Biological characterisation of tissue-engineered vascular grafts

This thesis describes the development of tissue-engineered (TE) autologous vascular prostheses, exclusively composed of biological materials and vascular cells, especially for small-diameter applications. A TE construct mimicking the media (middle layer) of a natural blood vessel in terms of morphology, mechanical strength 1 and appropriate cellular functions was produced. In addition, several methods to evaluate tissue homeostasis in the TE constructs were investigated

Physical characterization of vascular grafts cultured in a bioreactor

Tubular scaffolds of collagen and elastin (weight ratio 1:1) with interconnected pores were prepared by freeze drying and crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence or absence of a Jeffamine spacer (poly(propylene glycol)-bis-(2-aminopropyl ether), J230). The crosslinked and uncrosslinked matrices had porosities of 90% and average pore sizes of 131–151 μm. Smooth muscle cells (SMC) were cultured in the crosslinked and uncrosslinked tubular scaffolds under pulsatile flow conditions (mean flow rate 9.6 ml/min, 120 beats/min, pressure 80–120 mmHg). All the constructs could withstand cyclic mechanical strain in the absence of any mechanical support without cracking or suffering permanent deformation. After 7 d, SMC were homogeneously distributed throughout the uncrosslinked and EDC/NHS crosslinked constructs, whereas hardly any cell was observed on the luminal side of J230/EDC/NHS crosslinked matrices. Considering the better mechanical performance of EDC/NHS crosslinked matrices compared to non-crosslinked constructs after 7 d of culture, SMC were dynamically cultured in the former scaffolds for 14 d. During this period, the high strain stiffness of the constructs increased more than two-fold to 38±2 kPa, whereas the low strain stiffness doubled to 8±2 kPa. The yield stress and yield strain were 30±10 kPa and 120±20%, respectively. SMC were homogeneously distributed throughout the EDC/NHS crosslinked collagen/elastin constructs and collagen fibres tended to orient in the circumferential direction

Biological characterisation of vascular grafts cultured in a bioreactor

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications