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

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 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 CONTENTS ACKNOWLEDGEMENT I LIST OF TABLES III LIST OF FIGURES IV NOMENCLATURES AND ABBREVIATIONS IV ABSTRACT VII CHAPTER 1: INTRODUCTION 1.1 Overview .1 1.1.1 The process of wound healing on the skin .1 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 REFERENCES 53 II 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 III 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 Figure 3.13 Mask model on the peptri dish 46 IV 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 V NOMENCLATURES AND ABBREVIATIONS MSC ISCT PBS FBS ECM PDGF TGF-β FGF EGF DC DMSO DF USSCs PMSCs ASCs NSCs PCL PLGA PEG-PCL PLLA Ep D Sb Mesenchymal Stem Cells International Society for Cellular Therapy Phosphate - buffered saline Fetal Bovine Serum Extracellular matrix Platelet-derived growth factor Transforming growth factor-β Fibroblast growth factor Endothelial growth factor Direct current Dimethyl Sulfoxide Dilution factor Unrestricted somatic stem cells Plasma derived mesenchymal cells Adipose-derived mesenchymal cells Neural stem cells Poly(caprolactone) Poly(lactic-co-glycolic acid) Poly(ethyleneglycol)- poly(caprolactone) Poly-L-lactic acid Epidermis Dermis Subcutaneous VI 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 Key words: Electrospinning, nanobiomembrane, gelatin, Human Mesenchymal Stem Cells (MSCs), wound healing, fibers VII 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 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 Corrected OD540 (103 μg/mL) 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 (blank) 0.076 42 Sample Protein extraction (1× diluted) OD540 0.860 Corrected OD540 0.860 – 0.076= 0.784 (OD540 – blank) 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 Maker COL I β- actin - 145 kD - 42 kD (a) (b) 43 (c) 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 0.46 Band ratio    actin COLI 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) 44 (b) (a) (c) 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 45 Having nanobiomembrane Figure 3.13 Mask model on the peptri dish Day Day Day Control Jelly 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 46 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 HaCat Fibroblast MSC cells Control Samples 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 47 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 0.618 0.58 0.492 Figure 3.16 Ratio total cells counting between samples and controls 48 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 49 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 the culture medium The reason given that gelatin is a natural polymer, so it is not thermally stable at a high temperature Many authors have studied the combination 50 of natural and synthetic polymers to expand the applicability For instance, Binulal et al fabricated nanofibrous scaffolds with five different concentration PCL: gelatin ratios such as 90:10, 80:20, 70:30, 60:40, 50:50 wt.% [5] They use organic compounds to dilute like acetic acid and ethyl acetate As a results that the mixed nanofibers containing 30% and 40% gelatin showed an optimum combination of hydrophilic and biodegradability and also maintained the structural integrity of the scaffolding Also, the interaction and proliferation of hMSCs are the highest among synthetic materials containing gelatin 30 and 40% [5] Meanwhile, another study Ildeu et al combined PCL and gelatin through coaxial combustion and incubated in a bio-simulation solution containing 10 folds the concentration of calcium and phosphate ions in the human body [14] 51 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 In the future, we will continue to study further the interaction mechanism when combining cells and 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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:... number of total cell counting in control and samples 48 III 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... 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