Marloes Kamphuis

Flexible and elastic porous poly(trimethylene carbonate) structures for use in vascular tissue engineering

Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M(n) = 4.37 x 10(5)) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50-70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 microm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.

Effective seeding of smooth muscle cells into tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering

Porous tubular poly(trimethylene carbonate) (PTMC) scaffolds for vascular tissue engineering, with an inner diameter of 3 mm and a wall thickness of 1 mm, were prepared by means of dip-coating and subsequent leaching of NaCl particles. The scaffolds, with an average pore size of 110 μm and a porosity of 85%, showed a smooth muscle cell (SMC) seeding efficiency of only 10%. To increase the efficiency of cell seeding, these scaffolds were coated with a microporous PTMC outer layer with a thickness of 0.1-0.4 mm, an average pore size of 28 μm, and a porosity of 65%. Coating of the scaffolds with the microporous outer layer did not influence the inner pore structure or the mechanical properties of the scaffolds to a significant extent. The intrinsic permeability of the scaffolds decreased from 60 × 10(-10) m(2) to approximately 5 × 10(-10) m(2) after coating with the microporous outer layer. The latter value is still relatively high indicating that these scaffolds may facilitate sufficient diffusion of nutrients and waste products during cell culturing. The efficiency of SMC seeding determined after 24 h cell adhesion in the scaffolds increased from less than 10% to 43% after coating with the microporous outer layer. The cells were homogeneously distributed in the scaffolds and cell numbers increased 60% during culturing for 7 days under stationary conditions. It is concluded that coating of porous tubular PTMC scaffolds with a microporous PTMC outer layer facilitates effective cell seeding in these scaffolds.

Lowering caveolin-1 expression in human vascular endothelial cells inhibits signal transduction in response to shear stress.

Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm2 for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.

Poly(trimethylene carbonate)-Based Porous Tubular Structures for Tissue Engineering of Small Diameter Blood Vessels

For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. In this study, we prepared crosslinked porous tubular structures from poly(trimethylene carbonate) (PTMC), in which smooth muscle cells (SMCs) were seeded and cultured in a pulsatile bioreactor mimicking the physiological conditions. PTMC was synthesized and porous tubular structures were prepared by dipping coating, cross-linking by g-irradiation, and leaching. SMCs were seeded into the porous structures by perfusion and then the constructs were cultured in a pulsatile bioreactor system. The morphologies, mechnical properties were analyzed and SMCs attachment and proliferation were evaluated by histology studies and CyQuant. Flexible tubular structures were obtained by dip coating with 3mm inner diameter and 1mm wall thickness. The porosity of the structures in wet state reached 85 vol% and the pore sizes were 60-150 mm. PTMC tubular structures showed comparable tensile strength and higher elongation compared with natural blood vessels. A pulsatile bioreactor system mimicking the conditions in vivo (dynamic pressure 70 mmHg, 75 beats/min) was successfully built. Experiements showed 7-day dilation was <10% and variation of diameter at each pulse was <1%. SMCs were homogeneously seeded in the porous scaffolds by perfusion. SMCs proliferate well to form confluent cell layer during a time period of up to 14 days, leading to constructs with even better mechanical performance. PTMC Porous tubular structures were prepared with good microstructures, elasticity and biocompatibility. SMCs were seeded and proliferated well in pulsatile bioreactor system and significant improvement of mechnical strength was observed.