.. .AN EVALUATION OF THE EFFECTS OF STIFFNESS OF POLYCAPROLACTONE MEMBRANE ON CELL PROLIFERATION TAN PUAY SIANG (B.ENG, National University of Singapore) A THESIS SUBMMITED FOR THE DEGREE OF MASTER... cell viability test An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation Chapter Literature Review CHAPTER 2: LITERATURE REVIEW 2.1 Relationship of a cell and the stiffness. .. properties of such An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 19 Chapter Literature Review substrates has on cell migration, growth and cytoskeletal organization
AN EVALUATION OF THE EFFECTS OF STIFFNESS OF POLYCAPROLACTONE MEMBRANE ON CELL PROLIFERATION TAN PUAY SIANG NATIONAL UNIVERSITY OF SINGAPORE 2006 AN EVALUATION OF THE EFFECTS OF STIFFNESS OF POLYCAPROLACTONE MEMBRANE ON CELL PROLIFERATION TAN PUAY SIANG (B.ENG, National University of Singapore) A THESIS SUBMMITED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2006 PREFACE This thesis is submitted for the degree of Master of Engineering (Mechanical) in the Department of Mechanical Engineering at the National University of Singapore under the supervision of Prof Teoh Swee Hin. No part of this thesis has been submitted for other degree at other university or institution. To the author’s best knowledge, all the work presented in this thesis is original unless reference is made to other works. Parts of this thesis have been published or presented in the following: Journal Publications: P.S. Tan, S.H. Teoh. Effect of stiffness of polycaprolactone (PCL) membrane on cell proliferation. Materials and Engineering Science C. Vol 27, Issue 2: 304-308, 2007. Poster Presentations: P.S. Tan, S.H. Teoh. Effect of Stiffness of Polycaprolactone (PCL) Membrane on Cell Proliferation. 3rd International Conference on Materials for Advanced Technologies (ICMAT 2005). P.S. Tan, S.H. Teoh. An Evaluation of the Effects of Stiffness of Polycaprolactone (PCL) Membrane on Cell Proliferation. 2nd Materials Research Society of Singapore Conference on Advance Materials (MRSS-S 2006). i ACKNOWLEDGEMENTS The author would like to thank Professor Teoh Swee Hin, for all his guidance, invaluable advice, imparting of knowledge and skills for continued learning and utmost understanding to the student throughout the duration of the project. Prof Teoh have been a great FYP supervisor to the author in 2003/2004, a caring Master’s degree supervisor and mentor to the author since 2004-2006. The author is extremely grateful to Prof Teoh for the many golden opportunities that he has kindly given to her to jumpstart her since her Bachelor degree graduation in 2004. She thanks Professor Teoh for his teachings, to train her as a researcher with “Content, Contacts and Character”. As the author left NUS for work, Professor Teoh gave her another set of 3 Cs- “Concentration, Commitment and Character”. The author hopes that the chapter with Professor Teoh will not end just upon the Master’s degree graduation and wish that she will carry on with many of the 3 Cs in life that Professor Teoh has taught her, with one C never to change - “Character”. The author also wishes to express her gratitude towards Dr Chen Fulin, who has kindly started her training on cell culture. She thanks him for all his teachings and advice. She is also extremely thankful to Ms Bina Rai, for her kindness, patience, guidance, advice and rendering hand when the author was facing much difficulties in the cell culture work. ii The author must also thank Mr Mark Chong Seow Khoon, for his never ending help and support throughout the project. The author here expresses her most heartfelt gratitude towards Mark, when he has gladly offered to help the author to carry on with her cell culture assays when she had to be on MC for 2 weeks after being knocked down by a cement truck. The author is also extremely thankful for all the staffs: Dinah, Jackson, Kuan Ming, Jeremy, Chee Kong, Lin Yun, Kamal, Zhang Jing; post graduate students: Kay Siang, Fenghao, Alex, Erin, Junping; undergraduate students: Chen Ran, Kelvin, Galvin, Chin Seng, Kar Kit; and all who have come into her life for the duration of the whole course of study in NUS. Thank you for the great company and support given when help is needed. The author acknowledges her parents, for their unconditioned love for the author, and also their understanding and support for many of the stressful periods. She also thanks Siang Yong, for his unfailing help, patience, love, understanding and motivation to see the author through the whole course of the project. iii TABLE OF CONTENTS PREFACE Page i ACKNOWLEDGEMENTS ii-iii TABLE OF CONTENTS iv-vi SUMMARY vii-ix LIST OF FIGURES x-xiii CHAPTER 1 INTRODUCTION 1.1 Background 1.1.1 Biocompatibility of biomaterials 1.1.2 Applications of biomaterials 1.1.3 Uses of PCL in biomedical fields 1.1.4 Cell interactions with foreign surfaces 1.1.5 Role of substrate mechanics on cellular responses 1.2 Research Objectives 1.3 Research Scope 1 1 1 2 2 3 4 6 6 CHAPTER 2 LITERATURE REVIEW 2.1 Relationship of a cell and the stiffness of the matrix on which it resides 2.2 Cellular response to substrate of different stiffness 2.3 Stiffness of substrate 2.4 Effect of substrate stiffness on cell growth and proliferation 2.5 Effect of substrate stiffness on adhesion and cytoskeleton 2.6 Effects of stiffness of substrate on focal adhesion 2.7 Focal adhesion points in relation to cell proliferation 2.8 Formation of focal adhesion points 2.8.1 Marker of focal adhesions 2.9 Materials used for cell culture studies 2.9.1 Extracellular matrix and other natural hydrogels 2.9.2 Fibroblasts in collagen gels 2.9.3 Synthetic substrates: ligand-coated polyacrylamide gels 2.10 Specificity of cellular response to matrix compliance 2.10.1 Endothelial cells 2.10.2 Myoblast 2.10.3 Hepatocytes 2.10.4 Neurons and glial cells 2.11 Designing of tissue-engineering construct 2.12 Polycaprolactone 2.13 Principles of Two-roll Mills 7 7 7 9 10 12 14 17 18 19 19 20 20 21 22 23 24 25 26 27 28 31 iv 2.14 Advantages of Rolling Milling Process 2.14.1 Solvent-free PCL membrane 2.14.2 Breaking up of grain boundaries of individual PCL pellets 2.14.3 Cold drawing of PCL masses 2.15 Melt Pressing and Slow Cooling of PCL Solid Masses 2.16 Biaxial Stretching of PCL Films 2.17 Rational for slow cooling and biaxial stretching of PCL film 2.17.1 Changes of macrostructure of PCL membrane during biaxial stretching 2.17.2 Changes in the microstructure of PCL 32 32 32 33 34 34 35 36 CHAPTER 3 MATERIALS AND METHODS 3.1 Fabrication of ultra flat PCL Membranes 3.1.1 Heated Roll Milling 3.1.2 Melt Pressing 3.1.3 Biaxial Stretching 3.2 Sodium Hydroxide Treatment 3.2.1 Preparation of test samples 3.3 Self-designed O-rings 3.3.1 Design considerations of O-rings 3.3.2 To mount different thickness of PCL membrane firmly 3.3.3 To apply an equal amount of radial stress in all directions 3.3.4 No obstruction for water contact angle viewing 3.3.5 Versatility of the new O-ring design 3.3.6 Design constraints of the O-rings 3.4 Water Contact Angle Measurements 3.5 Stiffness Characterisation 3.6 In vitro studies 3.6.1 Loading of cells into wells with PCL membrane as the underlying surface 3.6.2 Focal Adhesion and Actin Cytoskeleton Staining 3.6.3 Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining 3.6.4 Cellular Proliferation Assay 1: AlamarBlue Assay 3.6.5 Cellular Proliferation Assay 2: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay 3.6.5.1 Standard curve 3.6.5.2 Test samples 44 44 44 45 45 47 48 48 49 51 52 52 53 55 57 59 60 61 CHAPTER 4 RESULTS AND DISCUSSIONS 4.1 Thickness of PCL film varies with pressure exerted by Melt Pressing 4.2 Improving Hydrophilicity of PCL Membranes 4.2.1 Sodium Hydroxide Treatment 4.2.2 Water Contact Angle Measurements 4.3 Stiffness Measurements 4.4 In vitro studies of NIH 3T3 Cells 68 68 39 62 64 65 66 66 67 69 69 70 71 73 v 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5 4.4.6 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 Phase Contrast Microscopy Focal Adhesion and Actin Cytoskeleton Staining Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining Quantitative study 1: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay Quantitative study 2: AlamarBlue Assay Conclusion for 3T3 studies In vitro studies of Pig’s Chondrocytes Phase Contrast Microscopy Focal Adhesion and Actin Cytoskeleton Staining Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining Quantitative study 1: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay Quantitative study 2: AlamarBlue Assay Conclusion for chondrocytes studies 73 75 77 79 81 83 84 85 87 94 99 103 106 CHAPTER 5 CONCLUSIONS 5.1 Final conclusions 5.1.1 Stiffness of PCL membrane controlled by its thickness 5.1.2 Increased wettability of PCL membrane by NaOH treatment 5.1.3 In vitro studies of NIH 3T3 cells 5.1.4 In vitro studies of Chondrocytes 5.1.5 Cell type specific response to PCL membrane 5.1.6 Optimal stiffness 108 108 108 109 109 109 110 110 CHAPTER 6 RECOMMENDATIONS 6.1 More cell types to be used to determine cellular response to PCL membrane of different stiffness 6.2 Further characterization of the stiffness of the PCL membranes to determine the optimal stiffness for cell specific growth 6.3 In depth study of the various kind of cellular response due to varying stiffness of PCL membranes 6.4 Nanoscale Enigneering at the surface 6.5 Studies to be carried out in a 3-D scaffolds 6.6 Nanoscale scaffold fabrication 6.7 PCL Blends 6.8 Plasma Treatment of PCL membranes of various stiffness 112 112 REFERENCES 117 APPENDIX 112 113 113 114 114 115 116 PUBLICATIONS vi SUMMARY Polycaprolactone (PCL) is a common biodegradable polymer that has emerged as a promising biomaterial in the recent years. It can be easily fabricated into thin membranes while maintaining its mechanical strength. It was reported that human keratinocytes could attach and proliferate well on solvent casted and biaxially stretched PCL membranes [Khor et al., 2002; Ng et al., 2000]. In addition, Ng et al showed that human dermal fibroblasts could grow well on such PCL substrates [Ng et al., 2001]. However, the use of solvent casted PCL membranes poses the concern of possible implications due to residual solvents in the membrane. In this study, the author has moved on to a solvent-free fabrication method for the PCL membranes. The fabrication of PCL into ultra thin and flat membranes has been well documented. The process, which consists of roll milling, followed by heat pressing and finally biaxially stretching, enables the production of solvent-free PCL membranes. In vitro studies performed in this work has proven the biocompatibility of PCL films. Water contact angle measurements were carried out to determine the effect of 5M sodium hydroxide (NaOH) has on PCL membrane. It has been found that by pre-treating the PCL membrane with 5M NaOH for a period of 3 hours could sufficiently lower its water contact angle from 84.9 ± 3.5o to 63.0 ± 4.0o, thus improving its hydrophilicity. vii Careful design considerations were done to ensure that the O-rings used in this study enabled water contact angle measurement of PCL membrane while transmitting equal amount of radial stress to it. The design of the O-rings also made them versatile for other works involving atomic force microscopy and coculturing of two different types of cells on PCL membranes. In the native environment, cells proliferate on matrices of different stiffness depending on the cell type [Discher, 2005]. For example, bone cells proliferate in hard environments while skin cells proliferate in softer environments. It is predicted that cells will grow better on a substrate that mimics more closely its physiological milieu. This study investigated two cell types, namely, chondrocytes and 3T3 cells. Results from stiffness characterization showed that stiffness of the PCL membrane is relatively proportional to its thickness. The stiffness of the biaxiallystretched PCL membranes was thus controlled by manipulating its thickness. The thickness was maintained in the range of 2 ± 0.01 to 30 ± 0.01 µm with corresponding stiffness in the range of 0.5 ± 0.01 to 0.55 ± 0.09 N/m. The effects of stiffness of PCL membrane on cell proliferation were evaluated via cell proliferation and viability studies conducted using Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining, Actin Cytoskeleton and Focal Adhesion Staining, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) and viii AlamarBlue assays. Results indicated that 3T3 cells demonstrated enhanced proliferation and viability on less stiff membranes while proliferation rate and viability of chondrocytes increased on stiffer membranes. Cytoskeleton staining revealed that fibroblasts were more spread out on less stiff membranes while chondrocytes proliferated faster on stiffer PCL membranes. In conclusion, stiffness of PCL membranes can affect cell proliferation. ix LIST OF FIGURES Figure Figure 1.1 Figure 1.2 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Descriptions Cell interactions with foreign surfaces are mediated by integrin receptors with absorbed adhesion proteins that sometimes change their biological activity when they absorb. The figure is schematic and not to scale [Ratner, 1996]. Progression of anchorage-dependent mammalian cell adhesion. (A) Initial contact of cell with solid substrate. (B) Formation of bonds between cell surface receptors and cell adhesion ligands. (C) Cytoskeletal reorganization with progressive spreading of the cell on the substrate for increased attachment strength [Ratner, 1996]. Strain distribution computed in a soft matrix beneath a cell. The circular cell has a uniform and sustained contractile prestress from the edge to near the nucleus [Disher, 2005]. Stress versus strain illustrated for several soft tissues extended by a force (per cross-sectional area). The range of slopes for these soft tissues subjected to a small strain gives the range of Young’s elastic modulus, E, for each tissue. Measurements are typically made on time scales of seconds to minutes and are in SI units of Pascal (Pa). The dashed lines (- - -) are those for (i) PLA, a common tissueengineering polymer (ii) artery-derived acellularized matrix; and (iii) matrigel [Disher, 2005]. An interplay of physical and biochemical signals in the feedback of matrix stiffness on contractility and cell signaling [Rottner, 1999]. (a): The arrows point to dynamic adhesions on soft gel and static focal adhesion on stiff gels [Pelham, 1997]. (b): Actin cytoskeleton on stiff and soft matrix [Discher, 2005]. Basic NIH 3T3 fibroblast morphological response to different extracellular matrix rigidity. Phase images of fibroblasts on soft (A) and stiff (B) fibronectin-coated polyacrylamide gels show that cells on stiff gels are less rounded and more able to extend processes than cells on softer gels. Fluorescence of images of fibroblasts stained with rhodamine-phalloidin against F-actin shows no articulated stress fibers in cells on Page 5 5 8 10 14 17 23 x Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Figure 2.10 Figure 2.11 Figure 2.12 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 soft gels (C), whereas on stiff gels (D) the stress fibers resemble those in a fibroblast on tissue culture plastic [Geroges, 2005]. Myoblasts on collagen-coated polyacrylamide gels of various rigidities were stained for myosin (green) and nuclei (blue). Multi-nucleated myotubes formed on each stiffness, but at 2 wk only intermediate stiffness gels supported formation of myosin striation. Bar = 20 µm. Egel, Young’s modulus of gel [Georges, 2005]. Structure of Polycaprolactone. Two roll mills counter rotate to provide laminar shear to the melted PCL mass [Powell, 1983]. Fringed-micelle model of crystallites in amorphous matrix [Powell, 1983]. (a) Unstretched polymer, chains are in coiled state. (b) Stretched polymer, chains are straightened out, causing polymer to elongate [Powell, 1983]. Typical stress-strain graph of a semi-crystalline polymer with corresponding macrostructural changes under tensile loading [Ashby and Jones, 1986]. Changes in microstructure of polymer under tensile loading [Daniels, 1989]. Two-Roll Mill Machine. Fully stretched PCL membrane in biaxial stretching Machine. Schematic diagram for the fabrication of PCL membranes. Top and bottom part of the O-ring and also an O-ring with PCL film mounted on it. Conventional O-ring will block the measurement of water contact angle. PCL mounted snugly like a drum onto the O-ring and arrow shows that enough height of the bottom part is designed to ensure that the well of the O-ring can have at least a volume of 500 µl. Mounting process of PCL membrane onto O-ring. a) PCL membrane placed over bottom part of O-ring. b-c) Top part of O-ring to sandwich PCL in between both parts. d) Top part is pressed down firmly by broad end of forceps. e) PCL membrane mounted. a) Accessory acts as a support when surface of PCL membrane is to be characterized by AFM. b) PCL membrane mounted on AFM support, ready for surface roughness measurement. 25 28 31 33 35 37 40 44 46 47 48 49 50 51 54 xi Figure 3.9 Figure 3.10 Figure 3.11 Figure 3.12 Figure 3.13 Figure 3.14 Figure 3.15 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 a) Accessory to be used for the co-culturing of two different types of cells. b) Mounting of PCL membrane onto the accessory. c-d) Volume of O-ring and accessory designed to accommodate at least 500 µl of medium. Dimensions of O-rings for fabrication. Enough allowance was given so that the O-rings can be easily removed from the well of a 12-well plate with a pair of forceps. Illustration of water contact angle on surface of a solid substrate. a) Machine to measure water contact angle. b) Close up of O-ring with PCL membrane mounted, on the platform of machine. c) A drop of 50 µl of de-ionised water dispensed out from machine. d) Water drop on PCL membrane, ready for water contact angle measurement. Instron Microtester for compression studies carried out to obtain stiffness of PCL membranes. Cell counter used to obtain an average amount of cells per ml of medium. [http://www.emsdiasum.com/microscopy/products/ma gnifier/counting.aspx#63560] Thickness of PCL film with pressure applied during melt pressing. PCL undergoing hydrolysis of its ester linkages. Drop in water contact angle with 5M NaOH treatment. Graph of Load Vs Extension for PCL membranes of different thickness. Stiffness of PCL membrane increases with its thickness. Phase Contrast Microscopy pictures of 3T3 cells seeded on membranes of different thickness, taken on Day 1 and Day 6. Scale bar represents 50 µm. Staining of NIH 3T3 cells cultured separately on 7 µm and 22 µm PCL membrane over a 9-day period. Staining of NIH 3T3 cells cultured separately on 8 µm and 18 µm PCL membrane over a 9-day period. MTT assay to obtain a standard curve for NIH 3T3. MTT assay of NIH 3T3 seeded on 2 µm, 8 µm and 21 µm PCL membrane over a 9-day period. AlamarBlue assay of NIH 3T3 seeded on 8 µm and 17 µm PCL membrane over a 9-day period. (a) Chondrocytes at Passage 1, showing a more rounded phenotype. (b) Chondrocytes at Passage 3, cells differentiating 54 56 56 58 58 59 62 69 70 71 72 73 74 76 78 80 81 83 85 xii Figure 4.13 Figure 4.14a Figure 4.14b Figure 4.14c Figure 4.14d Figure 4.15a Figure 4.15b Figure 4.15c Figure 4.16 Figure 4.17 Figure 4.18 Figure 6.1 into a fibroblastic phenotype. Scale bar represents 50 µm. Chondrocytes seeded on 4 µm, 10 µm and 20 µm PCL membrane. Scale bar represents 100 µm. Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 1. Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 3. Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 6. Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 9. FDA/PI Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 3. FDA/PI Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 6. FDA/PI Staining of chondrocytes at Passage 1 cultured separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 9. MTT assay to obtain a standard curve for chondrocytes. MTT assay of Chondrocytes at Passage 1 seeded on 2 µm, 15µm and 27 µm PCL membrane over a 9-day period. AlamarBlue assay of Chondrocytes at Passage 1 seeded on 2 µm, 15 µm and 26 µm PCL membrane over a 9-day period. Scaffold architecture affects cell binding and spreading. (A-B) Cells binding to scaffolds with microscale architectures flatten and spread as if cultured on flat surfaces. (C) Scaffolds with nanoscale architectures have larger surface areas to absorb proteins, presenting many more binding sites to cell membrane receptors. The absorbed proteins may also change conformation, exposing additional cryptic binding sites [Stevens, 2005]. 86 90 91 92 93 96 97 98 102 103 106 115 xiii Chapter 1 Introduction CHAPTER 1: INTRODUCTION 1.1 Background This chapter aims to provide background information on the wide biomedical applications of PCL and the cellular responses such as growth, proliferation and also focal adhesion contact points when cells are seeded onto a substrate. These points of interest have led the author to research further to evaluate the effects of the stiffness of the biomaterial, Polycaprolactone membrane, has on the cells. 1.1.1 Biocompatibility of biomaterials In the last decades, there have been a wide variety of biomaterials being developed with different physico-mechanical, chemical and biochemical properties depending on the biomedical applications. Biocompatibility of a biomaterial is defined as “the quality of not having toxic or injurious effects on biological systems” [Williams, 1999]. Biocompatibility of a biomaterial is then directly related to its chemical and biochemical characteristics. Recently, as more research is done to take into considerations of the interactivity between the biomaterial and the host, biocompatibility is also considered as “the ability of a material to perform with an appropriate host response in a specific application” [Williams, 1999]. Advances in biomaterials research has led to the rapid emergence of tissue engineering. This new interdisciplinary field applies principles of An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 1 Chapter 1 Introduction engineering and life sciences towards the development of biological substitutes with many different applications. 1.1.2 Applications of biomaterials Prominent applications for biomaterials include: orthopedics, cardiovascular, ophthalmics and drug-delivery systems. Bioresorbable or non-bioresorbable polymers are used, depending on applications. Bioresorbable polymers are mainly used for applications that only require the temporary presence of a polymeric implant such as suture materials, periodontal membranes, temporary vascular grafts and drug-delivery systems [Serrano, 2004]. Among bioresorbable polymers are homopolymers and copolymers based on poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and polycaprolactone (PCL). 1.1.3 Uses of PCL in biomedical fields PCL is regarded as a soft and hard tissue compatible bioresorbable material [Khor et al., 2002] and has been considered as a potential substrate for wide applications, such as drug delivery systems [Zhong, 2001; Christine, 2000], tissue engineered skin [Ng et al., 2001], axonal regeneration [Koshimune, 2003] and scaffolds for supporting fibroblasts and osteoblasts growth [Hutmacher, 2001; Rai, 2004]. PCL has also been found to be a suitable substrate candidate for tissue-engineered skin [Venugopal, 2006; Venugopal, Tissue Engineering, 2005; Dai, 2004]. An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 2 Chapter 1 Introduction 1.1.4 Cell interactions with foreign surfaces Cellular interactions with foreign surfaces generally consist of four steps: 1) protein absorptions; 2) cells anchored to absorbed protein via cell integrins; 3) cells differentiate, multiply, communicate with other cell types and organize themselves; 4) cells and tissues in implant materials respond to mechanical forces [Ratner, 1996]. Firstly, when the biomaterials are implanted into the body, proteins are immediately absorbed ([...]... Progression of anchorage-dependent mammalian cell adhesion (A) Initial contact of cell with solid substrate (B) Formation of bonds between cell surface receptors and cell adhesion ligands (C) Cytoskeletal reorganization with progressive spreading of the cell on the substrate for increased attachment strength [Ratner, 1996] An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 5... 2.6 Effects of stiffness of substrate on focal adhesion The cell has adhesion points called focal adhesions that are anchorage points to the substrate on which they lie By tugging on the matrix at these focal An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 14 Chapter 2 Literature Review adhesions, the cell creates a tension within its membrane walls [Beningo, 2002] The. .. resulting in the directed An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 11 Chapter 2 Literature Review migration onto the rigid surface When the leading edge approached the soft side, local retractions took place, causing the cell to change its direction of movement The effects of substrate stiffness are not restricted to soft tissue cells like epithelial cells and fibroblasts... information on the wide biomedical applications of PCL and the cellular responses such as growth, proliferation and also focal adhesion contact points when cells are seeded onto a substrate These points of interest have led the author to research further to evaluate the effects of the stiffness of the biomaterial, Polycaprolactone membrane, has on the cells 1.1.1 Biocompatibility of biomaterials In the. .. proteins and cells Finally, cells and tissues respond to mechanical forces Two samples made of the same material, one a triangle shape and the other a disk, implanted in soft tissue, will show different healing reactions with considerably more fibrous reaction at the asperities of the triangle than along the circumference of the circle [Ratner, 1996] 1.1.5 Role of substrate mechanics on cellular responses... resistance that the cell senses, regardless of whether the resistance derives from normal tissue matrix, synthetic substrate, or even an adjacent cell 2.2 Cellular response to substrate of different stiffness Adherent cells can transmit forces, which are often referred to as traction forces to the substrate that they reside on, and thus causing wrinkles or An Evaluation of the Effects of Stiffness of. .. used for cell culture studies as natural or synthetic substrates The effects of the mechanical properties of such An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 19 Chapter 2 Literature Review substrates has on cell migration, growth and cytoskeletal organization has been extensively studied 2.9.1 Extracellular matrix and other natural hydrogels Protein-based extracellular... layer of cells then arises The answer to the above question can significantly affect how standard cell culture should be carried out and more importantly, give invaluable insights to An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 8 Chapter 2 Literature Review tissue repair strategies and also understanding in morphogenesis and disease processes [Discher, 2005] 2.3 Stiffness. .. into adhesions [Chrzanowska- An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 15 Chapter 2 Literature Review Wodnicka, 1996] Additionally, although microtubules have been proposed to act as ‘‘struts’’ in cells and thus limit wrinkling of thin films by cells [Pletjushkina et al., 2001], quantification of their contributions to cells on gels shows that they provide only a minor... only deform very soft gels [Bridgman, 2001] Neurons also branch more on softer substrates [Flanagan, 2002], perhaps because the cytoskeleton is more pliable, if less structured An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 16 Chapter 2 Literature Review a b Figure 2.4(a): The arrows point to dynamic adhesions on soft gel and static focal adhesion on stiff gels [Pelham,