Poly-ε-caprolactone scaffold and reduced in vitro cell culture: beneficial effect on compaction and improved valvular tissue formation.

Tissue-engineered heart valves (TEHVs), based on polyglycolic acid (PGA) scaffolds coated with poly-4-hydroxybutyrate (P4HB), have shown promising in vivo results in terms of tissue formation. However, a major drawback of these TEHVs is compaction and retraction of the leaflets, causing regurgitation. To overcome this problem, the aim of this study was to investigate: (a) the use of the slowly degrading poly-ε-caprolactone (PCL) scaffold for prolonged mechanical integrity; and (b) the use of lower passage cells for enhanced tissue formation. Passage 3, 5 and 7 (P3, P5 and P7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips. After 4 weeks of culture, compaction, tissue formation, mechanical properties and cell phenotypes were compared. TEHVs were cultured to observe retraction of the leaflets in the native-like geometry. After culture, tissues based on PGA-P4HB scaffold showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Tissue formation, stiffness and strength were increased with decreasing passage number; however, this did not influence compaction. Ovine PCL-based tissues did render less strong tissues compared to PGA-P4HB-based tissues. No differences in cell phenotype between the scaffold materials, species or cell passage numbers were observed. This study shows that PCL scaffolds may serve as alternative scaffold materials for human TEHVs with minimal compaction and without compromising tissue composition and properties, while further optimization of ovine TEHVs is needed. Reducing cell expansion time will result in faster generation of TEHVs, providing more rapid treatment for patients.

Quantification of the temporal evolution of collagen orientation in mechanically conditioned engineered cardiovascular tissues.

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network.

Straining mode-dependent collagen remodeling in engineered cardiovascular tissue.

Similar to native cardiovascular tissues, the mechanical properties of engineered cardiovascular constructs depend on the composition and quality of the extracellular matrix, which is a net result of matrix remodeling processes within the tissue. To improve tissue remodeling, and hence tissue mechanical properties, various mechanical conditioning protocols, such as strain-based or flow-based conditioning, have been applied to engineered cardiovascular constructs with promising results. We hypothesize that tissue remodeling is dependent on the mode of straining. Therefore, the effects of two modes of straining, being either static or dynamic, were quantified on several indices of tissue remodeling. Differences in matrix composition (collagen and glycosaminoglycans [GAGs]) and maturity (collagen cross-links) were quantified with time on gene expression and protein levels. In addition, the secretion of specific collagen remodeling markers (matrix metalloproteinase-1), collagen synthesis marker (procollagen type I carboxy-terminal propeptide, PIP), and collagen degradation marker (carboxyterminal telopeptide of type I, ICTP) was investigated with time. Static strain stimulated collagen gene expression and production with time. Dynamic straining resulted in (1) lower collagen gene expression and production, but (2) enhanced collagen cross-link expression and density, and GAG production, and (3) stimulated collagen remodeling processes, as expressed by enhanced production of remodeling markers. Thus, despite a lower collagen production, the quality of the neotissue was enhanced by a dynamic straining component. These straining mode-dependent remodeling responses allow us for the first time to balance collagen and cross-link production and, thus, to fine tune tissue mechanical properties via mechanical conditioning protocols. This is of utmost importance for cardiovascular tissue engineering, where insufficient mechanical properties are currently a main limiting factor for present in vivo application.

Intermittent straining accelerates the development of tissue properties in engineered heart valve tissue.

Tissue-engineered heart valves lack sufficient amounts of functionally organized structures and consequently do not meet in vivo mechanical demands. To optimize tissue architecture and hence improve mechanical properties, various in vitro mechanical conditioning protocols have been proposed, of which intermittent straining is most promising in terms of tissue properties. We hypothesize that this is due to an improved collagen matrix synthesis, maturation, and organization, triggered by periodic straining of cells. To test this hypothesis, we studied the effect of intermittent versus constrained conditioning with time (2-4 weeks), using a novel model system of human heart valve tissue. Temporal variations in collagen production, cross-link density, and mechanical properties were quantified in engineered heart valve tissue, cyclically strained for 3-h periods, alternated with 3-h periods rest. In addition, an innovative method for vital collagen imaging was used to monitor collagen organization. Intermittent straining resulted in increased collagen production, cross-link densities, collagen organization, and mechanical properties at faster rates, as compared to constrained controls, leading to stronger tissues in shorter culture periods. This is of utmost importance for heart valve tissue engineering, where insufficient mechanical properties are currently the main limiting factor.

Living heart valve and small-diameter artery substitutes--an emerging field for intellectual property development.

Cardiovascular diseases, such as heart valve dysfunction and coronary artery stenosis, are next to cancer the leading cause of death in the US. Treatments involve replacement of the heart valve or bypassing the obstructed coronary artery with a small-diameter vascular graft. The major limitation of currently used replacements is their inability to grow, adapt and repair in the patient. Considering the increasing age of the population and the subsequent increase in cardiovascular disease incidence, efforts to improve existing replacements and unraveling novel types of replacements are of paramount importance. Cardiovascular tissue engineering represents a rapid evolving field of research, providing living heart valve and small-diameter vascular substitutes with the ability to grow, adapt and repair after implantation. Various tissue engineering approaches are being employed, based on in vivo and/or in vitro tissue formation. This review provides an overview of the current heart valve and small-diameter vascular replacements and presents the status and future developments within the various tissue engineering approaches. The potential of tissue engineering for the development of living heart valve and small-diameter vascular substitutes is reflected in the numerous patents related to this emerging field of research.

Structure and torsion of the normal and situs inversus totalis cardiac left ventricle. I. Experimental data in humans.

In 1926, the famous American pediatric cardiologist, Dr. Helen B. Taussig, observed that in situs inversus totalis (SIT) main gross anatomical structures and the deep muscle bundles of the ventricles were a mirror image of the normal structure, while the direction of the superficial muscle bundles remained unchanged (H. B. Taussig, Bull Johns Hopkins Hosp 39: 199-202, 1926). She and we wondered about the implication of this observation for left ventricular (LV) deformation in SIT. We used magnetic resonance tagging to obtain information on LV deformation, rotation, and torsion from a series of tagged images in five evenly distributed, parallel, short-axis sections of the heart of nine controls and eight persons with SIT without other structural (cardiac) defect. In the controls, during ejection, the apex rotated counterclockwise with respect to the base, when looking from the apex. Furthermore, the base-to-apex gradient in rotation (torsion) was negative and similar at all longitudinal levels of the LV. In SIT hearts, torsion was positive near the base, indicating mirrored myofiber orientations compared with the normal LV. Contrary to expectations, torsion in the apical regions of SIT LVs was as in normal ones, reflecting a normal internal myocardial architecture. The transition zone with zero torsion, found between the apex and base, suggests that the heart structure in SIT is essentially different from that in the normal heart. This provides a unique possibility to study regulatory mechanisms for myocardial fiber orientation and mechanical load, which has been dealt with in the companion paper by Kroon et al.

Effect of strain magnitude on the tissue properties of engineered cardiovascular constructs.

Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues.

The role of collagen cross-links in biomechanical behavior of human aortic heart valve leaflets--relevance for tissue engineering.

A major challenge in tissue engineering of functional heart valves is to determine and mimic the dominant tissue structures that regulate heart valve function and in vivo survival. In native heart valves, the anisotropic matrix architecture assures sustained and adequate functioning under high-pressure conditions. Collagen, being the main load-bearing matrix component, contributes significantly to the biomechanical strength of the tissue. This study investigates the relationship between collagen content, collagen cross-links, and biomechanical behavior in human aortic heart valve leaflets and in tissue-engineered constructs. In the main loading direction (circumferential) of native valve leaflets, a significant positive linear correlation between modulus of elasticity and collagen cross-link concentration was found, whereas no correlation between modulus of elasticity and collagen content was found. Similar findings were observed in tissue-engineered constructs, where cross-link concentration was higher for dynamically strained constructs then for statically cultured controls. These findings suggest a dominant role for collagen cross-links over collagen content with respect to biomechanical tissue behavior in human heart valve leaflets. They further suggest that dynamic tissue straining in tissue engineering protocols can enhance cross-link concentration and biomechanical function.

Cardiac fiber orientation and the left-right asymmetry determining mechanism.

The invariant nature of body situs within and across vertebrate species implies that a highly conserved pathway controls the specification of the left-right (L/R) axis. Situs-specific morphogenesis begins at the end of this pathway and leads to normal organ arrangement, also known as situs solitus. Occasionally, individuals have a complete, mirror image reversal of this asymmetry, called situs inversus totalis (SIT). In these individuals, gross anatomy is mirror imaged. However, the helical myofiber pattern within the left ventricle (LV) wall is only partially mirror imaged: apical and superficial basal fiber orientation are as in normal persons, whereas the deeper basal layers have an inverted fiber orientation. Because of this bivalent fiber orientation pattern, LV deformation in humans with SIT is mirror imaged only near the base, but near the apex it is as in normal subjects. Apparently, the embryonic L/R controlling genetic pathway does determine situs-specific gross anatomy morphogenesis, but it is not the only factor regulating fiber architecture within the LV wall.