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

First, the gelatin nanofibers were fabricated by using the electrospinning method.. Key words: Electrospinning, nanobiomembrane, gelatin, Human Mesenchymal Stem Cells MSCs, wound healin

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

Ha Noi, 2020

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

Ha Noi, 2020

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 2 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

Thirdly, I would like to thank Assoc Prof Bui Thanh Tung, Dr Luu Manh Quynh, MSc Nguyen Thi Van Khanh, MSc Nguyen Nhu Cuong spending their precious time to point out for technical guide for me and give me advices to improve my thesis Finally, there are my family and my friends, who always stay by my side, motivate and encourage me from the beginning until the end of my studying

II

CONTENTS

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

CHAPTER 1: INTRODUCTION 1

1.1 Overview 1

1.1.1 The process of wound healing on the skin 1

1.1.2 Membrane fabrication methods for wound healing 4

1.1.3 Materials used for fabricating the membrane 9

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

REFERENCES 53

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

Table 3.7 A number of total cell counting in control and samples 48

IV

LIST OF FIGURES

Figure 1.1 Images for a process of wound healing on the skin [13] 2

Figure 1.2 Wound healing diagram by ECM Synthesis [3] 5

Figure 1.3 Electrospinning method creates structure similar to ECM [26] 6

Figure 1.4 The phenomena of electrospraying and electrospinning occur when the electrostatic repulsive forces overcome the surface tension of the liquid [1] 8

Figure 1.5 A schematic view of the electrospinning (a) downward electrospinning setup; (b) Upward electrospinning setup; (c) Horizontal electrospinning setup [1] 8

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 5 min; UV light at 135 min 45

Figure 3.13 Mask model on the peptri dish 46

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

dish 46

Figure 3.15 Images of cells when cultured in the control and membrane samples

The scale bar is 100 µm 47

Figure 3.16 Ratio total cells counting between samples and controls 48 Figure 3.17 The cell viability of three types of cells such as HaCat, Fibroblast, MSCs

49

VI

NOMENCLATURES AND ABBREVIATIONS

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 3 samples

Key words: Electrospinning, nanobiomembrane, gelatin, Human Mesenchymal

Stem Cells (MSCs), wound healing, fibers

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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

According to Shpichka et al, would healing on the skin is recognized as a systematic process which including four main phases (1) hemostasis, (2) inflammation, (3)

proliferation and (4) remodeling which can be seen in Fig 1.1

- 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), platelet-derived angiogenesis factor, serotonin, bradykinin, platelet-activating factor, thromboxane A2, platelet factor IV, prostaglandins, and histamine [32]

 Stage 2: Inflammation phase

This stage begins immediately after injuring and extending from 6 to 8 days [30] Firstly, platelets release the growth factors that diffuse into the tissue surrounding the wound Then, neutrophils move to enter the wound, while they excrete the bactericidal agents Secondly, the debris of macrophages is removed by activated neutrophils They release a large number of lysosomal enzymes such as elastase,

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 3 weeks after injury and up to 2 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

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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 3 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

The extracellular matrix (ECM) plays an integral important role in proliferation and remodeling phase of wound healing Thus, the question is how to fabrication ECM or similarity to ECM structure is an interest field of many scientists To date, many recent reports on tissue engineering have focused on the fabrication of materials which have biocompatibility and biodegradation similar to the extracellular matrix

(ECM)-like components as can be seen in Fig 1.2 These materials mimic properties,

structures and functions the complex structure of extracellular matrices (ECM) ECM molecules have a large of collagen fibers that intertwine together to support cell adhesion and biological activity Therefore, scaffold manufacturing with a structure that mimicking ECM molecules is a new and practical field of research in tissue engineering [33] Furthermore, the structure used for tissue engineering have to special properties of decomposition, porosity, microscopic structure and dimensions These characteristics are highly dependent on manufacturing methods

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Figure 1.2 Wound healing diagram by ECM Synthesis [3]

There are numerous fabrication techniques including drawing, template synthesis, temperature-induced phase separation, molecular self-assembly, and electrospinning for the tissue engineering application Up to now, the electrospinning technique has remained the most popular because of providing many take advantage such as high porosity; easy to adjust the pore size, and low weight Besides, a large surface area and porous structure allow nanomembrane to enhance the function of cells [3] Thanks to these properties, the ability to diffusion oxygen and nutrients from the outside into the scaffolding is improved Thereby, the growth rate and proliferation

of cells are also enhanced [26]

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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]

Until in 1969, Taylor discovered that at the tip of the capillary, the polymeric solution will create a cone, if the surface tensions balance by the applied potential, it is called Taylor cone In addition to, Taylor also suggested that the diameter of fibers smaller than the diameter of the capillary by reason of the emitted fibers jet on the tip of the cone during the electrospinning process [19] However, this method did not make a hit with until the 1990s From 1999 and 2014 numerous numbers of researches had accomplished on electrospinning through several of publications

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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

0



(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

After increasing the strength of the electric field to a critical point, in which the

electrostatic force overtakes the surface tension, and then the spherical droplet breaks

up to smaller droplets This process was called electrospraying, as shown in Fig 1.4

On the other hand, when using the higher molecular weight solution, instead of

formatting the small droplet, the fiber was created thanks to the sufficient chain

entanglements of the polymer solution This process had named as eletrospinning

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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].There are three main components to set up a common electrospinning, which composed of high-voltage power, a syringe filled with the polymer solution

connected to pumps, and a collector where contain nanofiber, following Fig 1.5

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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

Fard et al suggested that to controll the membrane fabrication efficiency, it was necessary rely on the two main factors such as structure and materials as shown in

Fig 1.6 Material is chosen for no accidental, but it has to base on its defining

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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]

Thanks to the outstanding features of fiber materials such as high volume ratio, porosity, and low weight The membrane has been used to create

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

Owing to the complex structure of collagen fibers, these fibers can be difficult to extract However, it can be easily broken through hydrolysis to gelatin [38].These gelatin fibers can be broken down into chains of amino acids with the small structure, thus, gelatin becomes a biodegradable material with low immunity, and the most noticeable Gelatin is amphoteric due to the presence of alkaline and acid amino residues lead to form a thermally reversible network in water [9] Thereby, the mechanical properties of gelatin materials can be further adjusted through chemical

or physical cross-linking, it is essential due to the instability of natural biopolymer in the water at body temperature [4] In addition, collagen is extremely expensive, while

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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

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

- Positive expression (≥ 90%) such as CD105, CD90, CD73

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

 The application potential of mesenchymal stem cells in wound healing

Since the discovery of MSC that can generate stable cell lines, studies have increased significantly Specifically, the circulation of MSC in peripheral blood increased when injured Nakagawa et al earlier studied for implanted porcine skin equivalents into wounds made on the dorsal skin mice There are two models proposed such as a contain humans (hMSCs) or a combination of hMSC and bFGF Both treatment models healed wounds after 7 days The authors argued that hMSC differentiates into keratinocytes to heal wounds, while bone marrow derivatives (BM-MSC) also contribute to wound healing on the skin [24] In addition, some models of animal wounds after treatment with MSCs increased significantly the rate of healing, while other studies use BM-MSC Many studies also show that adipose tissue and umbilical cord- derived MSCs also improve the skin healing [3, 4].With the model uses MSC

to treatment on the human skin, resulting in increased re-epithelialization rate, skin

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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]

Gelatin is one of the most interesting fiber forming agents for its characteristic features Gelatin properties depend on some characteristic such as the source of collagen, type of collagen, age of animal, type of conversion of collagen to gelatin as acidic or hydrolysis as well as on conditions of final formation of scaffolds Until now, literature underlines the importance of gelatin as scaffold component mostly because of its biocompatibility, bioactivity, and hydrophilicity [37] Likewise, with these benefits, gelatin nanomembrane also plays an important role in covering, protecting, and supporting the skin regeneration Also, it is a place for cells and growth factors attach to the growth hMSCs can create fibers to stick similar to the native human body, the necessary is the adhesion to nanofiber for the proliferation

Up to date, the most well-known techniques for producing nanofibrous structures are self-assembly, phase separation, and electrospinning [37] Moreover, the

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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

 Cytotoxicity assessment of nanomembrane on the different types of cells

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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

PBS (Phosphate Buffered

Immun-Blot® low

ECL blocking agent

17

Anti-Rabbit IgG, horseradish

2.1.2 Equipment

Table 2.2 Equipment used in the laboratory

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

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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]

(1) A base region in which the nozzle end contains the charged surface of the solution

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(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

i Solution Properties

Concentration Increase in fiber diameter with increase of molecular

Molecular Reduction in the number of beads and droplets with increase of

molecular weight

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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)

Applied voltage Decrease in fiber diameter with increase in voltage

Feed rate/ Flow

Humidity High humidity results in circular pores on the fibers

Temperature Increase in temperature results in decrease of fiber diameter

To obtain nanofibers with the desired morphologies, there are three main parameters optimized during the process of electrospinning including environmental conditions, solution properties, and operation parameters The solution properties relate to the molecular weight of polymetric material, viscosity, surface tension, and the conductivity of the polymetric solution Moreover, the characteristics of electric

current also affect significantly the formation of fibers Looking at the Table 2.4 in

the detail, some parameters are applied such as flow rate, high voltage, and the distance between the tip of the spinneret and the collector The environmental conditions impact the characteristics of fibers due to changing conditions such as

relative humidity and temperature, relating to the evaporation rate of the solvent

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 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 ( o C)

Distance (cm)

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

45 min

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 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 and 120h cell morphology was observed and captured using a phase contrast microscope (Olympus, CK40) with an objective lens of 4× magnification in five

different positions of culture well as shown in Fig 2.5 The number of cells of each

captured image was counted by Image-J software on the computer This number used

to determine a cell density in each well of culture vessel