Development of Cell-Sheet Constructs for Layer-by-Layer Tissue Engineering Using the Blood Vessel as an Experimental Model Chong Seow Khoon, Mark (B.Eng, National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRAUDATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSTY OF SINGAPORE 2009 Preface Preface This thesis is submitted for the degree of Doctor of Philosophy in the Graduate Program in Bioengineering at the National University of Singapore under the supervision of Professor Lee Chuen Neng, Professor Teoh Swee Hin and Dr Jerry Chan No part of this thesis has been submitted for other degree or diploma at other universities or institutions 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: International Journal Publications Chong, MSK, CN Lee and SH Teoh (2007) "Characterization of smooth muscle cells on poly([epsilon]-caprolactone) films." Materials Science and Engineering: C 27(2): 309-312 Chong, MS, J Chan, M Choolani, CN Lee and SH Teoh (2009) "Development of cell-selective films for layered co-culturing of vascular progenitor cells." Biomaterials 30(12): 2241-51 Zhang, ZY, SH Teoh, MS Chong, JT Schantz, NM Fisk, MA Choolani and J Chan (2009) "Superior Osteogenic Capacity for Bone Tissue Engineering of Fetal Compared To Perinatal and Adult Mesenchymal Stem Cells." Stem Cells 27(1): 126-37 Lee, ES, J Chan, B Shuter, LG Tan, MS Chong, DL Ramachandra, GS Dawe, J Ding, SH Teoh, O Beuf, A Briguet, KC Tam, M Choolani and S-C Wang (2009) "Microgel Iron Oxide Nanoparticles For Tracking Human Fetal Mesenchymal Stem Cells Through Magnetic Resonance Imaging." Stem Cells (In press) Tan K, Tang K, Huang S, Putra A, Lee T, Ng S, Chan J, Tan L, Chong M (2009) Ex-Utero Harvest of Haematopoietic Stem Cells from Placenta/Umbilical Cord with an Automated Collection System IEEE Trans Biomed Eng Chong, MS, SH Teoh, EYL Teo, CN Lee, S Koh, M Choolani, and J Chan "CD34 antibody conjugation onto µXPCL film surfaces contributes to haemocompatibility by reducing procoagulatory responses" Biomaterials (submitted) Book Chapters Chan J, Chong MS Lentiviral vector transduction of human fetal mesenchymal stem cells In: Federico M, editor Lentivirus Gene Engineering Protocols; 2009 i Teoh SH, Rai B, Tiaw KS, Chong MSK, Zhang Z, Teo YE Nano-to-macro architectures polycaprolactone-based biomaterials in tissue engineering In: Tateishi T, editor Biomaterials in Asia; 2009 ii Abstract Cell sheet tissue engineering approaches have recently emerged as a method to generate stratified tissue and better recapitulate native architecture However, completely biological cell sheet approaches are limited by poor mechanical strength and appropriate substrate cues for modulating cell responses The development of microthin polycaprolactone (PCL) films raised the possibility of using these films as scaffolds to complement the cell sheet approach The aim of the present thesis was to investigate the use of PCL films as scaffolds in layered tissue engineering, using the blood vessel as a model The scope covers the engineering of microthin PCL films using bi-axial stretching and surface modification, isolation of vascular progenitor cells and in vitro evaluation of the engineered films It was found that solvent cast and electrospun microthin PCL films did not compare favourably against biaxially stretched PCL films (µXPCL) The latter was found to possess the highest tensile strength and resistance to chemical degradation as determined by erosion assays Tubular constructs formed from µXPCL were found to possess adequate burst pressure and slow degradation (as determined by DSC and SEM studies), and were predicted to retain tensile properties capable of withstanding physiological loads for up to twelve weeks in vivo A novel glow-discharge plasma immobilisation technique was used to functionalise the µXPCL film with carboxyl or amine groups, which allowed the conjugation of a range of biological moieties Conjugation of common model proteins, including iii collagen and cell-capture antibodies, was demonstrated, underlining the possibility of generating customised surfaces for layered tissue engineering Subsequently, perivascular cells derived from umbilical cord (UCPVC) were found suitable for reconstruction of the perivascular compartment for vascular tissue engineering Endothelial progenitor cells (EPCs) were found to exist in perinatal haematopoietic tissue, including two novel sources (fetal blood and liver) In particular, fetal blood derived EPC suggest superior vasculogenic capacity in Matrigel and microarray studies However, umbilical cord blood EPC (UCB-EPC) were deemed the most suitable for vascular tissue engineering due to lineage stability The feasibility of microthin PCL films for layered vascular tissue engineering was explored µXPCL film surfaces were modified to support UCPVC and UCB-EPC for the perivascular and intimal compartments respectively In addition, the intimal surface modification was found to improve haemocompatibility Finally, sequential cell seeding was employed to generate a layered vascular analogue In conclusion, the thesis demonstrated the application of µXPCL films for layered tissue engineering This has important implications in the generation of stratified composite tissue, such as skin, blood vessels and myocardium, a step closer to restoring tissue to normality ii Acknowledgements Acknowledgements The work detailed in this thesis was carried out in the BIOMAT Centre and the Experimental Fetal Medicine laboratory in the National University of Singapore, with funding provided by the National University Hospital (NUH) Endowment Fund, NUH Clinician Scientist Unit (CSU) and National Healthcare Group SIG Fund This work was made possible by the serendipitous meeting of my three supervisors: Professor Lee Chuen Neng, to whom I am indebted for his generous and unfailing support, Professor Teoh Swee Hin, with whom I first embarked in the fascinating realm of Biomaterials a decade ago, and last, but definitely not least, Dr Jerry Chan, who has taught me about asking the right questions, and continues to astound me with his energy, dedication and passion for research I would like to thank the students and staff of BIOMAT Centre, past and present, for their help and the pleasure of their company: Chee Kong, Fenghao, Kay Siang, Dinah, Puay Siang, Erin, Wen Feng, Jackson, Yuchun, Bindu, Jinxing, Ai Hoon, Justin, Thomas, Kar Kit and Xiangyu I am grateful for the administrative and technical support from the Department of Obstetrics and Gynaecology In particular, I’d like to thank Ginny and Lay Geok for their administrative support, and Dr Stephen Koh and staff from the Coagulation Lab for their help in the haemocompatibility tests I’d like to reserve special mention for the Experimental Fetal Medicine group: Eddy, Zhiyong, Lay Geok, Yiping, Citra, Praveen, Niraja and Darice for the camaraderie, motivation and inspiration I am privileged to have been a part of the GPBE, which has played a large part in the formative years of my scientific training In particular, I am grateful to the founding chairpersons, Professor Teoh and Professor Hanry Yu, as well as students of the 2003 cohort I would like to acknowledge the technical support from various facilities: Miss Satinderpaul Kaur from the Healthcare and Energy Materials Laboratories for assistance with materials characterization, Mr Toh Kok Tee, Miss Hu Xian and Mr Zhang Jie from NUMI for help with flow cytometry and confocal microscopy, Dr Zhong Shaoping from NUSNNI for help with the AFM and Mdm Deborah Loh from the EM Unit This work would not have been possible without the participation of the staff and patients of the National University Hospital, for whom I am grateful I would like to thank A/Prof Mahesh Choolani and Dr Jerry Chan for supporting the completion of my work, and the writing of this thesis Most importantly, I would like to thank my family: My parents, Chong Chee Poh and Susan Chong, and my sister, Sharon Chong for their unquestioning love and support all this while Finally, I am most grateful to my wife, Julie, and son, Ethan, my constant source of support and motivation Words cannot express my happiness for having both of you in my life iii Table of Contents Table of Contents Preface i Abstract iii Acknowledgements iii Table of Contents iv Table of Figures xiv List of Tables xvi List of Abbreviations xvii Introduction .1 1.1 Tissue Engineering as a Solution to Organ Shortage 1.1.1 Classical Tissue Engineering .3 1.1.1.1 Early success in skin and osteoarticular tissue 1.1.1.2 Recent advances .4 1.1.1.3 Limitations 1.1.2 Cellular Assembly 1.1.2.1 Cell printing .7 1.1.2.2 Cell sheet engineering 1.1.3 Layer-By-Layer Tissue Engineering Using Cell-Scaffold Hybrid Constructs 11 1.1.3.1 1.2 Microthin films for use in layer-by-layer tissue engineering 12 Vascular Tissue Engineering .13 1.2.1 Clinical Need For Vascular Tissue Engineering 14 1.2.2 Structure and Organisation of Blood Vessels 15 1.2.3 Intimal Compartment .15 iv Table of Contents 1.2.3.1 Regulation of haemostasis .15 1.2.3.2 Regulation of smooth muscle cell activity .18 1.2.4 Medial Compartment .18 1.2.4.1 1.2.5 Contractile function 19 Approaches and Strategies In Vascular Tissue Engineering 20 1.2.5.1 Acellular approaches 20 1.2.5.2 In vitro tissue engineered vascular grafts .22 1.2.5.3 Completely biological approaches 24 1.2.5.4 Summary of limitations in current vascular tissue engineering approaches 25 1.2.6 Cell Source .27 1.2.6.1 Endothelial cells (EC) 27 1.2.6.1.1 Mature endothelial cells 27 1.2.6.1.2 Differentiation from stem cells 28 1.2.6.1.3 Endothelial Progenitor Cells (EPC) 31 1.2.6.2 Smooth muscle cells 33 1.2.6.2.1 1.2.6.2.2 Smooth muscle progenitors 35 1.2.6.2.3 1.3 Immortalised smooth muscle cells 34 Umbilical cord perivascular cells .35 Scaffolds for Layer-By-Layer (LBL) Tissue Engineering 36 1.3.1 Scaffold Requirements 36 1.3.2 Bioresorbable Polymers 38 1.3.2.1 Synthetic bioresorbable materials 39 1.3.2.1.1 Polyglycolic Acid .41 1.3.2.1.2 Polylactic Acid 42 1.3.2.1.3 Polycaprolactone 43 v Table of Contents 1.3.2.1.4 Polyhydroxyalkanoates 44 1.3.2.1.5 Polyurethanes 46 1.3.2.2 1.3.3 Fabrication .49 1.3.3.1 1.4 Degradation and erosion 47 Microthin film fabrication 49 Surface Modification of Scaffolds in Tissue Engineering 51 1.4.1 Biological Responses to Material Surfaces 51 1.4.2 Improvement Of Surface Wettability .52 1.4.2.1 Surface hydrolysis 53 1.4.2.2 Grafting co-polymerisation 54 1.4.2.3 Plasma gas processes .55 1.4.3 Bio-Functionalisation Of Polymeric Surfaces 57 1.4.3.1 Surface modification to improve blood compatibility .58 1.4.3.1.1 Blood compatibility assays 59 1.4.3.2 Adhesion cues 62 1.4.3.3 Growth factors 63 1.4.3.4 Cell-capture surfaces 64 1.5 Summary 65 1.5.1 Hypotheses .67 Materials and Methods 68 2.1 Development of Scaffolds for Vascular Tissue Engineering .69 2.1.1 Film Fabrication .69 2.1.1.1 µXPCL film synthesis 69 2.1.1.2 Electrospinning 71 2.1.1.3 Solvent casting 72 vi Table of Contents 2.1.2 Preliminary Evaluation For Layer-By-Layer Tissue Engineering Application 72 2.1.2.1 Tensile test .72 2.1.2.2 Suture retention 72 2.1.2.3 Accelerated erosion studies 73 2.1.3 Preliminary Evaluation of µXPCL Films for Vascular Engineering Applications 73 2.1.3.1 Vessel formation 73 2.1.3.2 Burst pressure 74 2.1.3.3 Oxidative degradation 74 2.1.3.4 Mechanical testing 75 2.1.3.5 Differential Scanning Calorimetry (DSC) .75 2.1.3.6 Scanning Electron Microscopy (SEM) 75 2.1.4 Surface Functionalisation .76 2.1.4.1 Plasma immobilisation of polyacrylic acid 76 2.1.4.1.1 Quantification of surface carboxyl groups .76 2.1.4.1.2 Water contact angle 77 2.1.4.2 2.1.5 Plasma immobilisation of polyethylene imine .78 Heparin Conjugation 78 2.1.5.1 Heparin immobilisation method 78 2.1.5.2 XPS 78 2.1.6 Surface Conjugation Of Fluorescent Labels 79 2.1.7 CD34 Antibody Conjugation 79 2.1.7.1 CD34 antibody immobilisation method 79 2.1.7.2 EDC/NHS optimisation 80 vii Chapter Isolation and Characterisation Of Vascular Progenitors (a) (b) DSC Tensile Strength 150 Fenton's reagent H2O 55 H2O Fenton's reagent 100 50 MPa 50 45 40 Number of Weeks 12 10 12 10 0 % Crystallinity 60 Number of Weeks (c) weeks weeks weeks 12 weeks Figure 3-5: Hydrolytic and oxidative degradation of µXPCL in Fenton’s Reagent (a) Crystallinity as determined by Differential Scanning Calorimetery (DSC) over 12 weeks (b) Tensile strength over 12 weeks (c) SEM images (47,000x) illustrating crack formation on film surface as a result of immersion in Fenton’s reagent Similar images were obtained from samples immersed in deionised water Scale bars represent 200nm 3.3 Discussion 3.3.1 Summary Of Results Existing cell sheet technologies can be complemented by the use of polymeric scaffolds to provide mechanical stability, as shown in recent studies where cellscaffold sheet constructs are generated and assembled in layer-by-layer approaches 106 Chapter Isolation and Characterisation Of Vascular Progenitors Thus, I investigated the properties of PCL scaffolds for use in layer-by-layer tissue engineering PCL is easily processible and a range of methods can be employed to generate microthin films (Tiaw 2007) I selected three processing methods for comparison µXPCL films was found to possess the highest specific mechanical strength Further support for the use of µXPCL was provided by suture retention tests, with µXPCL having significantly improved suture retention strength over PCL-SC and PCL-NF Based on mechanical considerations therefore, µXPCL films present the best option for layer-by-layer vascular tissue engineering To evaluate the application of µXPCL in vascular tissue engineering, I first formed tubes out of µXPCL Heat welding was found to be a feasible method, and the generated conduits possessed burst pressures nine-folds in excess of physiological loads When the films were subjected to oxidative degradation, deterioration of mechanical properties was observed over 12 weeks 3.3.2 Mechanical Strength Although native PCL has a ten-fold lower tensile strength than that of other commonly used materials, such as PLA (Hutmacher 1996), biaxial stretching can be employed to improve mechanical properties of the film through molecular realignment arising from the drawing process (Ng 2000; Tiaw 2007) In my studies, I found that µXPCL has three-fold higher yield strength and six-fold higher tensile strength compared to PCL-MP Taking the blood vessel as a model, theoretical calculations suggest that µXPCL microthin films possess sufficient mechanical strength for vascular tissue engineering applications In the suture retention tests, I found that µXPCL films fall short of the previously quoted requisite of 2N (Lee 2008) 107 Chapter Isolation and Characterisation Of Vascular Progenitors albeit by less than 10% This suggests the possible need for considerations, such as suture rings and cuffs in the final design I found that electrospinning and solvent casting methods yielded highly porous films, compromising the strength of the microthin films Although electrospun conduits have previously been shown to have good burst pressures and suture retention strength (Lee 2008), the scaffolds used in these studies are typically 50-fold thicker and measure in the range of few hundreds micron thicknesses Such scaffolds are impenetrable to cells, and result in low cell densities, as well as poor cellular distribution through the engineered construct (Pham 2006) Mechanical properties notwithstanding, electrospun nanofibres provide several attractive features The nanotopography approximates deposited extracelluar matrix, and has been shown to be a favourable surface for cellular adhesion Topographical cues can be further used to manipulate cell responses, such as migration and alignment (Curtis 1997), features which are not provided by the smooth µXPCL films A possible method to marry the two technologies would be to modify µXPCL surface by nanofibre deposition (Chen 2007) 3.3.3 Erosion Studies Aside from robust mechanical strength, films for vascular tissue engineering should have suitably slow degradation kinetics in order to reduce the possibility of inflammatory responses to degradation by-products of the films (Sung 2004; Yang 2005) PCL is known to have long degradation rates, surpassing even that of polylactic acid Previous studies have shown no significant mass loss following 108 Chapter Isolation and Characterisation Of Vascular Progenitors immersion in saline for 12 months (Lam 2008; Lam 2008) Consequently, I chose to use an accelerated degradation test as a means to compare the various groups While the conditions in accelerated degradation assay are not physiological, it is a recognised method to provide comparative assessments (Htay 2004) The kinetics of erosion is highly dependent on the manufacturing process In line with previous studies, solvent-cast structures have been shown here to be more porous than melt-processed polymers and consequently less resistant to hydrolysis (Mathiowitz 1993) Consequently, PCL-NF and PCL-SC eroded faster than µXPCL In particular, we show PCL-NF to be particularly susceptible to degradation Erosion is known to be affected by the diffusion rate of water into the polymer bulk (von Burkersroda 2002), which is accelerated in open structures such as that of PCL-NF films Furthermore, the large surface area-to-volume ratio in the nanofibrous architecture allows for rapid absorption of water, followed by bulk erosion and fragmentation Consequently, rapid mass loss was found in the PCL-NF group, suggesting poor chemical resistance In comparison, mass loss of PCL-SC was initially low, with the second phase having a similar rate of erosion to that of PCL-NF PCL-SC films have a lower surface areato-volume ratio, which would account for the initial phase of hydrolytic degradation and erosion being confined to the material surface, with the introduction of crevices Following partial surface degradation, the resultant PCL-SC structure approaches that of PCL-NF, where it rapidly absorbs water and similarly undergoes bulk erosion and fragmentation 109 Chapter Isolation and Characterisation Of Vascular Progenitors In contrast, µXPCL has a dense microstructure, with very much reduced surface area to volume ratio, and consequently, mass loss was gradual Work by Mochizuki et al has further demonstrated that drawn polymers, including PCL, are more resistant to degradation (Mochizuki 1995; Mochizuki 1997; Tsuji 2007) I also noted that the geometry of µXPCL films was preserved throughout the period of study, and erosion was marked by surface roughening without bulk defects It thus follows that µXPCL degradation is surface dominated, resulting in the linear mass loss profile Such profiles are more predictable and reproducible, and desirable in applications such as drug delivery and tissue engineering, where one aims to predict and control the rate of degradation in vivo to match the physiological process of remodelling, as illustrated in Figure 3-6 Figure 3-6: Proposed model for ideal scaffold degradation Ideal progression of scaffold degradation and remodeling should coincide with native cells and extracellular matrix (ECM) deposition in vivo Degradation should be engineered to match remodeling to achieve stable mechanical properties through the process 3.3.4 Blood Vessel Application Of Microthin µXPCL Films I then proceeded to evaluate the suitability of µXPCL films for vascular tissue engineering Burst pressure testing demonstrates eight folds in excess of the upper 110 Chapter Isolation and Characterisation Of Vascular Progenitors limit of normal of systolic blood pressures (1,230 vs 140 mmHg) This augurs well for its utility in such applications However, we anticipate that the mechanical properties of the material will deteriorate in vivo following implantation where the polymer is placed in a dynamic environment with elevated temperatures, cyclical stresses and oxidative elements Furthermore, leukocytic and phagocytic infiltration of the materials can contribute to oxidative degradation of biological implants Together, these factors potentially contribute to accelerated degradation of the vascular graft scaffold material, and hence, premature and catastrophic failure prior to tissue remodeling Thus, it was necessary to test the response of µXPCL films to oxidative stresses My studies showed that degradation in aqueous media was found to occur over two weeks, with an increase in crystallinity, possibly due to preferential and rapid attack on the amorphous regions throughout the film This suggests swelling had taken place, with uptake of water through the polymer bulk and appeared to contradict my accelerated erosion studies, which suggested a surface dominated and slow erosion process Similar work on PCL scaffolds immersed in PBS show little mass loss to occur over ten months (Lam 2008; Lam 2008) Moreover, I found significant loss of tensile strength within the first four weeks, seemingly inconsistent with a slow model of degradation Similarly, Lam et al found little change in modulus or strength over a duration of ten months in saline (Lam 2008) I attribute these observations to the following factors Firstly, because the µXPCL films contain micro-sized dimensions, water diffusion into the polymer bulk may occur, leading to preferential degradation of the amorphous regions However, the 111 Chapter Isolation and Characterisation Of Vascular Progenitors degradation by-products remain trapped within the crystalline matrix, and consequently, no mass is lost It is important to note here that erosion is a physical phenomenon involving the removal of degradation byproducts, and that degradation is a chemical process arising from bond cleavage to yield lower molecular weight products Here, the degraded amorphous domains are degraded, but, rather than clearance of the cleaved products, the increased chain mobility and incubation at elevated temperatures can result in re-alignment and increase in crystallinity (Lam 2008) Next, it was observed that microcracks could be located on the film surface, coinciding with the drop in tensile strength These cracks serve as stress concentration points and crack initiation sites In this particular case of microthin geometry, the cracks significantly reduce the load bearing area, further reducing the apparent strength Thus, it is the emergence of cracks, rather than bulk degradation, that resulted in the drastic drop in tensile properties In contrast to the testing method here, Lam et al employed compressive test which is less sensitive to crack formation (Lam 2008) In comparing the two groups, the degradation process was, indeed, found to be accelerated by the presence of oxidative radicals Although a 62.7% drop of tensile strength was recorded after 12 weeks, related work has shown blood vessel would have sufficiently remodelled in vitro to assume physiological loads in three months (L'Heureux 2006), and will presumably perform similarly in vivo A rabbit model was attempted to validate this, but was abandoned due to technical difficulties and limitations of time 112 Chapter Isolation and Characterisation Of Vascular Progenitors Having identified a suitable scaffold material, I then proceeded to study possible cellular sources for the population of the vascular construct 113 Chapter Isolation and Characterisation Of Vascular Progenitors Isolation and Characterisation Of Vascular Progenitors 114 Chapter Isolation and Characterisation Of Vascular Progenitors 4.1 Introduction Following the advent of vascular tissue engineering (Niklason 1999), a lack of suitable cell sources became apparent (Poh 2005) Functional adult cells harvested from patients for autologous reconstruction of blood vessels have been found to exhibit limited capacity for expansion in vitro, severely compromising the clinical applicability Consequently, significant research has gone into studying alternative sources for smooth muscle (Gong 2006) and endothelial cells (Kim 2008), including differentiation from self-renewable adult stem cell sources (Oswald 2004; Gong 2008) or genetic engineering of terminally differentiated cells for increased life span (McKee 2003) However, the safety and efficacy of such approaches are still poorly understood, and need to be addressed before clinical translation Perinatal sources have emerged as attractive options for cardiovascular tissue engineering (Kadner 2002) Compellingly, tissues such as placenta and the umbilical cord are currently considered medical waste and thus available in abundance In addition, cells from umbilical cord tissue, including the cord and blood, are currently being banked, providing an avenue for the engineering of autologous tissue in the future Furthermore, compared to the adult counterparts, the more primitive umbilical cord derived cells are more primitive and have greater propensity for expansion (Zhang 2009) and integration into host tissue (Au 2008) Apart from umbilical cord tissue, fetal tissue has been proposed as a viable cell source Fetal tissue, obtained from abortuses, are similarly considered medical waste Mesenchymal stem cells, neural stem cells and primitive hepatocytes have all previously been isolated from fetal tissue and shown to have great expansion and 115 Chapter Isolation and Characterisation Of Vascular Progenitors differentiation potential, supporting their utility in stem cell therapy (Kelly 2004; Dan 2006; O'Donoghue 2006) Additionally, these cells are retrieved during the stage of fetal development, and conceivably better able to recapitulate the developmental events, in line with biomimetic tissue engineering principles (Ingber 2006) Indeed, fetal bone marrow derived stem cells have proved to be superior for bone tissue engineering applications (Zhang 2009) Moreover, fetal derived stem cells demonstrate reduced immunogenicity and display immunomodulatory effects, underpinning their potential application for banking and autologous, off-the-shelf use (Gotherstrom 2003) Thus, in this chapter, I describe my studies on the isolation and characterisation of vascular progenitors from perinatal tissue I have described in Chapter the rationale for the use of committed progenitor cells for tissue engineering applications Thus I first investigated the use of perivascular cells from the umbilical cord artery for the generation of a tunica media I then proceeded to isolate and characterise endothelial progenitor cells from umbilical cord blood, as well as fetal haematopoietic tissue, including fetal blood and liver 116 Chapter Isolation and Characterisation Of Vascular Progenitors 4.2 Isolation and Characterisation Of Vascular Progenitors 4.2.1 Umbilical Cord Perivascular Cells (UCPVC) Following enzymatic dissociation of the pervascular region, the retrieved cells were plated and adherent colonies began to appear overnight, which expanded over a few days to confluence The cells are typically spindle-shaped and capable of expansion up to ten passages without senescence (Figure 4-1a) UCPVC were successfully harvested from all samples, following optimisation of harvesting conditions Characterisation of the cells by immunocytochemical staining with monoclonal antibodies in some of the samples at passage (Figure 4-1b) revealed the expression of smooth muscle markers Smooth muscle actin (SMA) staining was widespread In contrast, calponin was found only on a small proportion of cells Smooth muscle myosin heavy chain was absent (smMHC) Following serum starvation, cell proliferation was suppressed, but a larger proportion of SMA and calponin expressing cells were found smMHC was found to be expressed after serum starvation In line with a previous report by Sarugaser et al (Sarugaser 2005), UCPVC expressed markers with a mesenchymal stem cell (MSC) phenotype, such as vimentin, CD73, CD44, CD105 and did not express haematatopoietic nor endothelial markers CD34, CD45, CD14, CD31 and von Willebrand Factor (vWF) In addition, these cells have a doubling time of 54.7±4.3 hours, observed over seven population doublings, and exhibit clonegenicity rates of 47.5±7.5% (Zhang 2009) 117 Chapter Isolation and Characterisation Of Vascular Progenitors Figure 4-1: Isolation of perivascular cells from umbilical cord arteries Umbilical cord perivascular cells demonstrate a fibroblastic morphology Expanded cells express smooth muscle actin and calponin but not myosin heavy chain In contrast, in response to serum starvation, cell proliferation was reduced but expression of smooth muscle markers were increased 118 Chapter Isolation and Characterisation Of Vascular Progenitors Figure 4-2: Isolation of MSC from fetal bone marrow Fetal MSC cells similarly demonstrate a fibroblastic morphology Expanded cells express some smooth muscle actin and calponin but not myosin heavy chain Similar to UCPVC, in response to serum starvation, cell proliferation was reduced but expression of calponin was increased However, SM MHC was not appreciably expressed 119 Chapter Isolation and Characterisation Of Vascular Progenitors 4.2.2 Endothelial Progenitor Cells 4.2.2.1 Adhesion culture When mononuclear cells from umbilical cord blood were plated in EGM2 supplemented with 10% FBS onto collagen coated tissue-culture plates, loose adherent colonies began to appear within one to two weeks The initially spindle shaped cells appeared heterogeneous in nature, eventually forming cobblestone colonies within three weeks, and rapidly proliferated to confluence Cells were lifted by trypsinisation and collectively termed UCB-EPC Subjecting fetal blood mononuclear cells to similar conditions resulted in adherent colonies beginning to appear within one week of culture Over the course of two weeks, more outgrowth colonies emerged, with heterogeneous phenotypes, and rapid proliferation to confluence (Figure 4-3b) Cells were then harvested and collectively termed FB-EPC To further study the differences between fetal blood and umbilical cord blood derived outgrowth cells, I carried out a colony forming assay by enumerating the outgrowth colonies formed after ten days of culture (Figure 4-4) 0.16 colonies were generated per 106 mononuclear cells seeded from cord blood In contrast, 8.6 colonies were generated from 106 mononuclear cells seeded from fetal blood However, it was also noted that the colonies adopted vastly different morphologies, ranging from spindle-shaped, fibroblastic morphologies to cobblestone colonies similar to cord blood derived outgrowth cells In contrast, the culture of cells from all fetal liver using similar methodology generated homogeneously fibroblastic cells with spindle-shaped morphology that 120 ... 11 1. 1.3 .1 1.2 Microthin films for use in layer- by -layer tissue engineering 12 Vascular Tissue Engineering .13 1. 2 .1 Clinical Need For Vascular Tissue Engineering 14 1. 2.2... 1. 1.2 Cellular Assembly 1. 1.2 .1 Cell printing .7 1. 1.2.2 Cell sheet engineering 1. 1.3 Layer- By -Layer Tissue Engineering Using Cell- Scaffold Hybrid Constructs. .. Organ Shortage 1. 1 .1 Classical Tissue Engineering .3 1. 1 .1. 1 Early success in skin and osteoarticular tissue 1. 1 .1. 2 Recent advances .4 1. 1 .1. 3 Limitations 1. 1.2