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Novel Mechanical stimulations for stem cells and cartilage engineering

줄기세포 및 연골조직공학에 기계적 자극이 미치는 영향

  • Woo Hee Choi (최우희, 아주대학교 일반대학원 분자과학기술학과)

초록/요약

During the cells or tissue cultivation in vitro, the culture condition is known to be important for regulation of the cell fate. To control the culture microenvironment, biological factors such as growth factors, cytokines, extracellular matrix (ECM) components) are generally used. However, these biological factors can’t be free of safety issues in clinical applications due to possibility of their immunogenic response. They also have a disadvantage which require a high cost to purity. On the other hand, mechanical stimuli such as stretch/shear stress and compression is also one of the factors that could be affecting the cell fate in vitro microenvironments. Many studies reported that mechanical stimuli can induce proliferation, differentiation or migration of various cell types, and their signals are transmitted into intracellular through transmembrane proteins much like biological factors. However, these stimuli could be exposed to contamination, because of their direct application system on the cells. In contrast, ultrasound, a special type of sonic waves with a high frequency, can be maintained the contamination-free condition during stimulation in vitro or applied on in vivo model, due to their nature that could be passed through substrate for culture or skin, therefore it was used as a new mechanical stimuli. But, the molecular mechanisms of ultrasound are unclear compared to other stimuli. Sometimes, two or more composite mechanical stimuli can be multiply applied in bioreactor systems to control culture environment with biochemical signals. In actuality, cells or tissue within the body are exposed to various mechanical stimuli at once, therefore bioreactor system have been developed towards imitated situation of in vivo tissue. Many studies reported that these combination stimuli enhance the cellular activity or tissue formation through anabolic responses on various cell/tissue types. But, stimuli conditions for tissue organization of microstructure are not optimized. The aim of this study is to demonstrate the biological effect of these mechanical stimuli on cell adhesion/migration or cartilage tissue zonation using low-intensity ultrasound or shear/compression combined joint mimicking system, respectively. In chapter I, we investigated that low-intensity ultrasound (LIUS) could increase the yield of mesenchymal stem cells (MSCs) during primary culture from the bone marrow. Cells were treated with LIUS for 10 min/day during the first 6 days after initial plating of BM mononuclear cells. After 12 days, the colony-forming unit-fibroblasts assay was performed, and the colonies were culture-expanded for further analyses. LIUS stimulation showed a significant increase in the colony-forming ability of MSCs during the early stage of primary culture, without affecting their phenotypes (such as CD29, CD90, CD73 and CD105) and multi-potency. The LIUS stimulation also showed an increase in the expression of integrins, fibronectin, and paxillin, and induced the formation of focal adhesions in MSCs, all of which involved in the cell adhesion process. In chapter II, we confirmed the underlying mechnotrasduction of LIUS on adhesion and migration of human fibroblasts cell line (HFF-1) to explain cell adhesion process by LIUS in chapter I. Before stimulation at intensity of 50mW/cm2, HFF-1 was pre-treated with 0.5ug/ml cytochalasin D for 1 hour to disruption of actin polymerization or with 1ug/ml mitomycin C for 1 hour to remove migration by proliferation, respectively. LIUS increased polymerization of F-actin and the number of focal adhesions and migration. Western blot analysis showed that LIUS preferably induced phosphorylation of FAK at Tyr925, but not autophosphorylation-Tyr397. These results suggested that LIUS appeared to activate mechanotransduction pathways through phosphorylation of FAK-tyr925 instead of autophosphorylation at FAK-tyr397 which induced by biological cue or other mechanical stimuli. In chapter III, we investigated joint mimicking loading system which combined stimuli composed of compression and shear stress, and examined that effect of the stimuli on cartilage zonal organization which is important to maintain cartilage functions such as lubrication and cushion. Cylinder shaped cartilage plugs (d= 5mm, h= 4mm) were taken from a young porcine (2 weeks old ) femoral head, placed under the device that was similar to the shape of a femoral condyle in human knee, and stimulated with joint mimicking loading system for 1 hour per day. After 4 weeks, larger amounts of GAG were expressed on the stimuli group, while it degraded on the control group (static cultivation). And, cells were arranged horizontally paralleled to the surface, but control group was not. The result of this study suggests that complex stimuli could affect the cartilage zonal organization. Our loading condition that imitated joint loading movement could be a useful system in manufacturing the native mimic artificial cartilage using cells/scaffold. In conclusion, we developed a novel mechanical stimuli system as LIUS and joint mimicking loading system, and were trying to prove the biological effects on cells or tissue by the stimulations. First, we have examined that the effect of low-intensity ultrasound (LIUS) on adhesion of BM-MSCs (Chapter I), and confirmed the phosphorylation of focal adhesion kinase (FAK) known as the key factors of adhesion signaling pathway on fibroblasts (Chapter II) to explain the mechanotransduction of cell adhesion by LIUS. Second, we confirmed the cartilage zonal differentiation by complex stimuli mimicking joint movement (Chapter III) to demonstrate the biological effects of mechanical stimulus during tissue cultivation. MSCs and cartilage tissue are representative elements for cell/tissue engineering, and our results suggest that developed mechanical stimuli systems could be a useful tool for various therapeutic applications instead of biological factors on regenerative medicine field.

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TABLE OF CONTENTS

TABLE OF CONTENTS................................................. i
LIST OF FIGURES........................................................iv
LIST OF TABLES.........................................................vi
ABBREVIATIONS.........................................................vii
ABSTRACT................................................................viii

-BACKGROUND-
1. Culture environment on cell and tissue engineering......02
2. The control of cellular activity by mechanical stimuli....05
2.1 Shear stress........................................................05
2.2 Stretch stress.......................................................07
2.3 Compression.......................................................09
2.4 Low intensity ultrasound (LIUS)..............................09
3. The bioreactor systems mimicking in vivo condition.....12
4. Aim and title of this study..........................................15

-CHAPTER I; Low-Intensity Ultrasound (LIUS) Increased Colony Forming Units-Fibroblast (CFU-Fs) of Mesenchymal Stem Cells during Primary Culture-

1. Introduction..............................................................17
2. Materials and Methods..............................................19
2.1 Isolation of BM-MNCs and culture of MSCs.............19
2.2 Ultrasound stimulation...........................................19
2.3 Colony-forming unit-fibroblasts (CFU-Fs) assay......20
2.4 Analysis of cell proliferation...................................20
2.5 Flow cytometry for the expression of cell surface antigens.....................................................................20
2.6 Differentiation of MSCs..........................................23
2.7 Reverse transcriptase-polymerase chain reaction (RT-PCR).........................................................................23
2.8 Immunostaining...................................................23
2.9 Western blot analysis...........................................25
2.10 Statistical analysis.............................................25
3. Results..................................................................26
3.1 LIUS increased the number of CFU-Fs in the primary culture of MSCs from bone marrow..............................26
3.2 LIUS stimulation did not change the original phenotypes of MSCs...................................................29
3.3 LIUS induced cellular events related to cell adhesion....................................................................35
4. Discussion..............................................................38

-CHAPTER II; Low-Intensity Ultrasound (LIUS) Enhances Adhesion and Motility of Fibroblasts through the activation of Integrin-Focal Adhesion Pathway-

1. Introduction.............................................................42
2. Materials and Methods.............................................45
2.1 Cell culture..........................................................45
2.2 Ultrasound stimulation...........................................45
2.3 Immunofluorescence staining.................................47
2.4 Protein extraction and Western blot analysis.............47
2.5 Wound healing and Cell migration...........................48
3. Results...................................................................49
3.1 LIUS enhances that cytoskeleton polymerization and focal adhesion formation..............................................49
3.2 LIUS signal was transmitted via focal adhesion kinase (FAK).........................................................................51
3.3 LIUS enhance the cellular motility...........................54
4. Discussion..............................................................56

-CHAPTER III; The Effect of Joint Mimicking Loading System on Zonal Organization of Cartilage-

1. Introduction..............................................................60
2. Materials and Methods..............................................63
2.1 Self-manufactured equipment for stimuli..................63
2.2 Isolation and culture of cartilage explants.................63
2.3 Complex loading on cartilage explants....................66
2.4 Histological analysis.............................................66
2.5 Analysis of chemical components..........................66
2.6 Split-Line analysis................................................67
2.7 Scanning Electron Microscope for collagen linking fiber............................................................................67
2.8 Statistical analysis................................................68
3. Results....................................................................69
3.1 Gross observation and biochemical analysis...........69
3.2 The histological change of articular cartilage after 4wks cultivation...........................................................71
3.3 The changes of collagen fiber network by stimuli.....75
4. Discussion..............................................................78

CONCLUSION.............................................................81
REFERENCE...............................................................82
국문요약.....................................................................95

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