Several biological changes will occur in the arterial wall as a consequence of cardiological interventions, like balloon angioplasty or brachytherapy, in the treatment of stenosis (narrowing) in arteries. In many cases, these changes lead to restenosis and cause a decrease in the effect of the treatment. Brachytherapy is a local irradiation of the arterial wall and is thought to in??uence the changes in the arterial wall and decrease the negative effects of balloon angioplasty. To understand the adaptive processes that play a role in the response to cardiological interventions and to predict the effects of balloon angioplasty or brachytherapy, a mechano-biological adaptation model that describes the cellular, morphological, and mechanical response of the arterial wall can be helpful. Before such a model can be developed, the response of the arterial wall to those cardiological interventions must be studied and quanti??ed. Here, the mechano-biological adaptation is studied in an in-vitro model. In such a model, an excised arterial segment can be kept viable for longer time by clamping it in an experimental set-up in which the relevant physiological circumstances, like temperature, blood pressure, blood ??ow, and wall stretch, are mimicked and controlled. This in-vitro model is designed and coronary arteries excised from porcine hearts that are obtained from the slaughterhouse are used. Furthermore, methods to determine and quantify the cellular and morphological state of the arterial tissue are developed. These methods are used to study the biological characteristics of the arterial wall before and after cardiological interventions. Therefore, a number of experiments are performed to test the in-vitro model and to determine whether the cellular and morphological state of the arterial segment after culturing in the set-up, without interventions, is similar to the state it is in just after excision. Subsequently, experiments are performed in which cultured arterial segments are treated with balloon angioplasty, either or not followed by brachytherapy. Finally, to study whether the results found can be translated in to a mechano-biological adaptation model the in-vitro model is used to determine the mechanical characteristics of the arterial wall, with or without vascular tone. In chapter 1, a concise outline is presented of atherosclerosis, its treatment, and the speci??c characteristics and problems related to interventional techniques. In chapter 2, the in-vitro model that has been developed is described in detail together with the protocol that has been used for the preparation and nutrition of porcine coronary arterial segments. The main limitation of the model is that wall shear stress and the circumferential strain applied to the arterial wall are relatively low compared to the in-vivo situation. This may have in??uenced the cellular processes to be studied. Although this limitation is present, this model is assumed to be representative to imitate the physiological environment in a coronary artery for short term experiments and the global response to cardiological interventions could be studied by using methods determining morphology, mitotic activity, metabolic activity and the mechanical characteristics of the arterial wall. In the study described in chapter 3, it is demonstrated that standard functionality tests are not successful in porcine coronary segments. Furthermore, ??uorescencebased metabolic activity analysis is not useful because of the auto??uorescence of elastin that is present in the arterial wall. The morphological tests used in this study (H&E, MTC) stain the different components of the arterial wall, like cells, nuclei, and collagen. In metabolically active cells, a tetrazolium salt (MTT) is converted into another salt, named formazan. By measuring the concentration of formazan, the metabolic activity of the cells within the tissue is determined. In mitotically active cells, BrdU is incorporated into the newly formed DNA. With immunohistochemical techniques, the cells containing new DNA can be visualized. The morphological (H&E, Masson Trichrome), metabolic (MTT), and proliferation (BrdU) analysis provide suf??cient information on the response of the in-vitro cultured arterial segment. In the next part of the study, reported in chapter 4, the speci??city and sensitivity of the MTT assay are tested and it is shown that the left circum??ex coronary artery (LCX) can serve as a control for the left anterior descending coronary artery (LAD). It is found that the MTT assay has a biological variance of less than 20% and that the response of the LCX after three different culturing protocols is the same as that of the LAD. Therefore, it is concluded that the LCX could be used as a static control for the dynamically in-vitro cultured LAD. The effects of in-vitro culturing is further studied and described in chapter 5. The changes in metabolic activity after 72h of culturing are determined. Surprisingly, the statically cultured LCX shows more increase in metabolic activity than the dynamically cultured LAD. After prolonging the culturing time to 168h, this is reversed; the LAD shows a signi??cantly higher metabolic activity. It is not clear yet whether the metabolic activity resembles the in-vivo situation. In the same samples, also the changes in morphology and proliferation are determined. It is concluded that after 168h of culturing the arterial wall structure is maintained better by the application of physiological mechanical loads as exerted by the in-vitro model. Furthermore, it is shown that the proliferation of the vascular cells in the in-vitro set-up is increased compared to reference data for the in-vivo situation. This is even more pronounced in the dynamically cultured LAD. Therefore, it is concluded that dynamic in-vitro culturing bene??ts the maintenance of metabolic activity compared to static culturing. In the next part of this study, chapter 6, the cultured arterial segment is treated with balloon angioplasty. This does not increase the metabolic activity of the vascular cells but the proliferation increases more than 2-fold. The proliferating cells are mainly located at the internal elastic lamina (IEL) side of the media and resulted in local thickening of the media (neo-media formation), thereby decreasing the arterial lumen; a situation that might be a ??rst step towards restenosis. In chapter 7 it is shown that the sharp increase in proliferation as a result of balloon angioplasty is inhibited when the angioplasty is immediately followed by brachytherapy. No new cells are identi??ed near the IEL after brachytherapy. This treatment also decreases the metabolic activity of the vascular cells. In chapter 8, the mechanical characteristics of the coronary segments determined just after excision are presented. The pressure-diameter relationships are measured under normal or relaxed state of the vascular smooth muscle cells present in the arterial wall. It is shown that the mechanical characteristics of the arterial wall, expressed by the distensibility, are not different for the normal and relaxed state at physiological pressures, although the inner radius increases signi??cantly without vascular tone. The stress-strain relationship is described by a constitutive model in which the artery is represented as an distensible thick-walled tube that is made of a ??ber-reinforced matrix material. Although this model is presented as micro-structural, it becomes clear that given the accuracy of the measurements and using one loading protocol only, the computed material parameter set is not unique so that physiological interpretation of the parameters is hampered. In conclusion, the in-vitro model developed in this study is suitable to study the response to catheter-based cardiological interventions. Adequate protocols to test and quantify metabolic activity and cell proliferation are developed, although no speci??c information on individual cells lines is obtained yet. The global changes in metabolic activity, morphology and proliferation give a ??rst glimpse into the effects of in-vitro culturing and into the response to injury by balloon angioplasty or brachytherapy. They form the basis for the development of adaptation models, that can give a ??rst prediction of the impact of interventions on the arterial wall. Further studies extending the culture period to more than 7 days and to specifying the response of individual cell lines are mandatory for the further development of mechano-biological models.
|Kwalificatie||Doctor in de Filosofie|
|Datum van toekenning||20 apr 2005|
|Plaats van publicatie||Eindhoven|
|Status||Gepubliceerd - 2005|