Mechano-regulation of collagen architecture in cardiovascular tissue engineering

Abstract

Clinically available heart valve replacements consist of non-living materials, lacking the ability to grow with the patient. Therefore, several re-operations are necessary to replace the valve with a larger one. In addition, there is a large need for living blood vessel substitutes that overcome the drawbacks of current vascular prostheses. Cardiovascular tissue engineering focuses on the creation of living heart valves and blood vessels that have the potential to grow and remodel in vivo. In brief, cells are acquired from a patient and seeded on a biodegradable material or scaffold. The cell-seeded scaffold is cultured in a bioreactor where mechanical and biochemical stimuli are applied to stimulate tissue development. After several weeks, the scaffold is replaced by tissue produced by the patient’s own cells. Ideally, the tissue can be implanted in the patient to replace dysfunctional tissue. In order to be mechanically functional, such engineered cardiovascular tissues should incorporate load-bearing structures to withstand (changes in) the hemodynamic environment. The mechanical properties of cardiovascular tissues are dictated by a well organized network of collagen fibers. The collagen architecture is influenced by the mechanical environment of the tissue. Hence, mechanical conditioning is considered to be an important regulator to create engineered cardiovascular tissues with defined load-bearing structures and mechanical properties. The aim of the research presented in this thesis is to elucidate the effects of well-defined mechanical conditioning protocols on the collagen architecture in engineered cardiovascular tissues. In this thesis, three main aspects of the collagen architecture in engineered cardiovascular tissues are quantified: collagen amount, collagen cross-link density, and collagen fiber orientation. To systematically investigate the effects of mechanical conditioning on collagen architecture, a model system has been employed. The tissues consist of rectangular strips of rapidly degrading polyglycolic acid based scaffolds seeded with human vascular cells. The advantage of the model system is that it reduces the number of required cells and it allows for the application of pre-defined strain regimes to multiple engineered tissues simultaneously. Static conditioning is applied in longitudinal direction by constraining the tissues at their outer ends. In addition, different uniaxial straining protocols, including continuous dynamic strain (4%, 1Hz, for 10 days and 4 weeks) and intermittent dynamic strain (3 hours on/off, 4%, 1Hz, for 2, 3, and 4 weeks) are applied using a modified straining system. The temporal effects of static and dynamic conditioning on collagen amount and cross-links are assessed up to 10 days of culture from gene and protein measurements. Both conditioning modes upregulate collagen and cross-link expression and protein content with time. Dynamic strain results in lower collagen expression and content, but enhances collagen cross-link expression and density, when compared with static conditioning. To study the effects of static and dynamic conditioning on the mechanical properties of newly formed tissue, the culture period has been extended to 4 weeks. By that time, the initial scaffold has lost its mechanical integrity and the mechanical properties of the constructs are only determined by the newly formed tissue. Compared to 4 weeks of static conditioning, continuous dynamic strain results in similar collagen contents but higher cross-link densities, which correlate to improved mechanical properties. These findings indicate that, despite a similar collagen amount, the quality and structural integrity of the tissue can be improved by dynamic strain via an increase in collagen cross-link densities. A novel technique has been used to quantify the orientations of the newly formed collagen fibers, based on 3D vital imaging using two-photon microscopy combined with image analysis. These collagen fiber orientation analyses reveal that mechanical conditioning induces collagen alignment in the constrained and intermittently strained directions. Importantly, intermittent dynamic strain improves and accelerates the alignment of the collagen fibers in the straining direction compared to constraining only. In addition, intermittent dynamic strain results in increased collagen production, cross-link densities, and mechanical properties at faster rates compared to static conditioning, leading to stronger tissues at shorter culture periods. In conclusion, these studies show that, when compared to constrained tissue culture, continuous dynamic strain does not increase the amount of collagen in the tissue but does enhance cross-link densities and collagen fiber alignment. Intermittent dynamic strain increases and accelerates the production of collagen, cross-links, and collagen fiber alignment. Therefore, intermittent dynamic strain can be used to accelerate the creation of load-bearing tissues with a well organized collagen architecture. This is of considerable importance for cardiovascular tissue engineering, where a functional load-bearing capacity is a prerequisite for in vivo application.