Dörthe Schmidt

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.

Functional Growth in Tissue-Engineered Living, Vascular Grafts Follow-Up at 100 Weeks in a Large Animal Model

Background— Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model. Methods and Results— Vascular grafts fabricated from biodegradable scaffolds (ID 18±l mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for ≤100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography–angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries. Conclusions— This study provides evidence of growth in living, functional pulmonary arteries engineered from vascular cells in a full growth animal model.

Living autologous heart valves engineered from human prenatally harvested progenitors

Background— Heart valve tissue engineering is a promising strategy to overcome the lack of autologous growing replacements, particularly for the repair of congenital malformations. Here, we present a novel concept using human prenatal progenitor cells as new and exclusive cell source to generate autologous implants ready for use at birth. Methods and Results— Human fetal mesenchymal progenitors were isolated from routinely sampled prenatal chorionic villus specimens and expanded in vitro. A portion was cryopreserved. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable leaflet scaffolds (n=12) and conditioned in a bioreactor. After 21 days, leaflets were endothelialized with umbilical cord blood-derived endothelial progenitor cells and conditioned for additional 7 days. Resulting tissues were analyzed by histology, immunohistochemistry, biochemistry (amounts of extracellular matrix, DNA), mechanical testing, and scanning electron microscopy (SEM) and were compared with native neonatal heart valve leaflets. Fresh and cryopreserved cells showed comparable myofibroblast-like phenotypes. Genotyping confirmed their fetal origin. Neo-tissues exhibited organization, cell phenotypes, extracellular matrix production, and DNA content comparable to their native counterparts. Leaflet surfaces were covered with functional endothelia. SEM showed cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. Conclusions— Prenatal fetal progenitors obtained from routine chorionic villus sampling were successfully used as an exclusive, new cell source for the engineering of living heart valve leaflets. This concept may enable autologous replacements with growth potential ready for use at birth. Combined with the use of cell banking technology, this approach may be applied also for postnatal applications.

Prenatally Fabricated Autologous Human Living Heart Valves Based on Amniotic Fluid–Derived Progenitor Cells as Single Cell Source

Background— A novel concept providing prenatally tissue engineered human autologous heart valves based on routinely obtained fetal amniotic fluid progenitors as single cell source is introduced. Methods and Results— Fetal human amniotic progenitors were isolated from routinely sampled amniotic fluid and sorted using CD133 magnetic beads. After expansion and differentiation, cell phenotypes of CD133− and CD133+ cells were analyzed by immunohistochemistry and flowcytometry. After characterization, CD133− derived cells were seeded onto heart valve leaflet scaffolds (n=18) fabricated from rapidly biodegradable polymers, conditioned in a pulse duplicator system, and subsequently coated with CD133+ derived cells. After in vitro maturation, opening and closing behavior of leaflets was investigated. Neo-tissues were analyzed by histology, immunohistochemistry, and scanning electron microscopy (SEM). Extracellular matrix (ECM) elements and cell numbers were quantified biochemically. Mechanical properties were assessed by tensile testing. CD133− derived cells demonstrated characteristics of mesenchymal progenitors expressing CD44 and CD105. Differentiated CD133+ cells showed features of functional endothelial cells by eNOS and CD141 expression. Engineered heart valve leaflets demonstrated endothelialized tissue formation with production of ECM elements (GAG 80%, HYP 5%, cell number 100% of native values). SEM showed intact endothelial surfaces. Opening and closing behavior was sufficient under half of systemic conditions. Conclusions— The use of amniotic fluid as single cell source is a promising low-risk approach enabling the prenatal fabrication of heart valves ready to use at birth. These living replacements with the potential of growth, remodeling, and regeneration may realize the early repair of congenital malformations.

A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.

Current scientific attempts to generate in vitro tissue-engineered living blood vessels (TEBVs) show substantial limitations, thereby preventing routine clinical use. In the present report, we describe a novel biotechnology concept to create living small diameter TEBV based exclusively on microtissue self-assembly (living cellular re-aggregates). A novel bioreactor was designed to assemble microtissues in a vascular shape and apply pulsatile flow and circumferential mechanical stimulation. Microtissues composed of human artery-derived fibroblasts (HAFs) and endothelial cells (HUVECs) were accumulated and cultured for 7 and 14 days under pulsatile flow/mechanical stimulation or static culture conditions with a diameter of 3mm and a wall thickness of 1mm. The resulting vessels were analyzed by immunohistochemistry for extracellular matrix (ECM) and cell phenotype (von Willebrand factor, alpha-SMA, Ki67, VEGF). Self-assembled microtissues composed of fibroblasts displayed significantly accelerated ECM formation compared to monolayer cell sheets. Accumulation of vessel-like tissue occurred within 14 days under both, static and flow/mechanical stimulation conditions. A layered tissue formation was observed only in the dynamic group, as indicated by luminal aligned alpha-SMA positive fibroblasts. We could demonstrate that self-assembled cell-based microtissues can be used to generate small diameter TEBV. The significant enhancement of ECM expression and maturation, together with the pre-vascularization capacity makes this approach highly attractive in terms of generating functional small diameter TEBV devoid of any foreign material.

Tissue engineering of heart valves using decellularized xenogeneic or polymeric starter matrices

Heart valve replacement represents the most common surgical therapy for end-stage valvular heart diseases. A major drawback that all contemporary heart valve replacements have in common is the lack of growth, repair and remodelling capability. In order to overcome these limitations, the emerging new field of tissue engineering is focusing on the in vitro generation of functional, living heart valve replacements. The basic approach uses starter matrices either of decellularized xenogeneic or polymeric materials configured in the shape of the heart valve and subsequent cell seeding. This manuscript will give a detailed overview of these two concepts without giving favour to one or the other. The concluding discussion will focus on current limitations and studies as well as future challenges prior to safe clinical application.

Umbilical cord cells as a source of cardiovascular tissue engineering.

There is increasing scientific evidence that human umbilical cord cells are a valuable source of adult stem cells that can be used for various implications including regenerative medicine and tissue engineering. The review describes the role of progenitor cells (mesenchymal, endothelial, prenatal) for the use in cardiovascular tissue engineering, i.e., the formation of large vessels and heart valves from umbilical cord cells.Currently used replacements in cardiovascular surgery are made of foreign materials with well known drawbacks such as thrombo-embolic complications, infection, loss of functional and biological properties, and others. Especially in the field of replacements in congenital cardiac defects, there would be a need of materials which have the advantage of optimal biological and mechanical properties. In the case of human umbilical cord cells, autologous cells can be used by minimally invasive procedures. The cells have excellent growth capacities and form a neo-matrix with excellent mechanical properties. For optimal growth and modeling, scaffolds are required with high biocompatibility and biodegradability, which allow cell attachment, ingrowth, and organization. Nutrients and waste must be easily transported and cells should be in entire contact with hosts body. Finally, regenerated materials can be fully incorporated and the scaffold is completely replaced. Besides these cell and scaffold requirements, feto-maternal conditions and risk factors concerning deriving stem cells are of major interest. There are still many open questions concerning whether and how maternal conditions such as infection (viral or bacterial) or gestational age of the newborn influence stem cell harvesting and quality. If these cells will be used for the construction of replacement materials, it is clear that very strict criteria and protocols be introduced enabling the promising step from isolated cells to a therapeutic device such as a new heart valve. It is hoped that it will be only a question of time until human umbilical cord cells will be used frequently as the source of cardiovascular tissues among others in the clinical setting of treating congenital heart defects.

A Three-Dimensional Engineered Artery Model for In Vitro Atherosclerosis Research

The pathogenesis of atherosclerosis involves dysfunctions of vascular endothelial cells and smooth muscle cells as well as blood borne inflammatory cells such as monocyte-derived macrophages. In vitro experiments towards a better understanding of these dysfunctions are typically performed in two-dimensional cell culture systems. However, these models lack both the three-dimensional structure and the physiological pulsatile flow conditions of native arteries. We here describe the development and initial characterization of a tissue engineered artery equivalent, which is composed of human primary endothelial and smooth muscle cells and is exposed to flow in vitro. Histological analyses showed formation of a dense tissue composed of a tight monolayer of endothelial cells supported by a basement membrane and multiple smooth muscle cell layers. Both low (LDL) and high density lipoproteins (HDL) perfused through the artery equivalent were recovered both within endothelial cells and in the sub-endothelial intima. After activation of the endothelium with either tumour necrosis factor alpha (TNFα) or LDL, monocytes circulated through the model were found to adhere to the activated endothelium and to transmigrate into the intima. In conclusion, the described tissue engineered human artery equivalent model represents a significant step towards a relevant in vitro platform for the systematic assessment of pathogenic processes in atherosclerosis independently of any systemic factors.

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity.

Background. Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-&agr; and hypoxia/reoxygenation in an in vitro model of I/R. Methods. MVEC were stimulated in vitro for 8 h with TNF-&agr;, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using 51Cr-release assays. Results. Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-&agr; but unaffected by hypoxia/reoxygenation in the absence of TNF-&agr;. ICAM-1 expression was further increased by a combination of TNF-&agr; and hypoxia/reoxygenation, whereas TNF-&agr;-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-&agr; and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-&agr; and slighty reduced by hypoxia/reoxygenation. Conclusions. Endothelial activation with TNF-&agr;, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

In Vitro Heart Valve Tissue Engineering

Heart valve replacement represents the most common surgical therapy for end-stage valvular heart diseases. A major drawback all contemporary heart valve replacements have in common is the lack of growth, repair, and remodeling capabilities. To overcome these limitations, the emerging field of tissue engineering is focusing on the in vitro generation of functional, living heart valve replacements. The basic approach uses starter matrices of either decellularized xenogeneic or biopolymeric materials configured in the shape of the heart valve and subsequent cell seeding. Moreover, in vitro strategies using mechanical loading in bioreactor systems have been developed to improve tissue maturation. This chapter gives a short overview of the current concepts and provides detailed methods for in vitro heart valve tissue engineering.