Simon P. Hoerstrup

Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering

OBJECTIVE In tissue engineering, three-dimensional biodegradable scaffolds are generally used as a basic structure for cell anchorage, cell proliferation and cell differentiation. The currently used biodegradable scaffolds in cardiovascular tissue engineering are potentially immunogenic, they show toxic degradation and inflammatory reactions. The aim of this study is to establish a new three-dimensional cell culture system within cells achieve uniform distribution and quick tissue development and with no toxic degradation or inflammatory reactions. METHODS Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human myofibroblasts cultures are suspended in fibrinogen solution and seeded into 6-well culture plates for cell development for 4 weeks and supplemented with different concentrations of aprotinin. Hydroxyproline assay and histological studies were performed to evaluate the tissue development in these fibrin gel structures. RESULTS The light microscopy and the transmission electron microscopy studies for tissue development based on the three-dimensional fibrin gel structures showed homogenous cell growth and confluent collagen production. No toxic degradation or inflammatory reactions could be detected. Furthermore, fibrin gel myofibroblasts structures dissolved within 2 days in medium without aprotinin, but medium supplemented with higher concentration of aprotinin retained the three-dimensional structure and had a higher collagen content (P<0.005) and a better tissue development. CONCLUSIONS A three-dimensional fibrin gel structure can serve as a useful scaffold for tissue engineering with controlled degradation, excellent seeding effects and good tissue development.

Fibrin gel – advantages of a new scaffold in cardiovascular tissue engineering

OBJECTIVE The field of tissue engineering deals with the creation of tissue structures based on patient cells. The scaffold plays a central role in the creation of 3-D structures in cardiovascular tissue engineering like small vessels or heart valve prosthesis. An ideal scaffold should have tissue-like mechanical properties and a complete immunologic integrity. As an alternative scaffold the use of fibrin gel was investigated. METHODS Preliminary, the degradation of the fibrin gel was controlled by the supplementation of aprotinin to the culture medium. To prevent tissue from shrinking a mechanical fixation of the gel with 3-D microstructure culture plates and a chemical fixation with poly-L-lysine in different fixation techniques were studied. The thickness of the gel layer was changed from 1 to 3 mm. The tissue development was analysed by light, transmission and scanning electron microscopy. Collagen production was detected by the measurement of hydroxyproline. Injection molding techniques were designed for the formation of complex 3-D tissue structures. RESULTS The best tissue development was observed at an aprotinin concentration of 20 microg per cc culture medium. The chemical border fixation of the gel by poly-L-lysine showed the best tissue development. Up to a thickness of 3 mm no nutrition problems were observed in the light and transmission electron microscopy. The molding of a simplified valve conduit was possible by the newly developed molding technique. CONCLUSION Fibrin gel combines a number of important properties of an ideal scaffold. It can be produced as a complete autologous scaffold. It is moldable and degradation is controllable by the use of aprotinin.

New Pulsatile Bioreactor for In Vitro Formation of Tissue Engineered Heart Valves

Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold. In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs. It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation. Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system. Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation. This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure. The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.

Tissue Engineering of Human Heart Valve Leaflets: A Novel Bioreactor for a Strain-Based Conditioning Approach

Current mechanical conditioning approaches for heart valve tissue engineering concentrate on mimicking the opening and closing behavior of the leaflets, either or not in combination with tissue straining. This study describes a novel approach by mimicking only the diastolic phase of the cardiac cycle, resulting in tissue straining. A novel, yet simplified, bioreactor system was developed for this purpose by applying a dynamic pressure difference over a closed tissue engineered valve, thereby inducing dynamic strains within the leaflets. Besides the use of dynamic strains, the developing leaflet tissues were exposed to prestrain induced by the use of a stented geometry. To demonstrate the feasibility of this strain-based conditioning approach, human heart valve leaflets were engineered and their mechanial behavior evaluated. The actual dynamic strain magnitude in the leaflets over time was estimated using numerical analyses. Preliminary results showed superior tissue formation and non-linear tissue-like mechanical properties in the strained valves when compared to non-loaded tissue strips. In conclusion, the strain-based conditioning approach, using both prestrain and dynamic strains, offers new possibilities for bioreactor design and optimization of tissue properties towards a tissue-engineered aortic human heart valve replacement.

Tissue engineering of heart valves: in vitro experiences.

BACKGROUND Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. METHODS We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. RESULTS It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. CONCLUSIONS Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells

BACKGROUND Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated. METHODS Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests. RESULTS Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker. CONCLUSIONS In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.

Autologous Human Tissue-Engineered Heart Valves Prospects for Systemic Application

Background— Tissue engineering represents a promising approach for the development of living heart valve replacements. In vivo animal studies of tissue-engineered autologous heart valves have focused on pulmonary valve replacements, leaving the challenge to tissue engineer heart valves suitable for systemic application using human cells. Methods and Results— Tissue-engineered human heart valves were analyzed up to 4 weeks and conditioning using bioreactors was compared with static culturing. Tissue formation and mechanical properties increased with time and when using conditioning. Organization of the tissue, in terms of anisotropic properties, increased when conditioning was dynamic in nature. Exposure of the valves to physiological aortic valve flow demonstrated proper opening motion. Closure dynamics were suboptimal, most likely caused by the lower degree of anisotropy when compared with native aortic valve leaflets. Conclusions— This study presents autologous tissue-engineered heart valves based on human saphenous vein cells and a rapid degrading synthetic scaffold. Tissue properties and mechanical behavior might allow for use as living aortic valve replacements.

Tissue-Engineering Bioreactors: A New Combined Cell-Seeding and Perfusion System for Vascular Tissue Engineering

One approach to the tissue engineering of vascular structures is to develop in vitro conditions in order ultimately to fabricate functional vascular tissues before final implantation. In our experiment, we aimed to develop a new combined cell seeding and perfusion system that provides sterile conditions during cell seeding and biomechanical stimuli in order to fabricate autologous human vascular tissue in vitro. The cell seeding and perfusion system is made of Plexiglas and is completely transparent (Berlin Heart, Berlin, Germany; University Hospital Benjamin Franklin, Berlin, Germany). The whole system consists of a cell seeding chamber that can be incorporated into the perfusion system and an air-driven respirator pump connected to the bioreactor. The cell culture medium continuously circulates through a closed-loop system. We thus developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfused bioreactor for long-term vascular conditioning. The cell seeding chamber can be easily connected to the bioreactor, which combines continuous, pulsatile perfusion and mechanical stimulation to the tissue-engineered conduit. Adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator allows various pulsatile flows and different levels of pressure. The whole system is a highly isolated cell culture setting, which provides a high level of sterility and a gas supply and fits into a standard humidified incubator. The device can be sterilized by ethylene oxide and assembled with a standard screwdriver. Our newly developed combination of a cell seeding and conditioning device provides sterile conditions and biodynamic stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered vessel.

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.

Evaluation of biodegradable, three-dimensional matrices for tissue engineering of heart valves.

A crucial factor in tissue engineering of heart valves is the type of scaffold material. In the following study, we tested three different biodegradable scaffold materials, polyglycolic acid (PGA), polyhydroxyalkanoate (PHA), and poly-4-hydroxybutyrate (P4HB), as scaffolds for tissue engineering of heart valves. We modified PHA and P4HB by a salt leaching technique to create a porous matrix. We constructed trileaflet heart valve scaffolds from each polymer and tested them in a pulsatile flow bioreactor. In addition, we evaluated the cell attachment to our polymers by creating four tubes of each material (length equals 4 cm; inner diameter, 0.5 cm), seeding each sample with 8,000,000 ovine vascular cells, and incubating the cell-polymer construct for 8 days (37 degrees C and 5% CO2). The seeded vascular constructs were exposed to continuous flow for 1 hour. Analysis of samples included DNA assay before and after flow exposure, 4-hydroxyproline assay, and environmental scanning electron microscopy (ESEM). We fabricated trileaflet heart valve scaffolds from porous PHA and porous P4HB, which opened and closed synchronously in a pulsatile bioreactor. It was not possible to create a functional trileaflet heart valve scaffold from PGA. After seeding and incubating the PGA-, PHA-, and P4HB-tubes, there were significantly (p < 0.001) more cells on PGA compared with PHA and P4HB. There were no significant differences among the materials after flow exposure, but there was a significantly higher collagen content (p < 0.017) on the PGA samples compared with P4HB and PHA. Cell attachment and collagen content was significantly higher on PGA samples compared with PHA and P4HB. However, PHA and P4HB also demonstrate a considerable amount of cell attachment and collagen development and share the major advantage that both materials are thermoplastic, making it possible to mold them into the shape of a functional scaffold for tissue engineering of heart valves.