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 host's 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 defect

Multiscale analysis of human tissue engineered matrices for heart valve tissue engineering applications

Human tissue-engineered matrices (hTEMs) have been proposed as a promising approach for in-situ tissue engineered heart valves (TEHVs). However, there is still a limited understanding on how ECM composition in hTEMs develops over tissue culture time. Therefore, we performed a longitudinal hTEM assessment by 1) multiscale evaluation of hTEM composition during culture time (2, 4, 6-weeks), using (immuno)histology, biochemical assays, and mass spectrometry (LC-MS/MS); 2) analysis of protein pathways involved in ECM development using gene set enrichment analysis (GSEA); and 3) assessment of hTEM mechanical characterization using uniaxial tensile testing. Finally, as proof-of-concept, TEHVs manufactured using 6-weeks hTEM samples were tested in a pulse duplicator. LC-MS/MS confirmed the tissue culture time-dependent increase in ECM proteins observed in histology and biochemical assays, revealing the most abundant collagens (COL6,COL12), proteoglycans (HSPG2,VCAN), and glycoproteins (FN,TNC). GSEA identified the most represented protein pathways in the hTEM at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks (ECM organization and maturation). Uniaxial mechanical testing showed increased stiffness and stress at failure, and reduction in strain over tissue culture time. hTEM-based TEHVs demonstrated promising in vitro performance at both pulmonary and aortic pressure conditions, with symmetric leaflet coaptation and no stenosis. In conclusion, ECM protein abundance and maturation increased over tissue culture time, with consequent improvement of hTEM mechanical characterics. These findings suggest that longer tissue culture impacts tissue organization, leading to an hTEM that may be suitable for high-pressure applications. STATEMENT OF SIGNIFICANCE: : It is believed that the composition of the extracellular matrix (ECM) in the human tissue engineered matrices (hTEM) may favor tissue engineered heart valve (TEHV) remodeling upon implantation. However, the exact protein composition of the hTEM, and how this impacts tissue mechanical properties, remains unclear. Hence, we developed a reproducible rotation-based tissue culture method to produce hTEM samples. We performed a longitudinal assessment using different analytical techniques and mass spectrometry. Our data provided an in-depth characterization of the hTEM proteome with focus on ECM components, their development, and how they may impact the mechanical properties. Based on these results, we manufactured functional hTEM-based TEHVs at aortic-like condition in vitro. These outcomes pose an important step in translating hTEM-based TEHVs into clinics and in predicting their remodeling potential upon implantation

Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering

A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplet

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 μg 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 aprotini

Endothelial Progenitor Cell-Based in vitro Pre-Endothelialization of Human Cell-Derived Biomimetic Regenerative Matrices for Next-Generation Transcatheter Heart Valves Applications

Hemocompatibility of cardiovascular implants represents a major clinical challenge and, to date, optimal antithrombotic properties are lacking. Next-generation tissue-engineered heart valves (TEHVs) made from human-cell-derived tissue-engineered extracellular matrices (hTEMs) demonstrated their recellularization capacity in vivo and may represent promising candidates to avoid antithrombotic therapy. To further enhance their hemocompatibility, we tested hTEMs pre-endothelialization potential using human-blood-derived endothelial-colony-forming cells (ECFCs) and umbilical vein cells (control), cultured under static and dynamic orbital conditions, with either FBS or hPL. ECFCs performance was assessed via scratch assay, thereby recapitulating the surface damages occurring in transcatheter valves during crimping procedures. Our study demonstrated: feasibility to form a confluent and functional endothelium on hTEMs with expression of endothelium-specific markers; ECFCs migration and confluency restoration after crimping tests; hPL-induced formation of neo-microvessel-like structures; feasibility to pre-endothelialize hTEMs-based TEHVs and ECFCs retention on their surface after crimping. Our findings may stimulate new avenues towards next-generation pre-endothelialized implants with enhanced hemocompatibility, being beneficial for selected high-risk patients

Next-generation tissue-engineered heart valves with repair, remodelling and regeneration capacity

Valvular heart disease is a major cause of morbidity and mortality worldwide. Surgical valve repair or replacement has been the standard of care for patients with valvular heart disease for many decades, but transcatheter heart valve therapy has revolutionized the field in the past 15 years. However, despite the tremendous technical evolution of transcatheter heart valves, to date, the clinically available heart valve prostheses for surgical and transcatheter replacement have considerable limitations. The design of next-generation tissue-engineered heart valves (TEHVs) with repair, remodelling and regenerative capacity can address these limitations, and TEHVs could become a promising therapeutic alternative for patients with valvular disease. In this Review, we present a comprehensive overview of current clinically adopted heart valve replacement options, with a focus on transcatheter prostheses. We discuss the various concepts of heart valve tissue engineering underlying the design of next-generation TEHVs, focusing on off-the-shelf technologies. We also summarize the latest preclinical and clinical evidence for the use of these TEHVs and describe the current scientific, regulatory and clinical challenges associated with the safe and broad clinical translation of this technology.</p