Mesenchymal stem cells in vascular tissue engineering A literature survey

Mesenchymal stem cells in vascular tissue engineering A literature survey M.G.J. Bongers October 2004 BMTE05.02 MSc-thesis Part I Supervisor: prof. dr. M. J. Post Eindhoven University of Technology Faculty of Biomedical Engineering

Contents Introduction 3 1 Arterial blood vessels 4 1.1 Introduction................................... 4 1.2 Structure and function of mature arteries.................. 4 1.3 Vessel wall and mechanical forces....................... 6 1.4 Embryonic development of blood vessels................... 7 1.4.1 Tube formation and stabilization................... 7 1.4.2 Branching and maturation....................... 8 2 Tissue engineering of arterial vessel substitutes 10 2.1 Introduction................................... 10 2.2 Tissue engineering of living grafts....................... 10 2.3 Stem cells and TEBVs............................. 11 3 Mesenchymal stem cells 13 3.1 Introduction................................... 13 3.2 Isolating and culturing MSC from human bone marrow.......... 13 3.3 Characterising MSC.............................. 14 3.4 MSC, endothelial cells and vascular smooth muscle cells have a common origin...................................... 15 3.5 Cytokine-induced in vitro differentiation of MSC.............. 17 3.6 Differentiation of MSC and mechanical stimuli............... 17 Discussion 18 2

Introduction Tissue engineering of blood vessel substitutes has gained interest since the mid-eighties of the past century. This was caused by the poor results which were achieved with the implantation of synthetic static grafts, associated with high occurrence of thrombosis. It was therefore that scientists focussed onto the construction of biologically functional vessel substitutes from vascular cells which allow remodelling in vivo. 23, 26, 31, 44 In recent years, several investigators have started pursuing the use of stem cells as cell source for living vessel grafts, because these undifferentiated cells allegedly can commit themselves to a wide spectrum of cell lineages, including vascular cells. 13, 22 A particular type of multipotent stem cell, the mesenchymal stem cell, seems to have potential for application in clinical vessel engineering because it is constantly and readily available in all humans. The question remains though, how its lineage commitment to vascular cell types can be controlled. In recent years several cytokines and growth factors have been identified which induce mesenchymal stem cells to differentiate into a specific (vascular) cell lineage. 29, 32, 43 Interestingly enough, recent studies indicate that the differentiation of stem cells is related to cytoskeletal tension which is in turn determined by the mechanical microenvironment. 28, 37 This relation being only recently established, science is still far away from the revolutionary practice of controlling stem cell differentiation by means of mechanical cues. This concept would have a major impact on vascular tissue engineering as well. The construction of a vessel graft incorporating endothelium, smooth muscle and fibroblasts by applying different mechanical stimuli to mesenchymal stem cells within a scaffold is of course an appealing idea and creates and opens new avenues for scientific exploration. This literature survey starts by describing the main structural and functional properties of mature arterial vessels. Furthermore, it is elucidated how these structures form from stem cells within the embryo. Thereafter the interplay between the in vivo mechanical environment and the functioning of cells in the vascular wall is discussed. Chapter 3 deals with the developments in vascular tissue engineering over the years and discusses the application of stem cells within this discipline. A general background on mesenchymal stem cells is provided by chapter 4 which also discusses the alleged relation between mesenchymal stem cell differentiation and mechanical cues. By means of a general discussion this leads to the formulation of a research question with associated hypotheses. This in order to contribute to the understanding of the relation between mechanical stimuli and lineage commitment of mesenchymal stem cells in a vascular context. 3

Chapter 1 Arterial blood vessels 1.1 Introduction The first organ to form within the embryo is the circulatory system, as oxygen and nutrient delivery and waste removal are essential to the development, functioning and survival of all other organs. The heart functions as the driving pump of blood flow, while the vessels distribute the blood among the different organ systems. The embryonic development of the vascular system is a complex yet not fully understood process, which this chapter tries to elucidate. First the function and structure of mature arteries is described both on a macroscopic and microscopic level. Thereafter, it is elucidated how these biological structures form within the embryo by the processes of vasculogenesis and angiogenesis. A basic notion of these events is fundamental to whomever would like to tissue-engineer blood vessels using stem cells. 1.2 Structure and function of mature arteries The arterial system basically serves as a transport and distribution system for blood towards organs. The proximal part of this system is formed by elastic arteries and functions as a compliance chamber which accomodates the large pressure and flow gradients imposed by the pumping heart. The distal part is a variable peripheral resistance which maintains a relative high diastolic blood pressure for essential organs such as brains and kidneys. Both functions are reflected within the structure of these vessels. An the macroscopic level all arteries and large arterioles share a typical three-layered wall structure consisting of an intima, media and adventitia. The innermost layer or intima, directly bordering the vessel lumen (see figure 1.1), is formed by a single layer of endothelial cells, called the endothelium and a thin layer consisting of collagen and elasting fibers, the basement membrane. 5 The endothelium s main functions are regulation of vascular permeability and prevention of thrombus formation. Various protein junctions between neighboring endothelial cells control the intercellular 4

Figure 1.1: The composition of a medium-size mature muscular artery in side view and cross section view. Pictures taken from Netter s Atlas of Human Physiology by J.T. Hansen and Molecular regulation of vessel maturation by R.K. Jain (Nature Medicine, 2003) space and in this way the ability of blood proteins and cells to enter the subendothelial space. 25 The antithrombogenic function of the endothelium is provided by the smooth endothelial surface itself, which averts activation of the clotting system. Secondly, the glycocalyx, a structure consisting of various glycosaminoglycans bound to the endothelial surface, repulses platelets and clotting factors. Finally, the protein thrombomodulin bound to the endothelium slows the clotting process by binding the clotting factor thrombin which circulates in the blood. The thrombomodulin-thrombin complex also inactivates other clotting factors. 5 Endothelial cells divide slowly under physiological conditions, about fifty times in a lifetime, but have a high potential for proliferation and migration when the endothelial surface has been damaged. 1 Within the media, the middle wall layer, elastic laminae alternate with layers of smooth muscle cells. Vascular smooth muscle lacks the typical striations of skeletal muscle that are caused by a longitudinal arrangement of its contractile filaments. Within smooth muscle, actin and myosin filaments are spirally arranged. For this reason, force generation by smooth muscle is not as powerful as by skeletal muscle. However, this typical configuration of the contractile system enables an increased shortening range compared to skeletal muscle. 1 From the promixal towards the distal side of the arterial system the amount of smooth muscle cells increases while the number of elastic laminae reduces. Within the promixal elastic arteries, smooth muscle cells regulate vessel stiffness by increasing and decreasing the separation of the elastic laminae. In this way blood flow is controlled. 20 The more distally located muscular arteries control the distribution of blood to the organ systems. They incorporate up to 40 layers of circumferentially orientated smooth muscle which regulate the tonus of the vessel wall tonus. Arterioles, the smallest vessels of the arterial system, mainly consist of smooth muscle cells. Smooth muscle contraction here results in lumen narrowing, which increases the peripheral resistance. 5

The outer layer, the adventitia, formes a connective tissue incorporating longitudinally orientated collagen and elastin, which support the vessel. This layer is present in all arterial vessels except the small arterioles. 20 The adventitia homes fibroblasts, which synthesizes collagen and elastin, the building materials for the extracellular matrix. 30 The other constituents of the adventitia are pericytes, mesenchymal precursor cells of vascular smooth muscle, vasa vasorum, which provide the vessel s own blood and nutrients supply and vasomotor neurons which branch through the adventitia and innervate smooth muscle within the media. 1.3 Vessel wall and mechanical forces The walls of the vessel system are continuously exposed to mechanical forces imposed by circulating blood. These mechanical loads have a cyclic character directly related to the rhythm of the pumping heart and can be divided into both time-dependent shear stress and transmural pressure, as depicted in figure 1.2. The magnitude of the wall shear stress has a periodic character, with maxima ranging from near 0 Pa proximally up to 3 Pa distally. 7, 25 Shear stress is elicited by blood flowing along the endothelium. This stretches the cell membrane of the endothelial cells. Changes within local flow patterns are sensed via receptors within the cell membrane and directly affect internal processes like reorganization of the cytoskeleton. 24, 25 Endothelial cells for example always orientate their longitudinal axis parallel to the direction of flow, i.e. stress vector, τ w (t). Transmural pressure includes hydrostatic pressure relative to the atmospheric pressure within the surrounding tissues, potential energy pressure induced by the pumping heart and dynamic pressure arising from moving blood. Taken together this results in a radial load on the vessel wall, with typical diastolic aortic value around 12 kpa and systolic peaks up to 18 kpa. 42 The transmural pressure circumferentially strains the vessel wall, up to 10% within the human aorta. 10 The resulting stress is buttressed by the medial wall layer, which is reflected in the circumferential orientation of smooth muscle cells. Longitudinally straining of the vessel is largely impeded by surrounding connective tissues. Minor longitudinal stress is buttressed by the longitudinally orientated collagen stress fibers within the adventitia. The mechanical forces experienced by the living constituents of the vessel wall change periodically on a time scale of seconds due to the rhythm of the pumping heart as well as chronically on time scale of years due to pathological conditions. Vascular cells will always try to counteract the changing mechanical conditions they experience. In a hypertensive situation for example, increased circumferential stress within the media induces smooth muscle cells to synthesize matrix. In this way the media enlarges, which reduces hoop stress. The response of both endothelial and smooth muscle cells to stress and strain has been and is being extensively studied both in vivo and in vitro. 17, 27, 39, 40 However, in vitro models do not necessarily resemble the in vivo situation. Straining of in vitro cultured smooth muscle cells for example results in protein synthesis and increased proliferation, whereas the stretching of intact aortic vessels results in smooth muscle cells producing proteins without proliferating. 24 Despite this discrepancy it is evident that the mechanic (micro)environment and vascular wall cells affect and control each other inextricably. 6

Figure 1.2: Illustration of the mechanical forces acting on a straight part of the vessel wall in a local coordinate system. p represents transmural pressure, τ w wall shear stress, ɛ φφ circumferential strain, ɛ zz longitudinal strain. 1.4 Embryonic development of blood vessels 1.4.1 Tube formation and stabilization Oxygen delivery to growing and developing embryonic tissues is initially sufficiently provided by diffusion solely. However, when embryonic tissues enlarge, tissue hypoxia develops while diffusion flux decreases with increasing diffusion distance. Driven by subnormal oxygen tension (< 21% O 2 ) cells increase the production of HIF-1α, which on its turn stimulates the production and secretion of vascular endothelial growth factor (VEGF). VEGF stimulates stem cells from the mesodermal germ layer to differentiate into endothelial cells by binding to their Flk1-receptors. 2 In addition VEGF associates with Flt1-receptors, which stimulates proliferation of the newly formed endothelial cells and their assembly into tubular structures (figure 1.3 A). This process of de novo generation of primitive blood vessels is called vasculogenesis. Endothelial tubes cannot exist without the presence of pericytes. 6, 8 It has been shown that nascent vessels lacking pericytes regress without VEGF being present due to apoptosis of endothelial cells (figure 1.3 D). Vessels associated with pericytes in contrast are no longer dependent on VEGF 6 (as indicated in figure 1, bottom). The growth factors PDGF-β (platelet-derived growth factor) and TGF-β (transforming growth factor) are pivots in this process of vessel stabilization. PDGF-β secreted by endothelial cells attracts mural cells and supports their proliferation, whereas TGF-β promotes genera- 7

Figure 1.3: Formation of immature vasculature. (A) Endothelial tubes form by vasculogenesis or angiogenesis. Long-term exposure to VEGF results in vessel regression (D). The decreasing dependence of VEGF with vessel maturation is indicated. Stabilization is accomplished by generation of basement membrane / ECM and association with mural cells (B). Stabilized vessels can degrade their ECM and loosen pericytes under influence of VEGF and Angiopoietin-2 for the purpose of vessel branching and remodelling. Picture modified from Blood vessel maturation: vascular development comes of age by Darland and D Amore (Nature, 1999). tion of extracellular matrix (ECM) and differentiation of mesenchymal stem cells into pericytes (figure 1.3 B). Binding of Angiopoietin-1 to Tie-2 receptors promotes the interaction between endothelial cells and mural cells. In this way the primitive vessels seal themselves against leaking (figure 1.3 C). 1.4.2 Branching and maturation The newly formed primitive vessels start branching in order to form a vascular network within their homing organ. This process involves proliferation, migration and apoptosis of pericytes and endothelial cells via and favoured by constituents of the ECM. Angiopoietin-2 competes with its antagonist Angiopoietin-1 for Tie-2 binding sites which counteracts the stabilizing Angiopoietin-1 / Tie -2 signal. At the same time the ECM is degraded in a controlled manner by combined action of proteases and protease inhibitors. Together these destabilizing events loosen pericytes and mural cells from the underlying endothelial layer. Proangiogenic factors like VEGF, which are released from matrix during its degradation, stimulate the uncovered endothelial cells to proliferate and migrate via the matrix and to form new vessel sprouts. The formation of vessels from existing ones is called angiogenesis. Vessel branching is further regulated by vessel pruning, which involves controlled cell apoptosis, and by mutual repellence of arterious and venous cells. 18 With time large-diameter vessels form by additional generation of 8

ECM, elastic laminae in case of an artery and the acquisition of extra layers of mural cells. Eventually, the complex process as described here leads to the establishment of a well-organized interconnected system of arteries, arterioles, capillaries, venules and veins. The full postnatal maturation of the arterial wall takes up to twenty years. 20 Angiogenesis however continues throughout a human s lifetime in physiological circumstances like wound healing as well as pathological conditions, for example tumor growth. 9

Chapter 2 Tissue engineering of arterial vessel substitutes 2.1 Introduction The need for arterial substitutes in the clinic is ongoing and unfortunately increasing, caused by the increasing occurence of atherosclerosis in recent years. Current therapies include replacement of occluded arteries by autologous veins or synthetic grafts. Unfortunately, both therapies have important drawbacks. Veins functioning under arterial environmental conditions will damage over time resulting in disfunction. (Coated) synthetic grafts on the other side are associated with problems such as inflammatory responses, infections and thrombosis. It is therefore that scientists resorted to the development of living grafts, which have a lower risk of thrombosis and allow growth, repair and remodelling, which marked the birth of vascular tissue engineering. In this chapter it is discussed where the developments within this discipline have led to thus far. 2.2 Tissue engineering of living grafts In 1986, Weinberg and Bell were the first to present a completely tissue engineered blood vessel (TEBV). 44 As is the common approach in tissue engineering, the construction of this TEBV involved the seeding cells within a stabilising matrix or scaffold which degrades over time. Its supporting function is gradually taken over by the extracellular matrix (ECM) that is deposited by the cells. Although this vessel possessed a typical three-layered arterial wall structure, its burst strength was not a physiological level, which would certainly impede clinical application. However, this study stimulated others to engineer similar TEBVs. A major goal hereby was the optimization of the mechanical integrity in order to enable long term in vivo functioning. Eventually it was shown that by optimizing the deposition of extracellular matrix by vascular cells a physiological burst strength could be achieved. In 1998, Heureux presented a TEBV able to withstand an internal pressure of 2000 mmhg, achieved by supplementing the culture media with ascorbic acid, which enhanced matrix deposition by SMCs and fibroblasts (see figure 10

Figure 2.1: Example of a tissue engineered blood vessel (TEBV) fabricated by Heureux et al. (The FASEB Journal, 1998) 2.1). 26 A year later, Niklason et al. showed that an EC/SMC seeded scaffold transformed into a TEBV with a rupture strength of 2150 mmhg within 8 weeks, by actively perfusing the graft with a medium that enhanced collagen synthesis by SMC. The engineered vessel structurally and functionally (i.e. contractive responsive) resembled native arteries. 31 A step forward towards the clinical relevance of TEBVs had certainly been made. However, at this point, with clinical application in mind, attention had to be paid to the biocompatibility of TEBVs. The usage of allogeneic cells is inevitably associated with inflammatory responses and graft rejection by the body. Autologous vascular cells can be harvested in situ from living vessels, which subsequently can be expanded in vitro and seeded on a scaffold. Yet, this means that intact vessels need to be disrupted, which of course is not preferential. Fortunately, the human body possesses a rather inexhaustible source of so-called stem cells, which can be harvested from bone-marrow or peripheral blood with relative ease and without inflicting severe damage to the body. The application of these cells in vascular tissue engineering till the present day forms the topic of the next section. 2.3 Stem cells and TEBVs Stem cells constitute a mixed population of cells and are generally distinguished from other cell types by two characteristics. First, stem cells possess a relatively unlimited capacity for self-renewal by means of cell division compared to the lifespan of animals. Secondly, they are able to differentiate, under appropriate conditions, from cells with an unspecialized phenotype into committed cells capable of performing specific functions. 1 If these properties can be exploited and controlled, these cells can constitute a perfect autologous cell source for tissue engineers of all disciplines. In recent years, several subpopulations of the stem cell pool have been used in the fabrication of TEBV s. Kaushal 11

et al. for example seeded decellularized grafts with endothelial progenitor cells (EPCs), a subset of the human stem cell population which can easily be isolated from peripheral blood. These grafts were preconditioned into a perfusion system and subsequently implanted in carotid position in a sheep model. The seeded TEBVs showed excellent antithrombogenic properties compared to unseeded grafts and the luminal surface proved to be functioning like endothelium up to 130 days after implantation. In addition the vessel had gained a medial layer incorporating functional smooth muscle cells. 22 While EPC are continuously circulating in the human blood, they form a readily available cell source for vascular tissue engineers. The usefulness of another type of autologous stem cells, so-called umbilical cord cells (UCC), in vessel engineering was investigated by Hoerstrup et al. They produced large caliber arterial conduits by seeding human umbilical cord cells on a scaffold and applying gradually increasing pulsatile nutrient medium flow. 13 TEBVs grown and conditioned in this way resembled the native pulmonary artery structure and tensile strength after 14 days of culture, which showed that umbilical cord stem cells are also promising candidates for vascular tissue engineering. However, the usage of UCCs in clinic will likely be associated with practical and financial problems, while the umbilical cord of individuals will have to be preserved at birth, should it serve as an autologous cell source later in life. Yet there is another subset of the human stem cell population which should receive attention by vascular tissue engineers, which are the mesenchymal stem or precursor cells, also referred to with bone-marrow stromal cells. Kadner et al. recently investigated the potential of bone-marrow stromal cells, as cell source for vascular tissue engineering and showed that constructs seeded with these cells cells developed a matrix composition similar to that of constructs seeded with vascular cells, 21 which is a promising result. From here on we will use the term mesenchymal stem cells or MSC to avoid confusion. The next chapter focusses on the intriguing properties of this type of stem cell. 12

Chapter 3 Mesenchymal stem cells 3.1 Introduction Several types of stem cells and precursor cells are present in many if not all adult tissues for the purpose of tissue renewal and repair. Tissue-specific precursor cells possess the ability to differentiate into the committed cells of the homing tissue, while multipotent stem cells, which are mainly located in the bone-marrow can commit themselves to a wider spectrum of cell types. The multipotent stem cell pool in the adult bone marrow compartment consists of two sub-populations, the hematopoietic stem cells, which are beyond the scope of this discussion, and the mesenchymal stem cells (MSC), which have our attention. Adult MSC from bone-marrow prove their true stem cell nature by the fact that a single MSC is self-renewing and that it can differentiate into a wide variety of lineages. 34 Namely, over the years it become evident by several in vitro and in vivo studies that adult MSC are able to form cells of various connective tissues, including adipocytes, osteocytes, skeletal myocytes, cardiomyocytes, fibroblasts, chondrocytes, tenocytes and bone-marrow stromal cells. 29, 33, 34, 41 Clinically, MSC have already been used for the treatment of children s osteogenesis imperfecta 3 and the regeneration of damaged articular cartilage 35 and tendon. 4 Worth noting is the phenomenon that mesenchymal stem cell are able to commit themselves to cell lineages which are not from mesenchymal origin. This socalled plasticity is a controversial and dubious phenomenon which has also been reported for other stem cell populations. 14, 16, 19, 29 Considering their (in vivo) multipotency, MSC truly have unexploited potential for tissue engineering in general, especially if we regard their easy isolation and expansion procedures, which are the topic of the next section. 3.2 Isolating and culturing MSC from human bone marrow Bone-marrow is aspirated from a donor s posterior iliac crest using a syringe filled with heparin in order to prevent blood clotting. This aspirate is washed in phosphate buffered 13

Figure 3.1: Monolayer culture of human MSC passage 3, day 1 saline (PBS) and subjected to density gradient centrifugation, a procedure which separates erythrocytes from nuclear cells. If the nuclear cells are carefully aspirated and cultured, mesenchymal stem cells will attach to the culture dish, spread like fibroblasts and form colonies within 4-6 days. Non-adherent cells are removed with medium changes. Before colonies reach other, cells are trypsinized and subcultured in a 1:3 ratio. From then on cells are passaged at 80-90% confluence. The serum which supplements the culture medium is carefully tested in advance in order to maintain an undifferentiated phenotype. It has been observed that after cell passage 7 the multi-potency of MSC devalues and that phenotype changes. However, a normal karyotype and telomerase activity are maintained up to passage 12. 29, 33 3.3 Characterising MSC MSC in in vitro culture are adherent, contact-inhibited cells with generally a spindleshaped morphology (see figure 3.1). The MSC phenotype can be confirmed by checking the chondrogenic, adipogenic and osteogenic potency of a batch of cells while for this purpose well-established protocols are available (see section 3.5). It is however difficult to come up with a generally accepted set of cellular antigens that uniquely identify MSC. The probing of MSC with antibodies against 70 different cellular antigens has indicated that these cells express several surface proteins associated with other committed cell types but in different combinations, 34 i.e. MSC express markers which are characteristic for endothelial, epithelial and muscle cells. 3, 41 It is common practice to distinguish MSC from contaminating hematopoietic cells by 14

selecting non-adherent cells which do not express specific hematopoietic markers. MSC freshly isolated by bone-marrow puncture, do not express the hematopoietic marker CD50. 41 Later on, after culture, the hematopoietic markers CD34, CD14 and CD45 disappear. 3, 34 The identification of MSC by bone-marrow punctures based on positive expression of markers is still problematic. There is however increasing support for the notion that expression of STRO-1, SH2, SH3 and SH4 identifies MSC in in vitro culture. 3 It is probably due to the ill-defined nature of MSC that investigators nowadays often use commercially available mesenchymal stem cell lines, which have a guaranteed purity. Others however isolate cells from animals or humans and characterize them using flow cytometry according to their own defined sets of markers. This makes it rather troublesome to compare the results of different studies. 3.4 MSC, endothelial cells and vascular smooth muscle cells have a common origin All cells and tissues of the adult body derive from the so-called embryonic germ layers. These three cell layers are present in the gastrula stage of embryonic development and represent three distinct cell lineages, denoted by endoderm (inner layer), mesoderm (middle layer) and ectoderm (outer layer) (see figure 3.2). Each layer gives rise to an unique set of adult tissues as illustrated by table 3.1. It appears from this table that adult bone marrow cells (including mesenchymal stem cells), endothelial cells, vascular smooth muscle cells and fibroblasts all derive from the mesoderm. This close embryonic relationship justifies studies focussed on guiding differentiation of adult mesenchymal stem cells into vascular cells. Figure 3.2: Cross-section of a frog gastrula 15

Table 3.1: Overview of adult tissues originating from the three germ layers Endoderm inner lining of digestive tract liver, pancreas inner lining of respiratory tract larynx, trachea, lung urinary bladder, urethra, vagina most glands Mesoderm vascular system bone (marrow), cartilage muscle (all types) several connective tissues gonads Ectoderm skin, hair, nails brain, nervous system eyes ears 16

3.5 Cytokine-induced in vitro differentiation of MSC For the differentiation of MSC into members of the connective tissue family (adipocytes, chondrocytes and osteoblasts) well-established protocols exists. Adipogenic differentiation requires postconfluent cultures and the presence of isobutylmethylxantine and insulin in serum-containing medium. MSC differentiate into chondrocytes when pelleted and provided with serum-free medium containing a member of the transforming growth factor-β (TGF-β) superfamily, i.e. TGF-β1, TGF-β2 or TGF-β3. Osteogenesis is stimulated by supplying monolayer-cultured MSC with medium containing serum, β- glycerol-phosphate, ascorbic acid-2-phosphate and dexamethason. Next to these generally accepted differentiating treatments there are scattered reports on differentiation of MSC into for examples tenocytes, bone marrow stromal cells, skeletal muscle cells, smooth muscle cells and cardiac muscle cells by culturing in specific supplemented media. 29 3.6 Differentiation of MSC and mechanical stimuli Well-established protocols for in-vitro differentiation of bone-marrow derived mesenchymal stem cells into pure populations of osteocytes, chondrocytes and adipocytes based on modification of the culture medium currently exist. 33 In recent years evidence has been provided that mechanical stimuli are also able to influence differentiation of stem cells in general. 11, 37 Simmons et al. recently showed that this is also valid for mesenchymal stem cells. They reported that equibiaxial cyclic strain applied to human mesenchymal stem cells cultured in osteogenic medium decreased proliferation and increased deposition of mineralized matrix, a late differentiation marker of osteogenesis. 38 Nonetheless, the control of mesenchymal stem cell differentiation by means of mechanical cues alone has received little attention. A literature search yielded only one study which investigated this topic. Recently, Huang et al. studied the isolated effect of cyclic compressive loading on the chondrogenic differentiation of rabbit bone-marrow derived MSC and compared this with typical TGF-β-induced chondrogenic differentiation of the same MSC. It was found that both treatments yielded a similar chondrocytic phenotype, which shows that compressive loading alone can induce chondrocytic differentiation of rabbit MSC. 15 This justifies further investigation of the relation between mechanical stimuli and differentiation patterns of MSC. 17

Discussion It appeared that several types of stem cells have potential for application in vascular tissue engineering. For the present study however we disregard endothelial precursor cells and umbilical cord cells, and concentrate on the usefulness of mesenchymal stem cells for vascular tissue engineering. Chapter 2 mentioned the study by Kadner et al. 21 which indicated that scaffold-seeded mesenchymal stem cells deposit an extracellular matrix with excellent mechanical properties for vascular application. Furthermore it appeared that they share a common embryonic origin with vascular wall cells. Therefore, this study should be continued by investigation focussed on the question how to control the transformation of these mesenchymal stem cells into functional vascular wall cells. Considering the strong postnatal influence of the mechanical environment on the functioning and remodelling of the vessel as discussed in chapter 2 it might be questioned if mechanical cues can influence the differentiation of adult mesenchymal stem cells within the vascular wall, for example for the purpose of remodelling or repair. It is conceivable that pericytes in the adventitia can differentiate into smooth muscle cells in response to typical local mechanical forces. For instance, differentiation into cartilage in response to compressive load has been shown. 15 Furthermore it is known that mesenchymal stem cells from bone-marrow circulate in the blood and nestle themselves in various tissues. 36 Most interestingly, these circulating mesenchymal stem cells migrate into the subendothelial space of the vessel wall and form endothelial cells, smooth muscle cells as well as fibroblasts. 9, 12 In this way these precursors contributed to the stabilization of myocardial infarction. The fluid flow patterns around such lesions and local forces in the vessel wall could accomplish this differential lineage commitment. For these reasons and in order to contribute to the understanding of lineage commitment of mesenchymal stem cells under influence of mechanical stimuli, I formulated the following research question: Can mechanical stimuli induce differentiation of mesenchymal stem cells into vascular wall cells? Explicitly, I hypothesize that the application of fluid shear flow to mesenchymal stem cells results in their differentiation into endothelial cells. Furthermore I hypothesize that the application of cyclic strain causes mesenchymal stem cells to differentiate into smooth muscle cells. The reader is referred to part II of this thesis, which discusses the experimental approach used to validate these hypotheses and answer the proposed research question. 18

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