive Control

0.92 ± 0.09 1.28 54.15

3.2.2 The determination of proteins in hMSCs

42

in whole-cell is 1.94× 103 μg/ml Additional, β-actin and COL I was observed by

taking 10 μgtotal protein of protein extraction loaded into each lane of SDS-PAGE with

15% precast gel

In the Western blotting, using ChemiDoc MP imaging system was detected of targeted proteins The molecular weight of β-actin and COL I bands were 42 kDa, 145 kDa, respectively According to Figure 3.9, band signals of targeted proteins were clearly shown at their specific molecular weights COL I had a large weight, therefore it would be on the top of the Supergel, while another protein on the middle Additional, the results of experiment group had no difference compare to a positive group which practiced in parallel during the experiment

Table 3.5 OD540 of standard BSA at varied concentration

Standard BSA concentration

(103 μg/mL)

OD540

Corrected OD540

(OD540-blank)

2.00 0.951 0.875

1.50 0.711 0.635

1.00 0.525 0.449

0.75 0.413 0.337

0.50 0.303 0.227

0.25 0.189 0.113

0.125 0.135 0.059

43

Sample Protein extraction (1× diluted)

OD540 0.860

Corrected OD540

(OD540 – blank)

0.860 – 0.076= 0.784

Protein concentration

0.784 − 0.0063 0.0004 = 1.94 × 103 μg/ml

Table 3.6 Calculation of total protein concentration in sample base on standard curve of BSA protein

Figure 3.8 Standard curve of BSA protein concentration

(a) (b) (c)

β- actin COL I

- 42 kD

- 145 kD

44

Figure 3.9 The images of COL I and β-actin band using ChemiDoc MP imaging system (a) β-actin; (b) Col-1; (c) Maker

Figure 3.10 Results of band ratio between β-actin, and COL I

Bands of targeted proteins, including β-actin, and COL I were detected at base on their specific molecular weights This result is an integral play in wound healing, because COL I is a part of the extracellular matrix

3.3 Results of the combination between bionanomembranes and cells 3.3.1 Nanobiomembrane sterilization

In order to be able to apply the manufactured membrane in in-vitro studies, there is imperative require that the sample have to completely sterile Normally, there are two basic methods for disinfecting samples that are not high heat resistance, usually use UV light or ethanol 70% However, the results of Fig 3.11 provide that these two methods not completely disinfect the microorganisms during the fibers forming process (The red arrow given that the microorganism is growing strongly after 24 h)

0.46

45

Figure 3.11 Examination the membrane sterilization by immerse in ethanol 70%, and UV lamp after 24 h incubation (a) untreated sample; (b) immerse in ethanol

70% within min; UV light at 135

From above result, it can be seen that the methods are not usable to the membrane sterilization Regarding to Fig 3.12, the microbiological infection only occurs at the determined stage in which after the membrane formation and taken to the biosafety cabinet Owing to in the previous stage, using electric fields with high voltage killed all microorganisms Hence, the mask model was created on the peptri dish as can be seen in Fig 3.13 and autoclaved at 121oC within 15 before placing on a collector

during the membrane fabricate

Figure 3.12 The electric spinning process did not place on the cleanroom

46

Figure 3.14 Sterilization results on membrane samples using mask model on the dish

As a consequence, the membrane sample is sterilized completely, and examined by the Jelly Agar It is can be used for in-vitro research, as well as the process is repeated three times to ensure the accuracy of the method

Control

Jelly

Day Day Day

Figure 3.13 Mask model on the peptri dish

47

In conclusion, during the manufacturing process, the enternal environment factors affects to the non-sterile membrane due to the contaminating microorganisms from outside, and the memnbrane can not be applied for further research This process occurs at the stage after electrospining Thereby, the mask model was fabricated to the sterile membrane and free from external microbial contamination The results record that the membrane can be used in the in-vitro studies

3.3.2 The effect of nanobiomembrane on the cell growth

Figure 3.15 Images of cells when cultured in the control and membrane samples The scale bar is 100 µm

There was significant difference between the experimental and the control group Firstly, after seeding for 24, and 48 h in the 6-well dish, a phase contrast microscope was used for examine the cells distribution on the culture surface The images illustrated that cells on the experiment attached on the dish followed by Fig 3.15. However, it is seen that a number of cells in the sample is less than the control sample in all three case Then, the total cells were calculated by using hemocytometer As a result, number of total cells of three experimental groups contain membrane were recorded as lower than control group as shown in Table 3.7

HaCat Fibroblast MSC cells

Control

48

On the other hand, at the time 48 h, a phase contrast microscope was used to demonstrate that almost cells more likely to extended in membrane position Furthermore, cells morphology of hMSCs more tended to like fibroblast, while Hacat cell is slightly round in shape and forms clusters of cells Finally, using Origin 8.0 software, ratio of total cells is counted between samples and controls as shown in Fig 3.16 The results showed that the total number of cells in the samples contain membrane was only approximately 50% compare to the control group in three types of cells

Table 3.7 A number of total cell counting in control and samples Cells Couting HaCat (cells) Fibroblast (cells) MSCs (cells)

Membrane 2.46 x 105 0.42 x 105 6.17x 105

Control 5.0 x 105 0.68x 105 10.63 x 105

Figure 3.16 Ratio total cells counting between samples and controls 0.492

49

3.3.3 The effect of nanobiomembrane on cell viability

Figure 3.17 The cell viability of three types of cells such as HaCat, Fibroblast, MSCs Regarding cell viability, following Fig 3.17, the value of cell viability is accepted owing to higher than 90 percent within the standard limit value in the both groups Especial in the experiment contain fibers is remarkable This provides that membrane samples did not kill three types of cells as Hacat, fibroblast, MSCs

50 DISCUSSION

In this study, we have successfully fabricated the nanofibers with gelatin 25% by the electrospinning method The size of fibers obtained from 50 to 150 nm in this research This result was considered as the difference compares to Marisa et al (2015) They suggested that the gelatin higher than 300mg/ ml with 25% acid acetic was can be optimal, while this study recorded the gelatin 25% with 50% acid acetic There are several differences in the electrospinning process High voltage 25kV was used to during the electrospinning, while they set up from 16 kV to 18 kV And, in the preparation, samples were stirred on 80oC within h, Marisa et al (2015) only stirred

on temperature (23oC) within 1h [12] However, FTIR results showed that both two

studies had received the pure gelatin nanofibers Meanwhile, the result of diameter fibers in this study was obtained similarly as Majid et al (2018), which have gelatin nanofibers with a diameter of 97 nm [23]

Regarding the cytotoxicity, there are limitations for research on cell viability when the combination of gelatin fibers and cells The cell viability of membranes and hMSCs as well as some other types of cells are optimistic results Results were achieved more than 90% of the great cell viability and there were no differences between all three types of cells, while Marisa et al (2015) also gained the similarity result [12] However, the combination of nanofibers and cells has a considerable effect on cell growth In particular, the total number of cells decreases significantly in all cell types Hacat cells were recorded the highest decrease, by contract the fibroblast cells is the lowest This result is similar to Marisa et al (2015), as well as the cell morphology of fibroblast for elongated, spindle shape Acid acetic was used to dissolve the gelatin, it makes the medium acidic so that this is the main factor influencing the results in the study [12]

On the other hand, although the natural polymer materials have good biological compatibility, low immune response, but they often unstable The in-vitro process, cell and fibers were incubated at 37oC, but the nanobiomembrane was dissolved in

51

52

CHAPTER 4: CONCLUSION

Natural polymers have outstanding characteristics such as biodegradation and biocompatibility Especially, gelatin has attracted many benefits for its use as a scaffold in tissue engineering From the results achieved above, there are three conclusions for this study Firstly, the gelatin nanobiomembrane has been initiated successfully fabricated by using SEM images The results of SEM images suggested a sample with 25% gelatin concentration which is suitable for in-vitro research The diameter of nanofibers gelatin is about 150 nm of large fibers and 50 nm of other types Secondly, the MSCs culture has been considered in this thesis, as well as COLI was detected by the Western blotting technique, it is a component of the extracellular matrix (ECM) for wound healing Finally, the toxicity test on three different cell lines as Hacat, fibroblast, hMSCs proved that membrane did not kill cells in all three cases shown as the cell viability was greater than 90% And, the fabricated membrane restricted the cell growth due to the total number of cells in the sample achieved only 50% compared to control groups

PERSPECTIVE

53

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