Anita Anita Driessen-Mol

Minimally-invasive implantation of living tissue engineered heart valves: a comprehensive approach from autologous vascular cells to stem cells.

OBJECTIVES The aim of this study was to demonstrate the feasibility of combining the novel heart valve replacement technologies of: 1) tissue engineering; and 2) minimally-invasive implantation based on autologous cells and composite self-expandable biodegradable biomaterials. BACKGROUND Minimally-invasive valve replacement procedures are rapidly evolving as alternative treatment option for patients with valvular heart disease. However, currently used valve substitutes are bioprosthetic and as such have limited durability. To overcome this limitation, tissue engineering technologies provide living autologous valve replacements with regeneration and growth potential. METHODS Trileaflet heart valves fabricated from biodegradable synthetic scaffolds, integrated in self-expanding stents and seeded with autologous vascular or stem cells (bone marrow and peripheral blood), were generated in vitro using dynamic bioreactors. Subsequently, the tissue engineered heart valves (TEHV) were minimally-invasively implanted as pulmonary valve replacements in sheep. In vivo functionality was assessed by echocardiography and angiography up to 8 weeks. The tissue composition of explanted TEHV and corresponding control valves was analyzed. RESULTS The transapical implantations were successful in all animals. The TEHV demonstrated in vivo functionality with mobile but thickened leaflets. Histology revealed layered neotissues with endothelialized surfaces. Quantitative extracellular matrix analysis at 8 weeks showed higher values for deoxyribonucleic acid, collagen, and glycosaminoglycans compared to native valves. Mechanical profiles demonstrated sufficient tissue strength, but less pliability independent of the cell source. CONCLUSIONS This study demonstrates the principal feasibility of merging tissue engineering and minimally-invasive valve replacement technologies. Using adult stem cells is successful, enabling minimally-invasive cell harvest. Thus, this new technology may enable a valid alternative to current bioprosthetic devices.

Decellularized homologous tissue-engineered heart valves as off-the-shelf alternatives to xeno- and homografts.

Decellularized xenogenic or allogenic heart valves have been used as starter matrix for tissue-engineering of valve replacements with (pre-)clinical promising results. However, xenografts are associated with the risk of immunogenic reactions or disease transmission and availability of homografts is limited. Alternatively, biodegradable synthetic materials have been used to successfully create tissue-engineered heart valves (TEHV). However, such TEHV are associated with substantial technological and logistical complexity and have not yet entered clinical use. Here, decellularized TEHV, based on biodegradable synthetic materials and homologous cells, are introduced as an alternative starter matrix for guided tissue regeneration. Decellularization of TEHV did not alter the collagen structure or tissue strength and favored valve performance when compared to their cell-populated counterparts. Storage of the decellularized TEHV up to 18 months did not alter valve tissue properties. Reseeding the decellularized valves with mesenchymal stem cells was demonstrated feasible with minimal damage to the reseeded valve when trans-apical valve delivery was simulated. In conclusion, decellularization of in-vitro grown TEHV provides largely available off-the-shelf homologous scaffolds suitable for reseeding with autologous cells and trans-apical valve delivery.

Off-the-shelf human decellularized tissue-engineered heart valves in a non-human primate model

Heart valve tissue engineering based on decellularized xenogenic or allogenic starter matrices has shown promising first clinical results. However, the availability of healthy homologous donor valves is limited and xenogenic materials are associated with infectious and immunologic risks. To address such limitations, biodegradable synthetic materials have been successfully used for the creation of living autologous tissue-engineered heart valves (TEHVs) in vitro. Since these classical tissue engineering technologies necessitate substantial infrastructure and logistics, we recently introduced decellularized TEHVs (dTEHVs), based on biodegradable synthetic materials and vascular-derived cells, and successfully created a potential off-the-shelf starter matrix for guided tissue regeneration. Here, we investigate the host repopulation capacity of such dTEHVs in a non-human primate model with up to 8 weeks follow-up. After minimally invasive delivery into the orthotopic pulmonary position, dTEHVs revealed mobile and thin leaflets after 8 weeks of follow-up. Furthermore, mild-moderate valvular insufficiency and relative leaflet shortening were detected. However, in comparison to the decellularized human native heart valve control - representing currently used homografts - dTEHVs showed remarkable rapid cellular repopulation. Given this substantial in situ remodeling capacity, these results suggest that human cell-derived bioengineered decellularized materials represent a promising and clinically relevant starter matrix for heart valve tissue engineering. These biomaterials may ultimately overcome the limitations of currently used valve replacements by providing homologous, non-immunogenic, off-the-shelf replacement constructs.

How to Make a Heart Valve: From Embryonic Development to Bioengineering of Living Valve Substitutes

Cardiac valve disease is a significant cause of ill health and death worldwide, and valve replacement remains one of the most common cardiac interventions in high-income economies. Despite major advances in surgical treatment, long-term therapy remains inadequate because none of the current valve substitutes have the potential for remodeling, regeneration, and growth of native structures. Valve development is coordinated by a complex interplay of signaling pathways and environmental cues that cause disease when perturbed. Cardiac valves develop from endocardial cushions that become populated by valve precursor mesenchyme formed by an epithelial-mesenchymal transition (EMT). The mesenchymal precursors, subsequently, undergo directed growth, characterized by cellular compartmentalization and layering of a structured extracellular matrix (ECM). Knowledge gained from research into the development of cardiac valves is driving exploration into valve biomechanics and tissue engineering directed at creating novel valve substitutes endowed with native form and function.

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.

Strain-dependent modulation of macrophage polarization within scaffolds.

Implanted synthetic substrates for the regeneration of cardiovascular tissues are exposed to mechanical forces that induce local deformation. Circulating inflammatory cells, actively participating in the healing process, will be subjected to strain once recruited. We investigated the effect of deformation on human peripheral blood mononuclear cells (hPBMCs) adherent onto a scaffold, with respect to macrophage polarization towards an inflammatory (M1) and reparative (M2) phenotype and to early tissue formation. HPBMCs were seeded onto poly-ε-caprolactone bisurea strips and subjected to 0%, 7% and 12% cyclic strain for up to one week. After 1 day, cells subjected to 7% deformation showed upregulated expression of pro and anti-inflammatory chemokines, such as MCP-1 and IL10. Immunostaining revealed presence of inflammatory macrophages in all groups, while immunoregulatory macrophages were detected mainly in the 0 and 7% groups and increased significantly over time. Biochemical assays indicated deposition of sulphated glycosaminoglycans and collagen after 7 days in both strained and unstrained samples. These results suggest that 7% cyclic strain applied to hPBMCs adherent on a scaffold modulates their polarization towards reparative macrophages and allows for early synthesis of extracellular matrix components, required to promote further cell adhesion and proliferation and to bind immunoregulatory cytokines.

A comparative analysis of the collagen architecture in the carotid artery: second harmonic generation versus diffusion tensor imaging.

Collagen is the main load-bearing component of the artery. The 3D arrangement of the collagen fibers is crucial to understand the mechanical behavior of such tissues. We compared collagen fiber alignment obtained by second harmonic generation (SHG) microscopy with the alignment obtained by diffusion tensor imaging (DTI) throughout the wall of a porcine carotid artery to check the feasibility of using DTI as a fast and non-destructive method instead of SHG. The middle part of the artery was cut into two segments: one for DTI and one for the SHG measurements. The tissue for SHG measurements was cut into 30μm tangential sections. After scanning all sections, they were registered together and the fiber orientation was quantified by an in-house algorithm. The tissue for DTI measurement was embedded in type VII agarose and scanned with an MRI-scanner. Fiber tractography was performed on the DTI images. Both methods showed a layered structure of the wall. The fibers were mainly oriented circumferentially in the outer adventitia and media. DTI revealed the predominant layers of the arterial wall. This study showed the feasibility of using DTI for evaluating the collagen orientation in native artery as a fast and non-destructive method.

Controlling matrix formation and cross-linking by hypoxia in cardiovascular tissue engineering.

In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O(2) (n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen α1(I) and α1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O(2) (n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O(2). Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen α1(I) and α1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O(2), LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O(2)). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development.

Synergistic protein secretion by mesenchymal stromal cells seeded in 3D scaffolds and circulating leukocytes in physiological flow

Mesenchymal stromal cells (MSC) play an important role in natural wound healing via paracrine and juxtacrine signaling to immune cells. The aim of this study was to identify the signaling factors secreted by preseeded cells in a biomaterial and their interaction with circulating leukocytes, in the presence of physiological biomechanical stimuli exerted by the hemodynamic environment (i.e. strain and shear flow). Electrospun poly(ε-caprolactone)-based scaffolds were seeded with human peripheral blood mononuclear cells (PBMC) or MSC. Protein secretion was analyzed under static conditions and cyclic strain. Subsequently, the cross-talk between preseeded cells and circulating leukocytes was addressed by exposing the scaffolds to a suspension of PBMC in static transwells and in pulsatile flow. Our results revealed that PBMC exposed to the scaffold consistently secreted a cocktail of immunomodulatory proteins under all conditions tested. Preseeded MSC, on the other hand, secreted the trophic factors MCP-1, VEGF and bFGF. Furthermore, we observed a synergistic upregulation of CXCL12 gene expression and a synergistic increase in bFGF protein production by preseeded MSC exposed to PBMC in pulsatile flow. These findings identify CXCL12 and bFGF as valuable targets for the development of safe and effective acellular instructive grafts for application in in situ cardiovascular regenerative therapies.

In situ heart valve tissue engineering: simple devices, smart materials, complex knowledge

Evolution of heart valve tissue engineering Since the implantation of the first artificial aortic heart valve by Hufnagel et al., the design and construction of heart valve prostheses has undergone continuous and drastic changes to improve the survival and quality of life of approximately 300,000 patients with end-stage valvular disease per year [1]. These include the development of bioprostheses consisting of animal or human tissue [2,3], and the more recent introduction of valve designs for transcatheter valve replacement [4]. The most revolutionary change, however, has been the introduction of living valves, obtained via the process of tissue engineering [5]. These valves can grow and adapt to physiological demand changes and offer the potential of true regeneration. As such, they can circumvent many existing prosthesis-related complications and last a lifetime, which is of particular relevance for the relatively young patient group with congenital heart disease currently receiving a mechanical prosthesis [6]. Classical in vitro heart valve tissue engineering (HVTE) strategies involve the harvest and expansion of autologous cells, cell seeding onto preshaped biodegradable carriers or scaffolds, and prolonged tissue culture outside the human body prior to implantation. Despite optimistic outlooks based on breakthrough results in sheep over a decade ago [7,8], in vitro HVTE has not yet been introduced in the clinic. This is mainly owing to the complexity of the procedure and suboptimal long-term in vivo performance – the biggest issue being valve leaflet retraction and consequent regurgitation [9,10]. Another aspect is significant inter-patient variability, preventing early prediction of HVTE and intervention outcome [11]. In the mean time, numerous modifications to the HVTE scheme have been explored to optimize valve performance and to simplify the procedure to accelerate clinical introduction. Important steps taken are the use of on-the-f ly harvest able cells to avoid cell and tissue culture (the so-called ‘one-step procedure’) [12], and percutaneous implantation of in vitro engineered valves [9].