Roman Schoenauer

Injectable living marrow stromal cell-based autologous tissue engineered heart valves: first experiences with a one-step intervention in primates

AIMS A living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates. METHODS AND RESULTS Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n = 6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118 ± 17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodelling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress-strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue. CONCLUSION Here, we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.

M-band: a safeguard for sarcomere stability?

The sarcomere of striated muscle is a very efficient machine transforming chemical energy into movement. However, a wrong distribution of the generated forces may lead to self-destruction of the engine itself. A well-known example for this is eccentric contraction (elongation of the sarcomere in the activated state), which damages sarcomeric structure and leads to a reduced muscle performance. The goal of this review is to discuss the involvement of different cytoskeletal systems, in particular the M-band filaments, in the mechanisms that provide stability during sarcomeric contraction. The M-band is the transverse structure in the center of the sarcomeric A-band, which is responsible both for the regular packing of thick filaments and for the uniform distribution of the tension over the myosin filament lattice in the activated sarcomere. Although some proteins from the Ig-superfamily, like myomesin and M-protein, are the major candidates for the role of M-band bridges, the exact molecular organisation of the M-band is not clear. However, the protein composition of the M-band seems to modulate the mechanical characteristics of the thick filament lattice, in particular its stiffness, adjusting it to the specific demands in different muscle types. The special M-band design in slow fibers might be part of structural adaptations, favouring sarcomere stability for a continuous contractile activity over a broad working range. In conclusion, we discuss why the interference with M-band structure might have fatal consequences for the integrity of the working sarcomere.

Tissue engineering on matrix: future of autologous tissue replacement

Tissue engineering aims at the creation of living neo-tissues identical or close to their native human counterparts. As basis of this approach, temporary biodegradable supporter matrices are fabricated in the shape of a desired construct, which promote tissue strength and provide functionality until sufficient neo-tissue is formed. Besides fully synthetic polymer-based scaffolds, decellularized biological tissue of xenogenic or homogenic origin can be used. In a second step, these scaffolds are seeded with autologous cells attaching to the scaffold microstructure. In order to promote neo-tissue formation and maturation, the seeded scaffolds are exposed to different forms of stimulation. In cardiovascular tissue engineering, this “conditioning” can be achieved via culture media and biomimetic in vitro exposure, e.g., using flow bioreactors. This aims at adequate cellular differentiation, proliferation, and extracellular matrix production to form a living tissue called the construct. These living autologous constructs, such as heart valves or vascular grafts, are created in vitro, comprising a viable interstitium with repair and remodeling capabilities already prior to implantation. In situ further in vivo remodeling is intended to recapitulate physiological vascular architecture and function. The remodeling mechanisms were shown to be dominated by monocytic infiltration and chemotactic host-cell attraction leading into a multifaceted inflammatory process and neo-tissue formation. Key molecules of these processes can be integrated into the scaffold matrix to direct cell and tissue fate in vivo.

Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation

Prenatal heart valve interventions aiming at the early and systematic correction of congenital cardiac malformations represent a promising treatment option in maternal-fetal care. However, definite fetal valve replacements require growing implants adaptive to fetal and postnatal development. The presented study investigates the fetal implantation of prenatally engineered living autologous cell-based heart valves. Autologous amniotic fluid cells (AFCs) were isolated from pregnant sheep between 122 and 128 days of gestation via transuterine sonographic sampling. Stented trileaflet heart valves were fabricated from biodegradable PGA-P4HB composite matrices (n = 9) and seeded with AFCs in vitro. Within the same intervention, tissue engineered heart valves (TEHVs) and unseeded controls were implanted orthotopically into the pulmonary position using an in-utero closed-heart hybrid approach. The transapical valve deployments were successful in all animals with acute survival of 77.8% of fetuses. TEHV in-vivo functionality was assessed using echocardiography as well as angiography. Fetuses were harvested up to 1 week after implantation representing a birth-relevant gestational age. TEHVs showed in vivo functionality with intact valvular integrity and absence of thrombus formation. The presented approach may serve as an experimental basis for future human prenatal cardiac interventions using fully biodegradable autologous cell-based living materials.

Myomesin 3, a novel structural component of the M-band in striated muscle

The M-band is the cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. Apart from the myosin tails and the C-termini of titin, only two closely related structural proteins had been detected at the M-band so far, myomesin and M-protein. However, electron microscopy studies revealed structural features that do not correlate with the expression of these two proteins, indicating the presence of unknown constituents in the M-band. Using comparative sequence analysis, we have identified a third member of this gene family, myomesin 3, and characterised its biological properties. Myomesin 3 is predicted to consist of a unique head domain followed by a conserved sequence of either fibronectin- or immunoglobulin-like domains, similarly to myomesin 3 and M-protein. While all three members of the myomesin family are localised to the M-band of the sarcomere, each member shows its specific expression pattern. In contrast to myomesin, which is ubiquitously expressed in all striated muscles, and M-protein, whose expression is restricted to adult heart and fast-twitch skeletal muscle, myomesin 3 can be detected mainly in intermediate speed fibers of skeletal muscle. In analogy to myomesin, myomesin 3 targets to the M-band region of the sarcomere via its N-terminal part and forms homodimers via its C-terminal domain. However, despite the high degree of homology, no heterodimer between distinct members of the myomesin gene family can be detected. We propose that each member of the myomesin family is a component of one of the distinct ultrastructures, the M-lines, which modulate the mechanical properties of the M-bands in different muscle types.

Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata

Aims Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. Methods and results We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). Conclusion Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps.

The molecular composition of the sarcomeric M-band correlates with muscle fiber type

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.

In vitro fabrication of autologous living tissue‐engineered vascular grafts based on prenatally harvested ovine amniotic fluid‐derived stem cells

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non‐allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow‐derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT‐3. Isolated ovine amniotic fluid‐derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small‐ and large‐diameter tissue‐engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre‐clinical in vivo assessment model for future amniotic fluid cell‐based therapeutic applications. Copyright

Engineering of living autologous human umbilical cord cell-based septal occluder membranes using composite PGA-P4HB matrices.

Interventional closure of intracardiac wall defects using occluder devices has evolved as a highly attractive treatment option. However, incomplete and delayed healing reactions often result in a major risk of residual defects, thromboembolism, or device fractures. Biodegradable living tissue engineered occluder membranes (TEOMs) could provide autologous thromboresistant implants with growth and remodeling capacities. PGA-P4HB composite matrices were seeded with human umbilical cord-derived cells or vascular-derived control cells and exposed to static (n = 19) or dynamic (n = 13) conditioning. Harvested TEOMs were integrated into occluder frameworks, exposed to crimping and delivered into pre-formed defects of juvenile porcine hearts. Dynamically conditioned TEOM constructs showed higher collagen formation in histology than static constructs with significantly higher stiffness moduli in uniaxial tensile testing. Grating interferometry revealed substantial but inhomogeneous cone-like degradation of the composite matrices in dynamic conditioning. The crimping and delivery procedures resulted in no significant changes in macroscopy, histo-morphology, cellular viability, DNA or hydroxyproline content, and scanning electron microscopy findings. Here, we present the in vitro fabrication, crimping and experimental delivery of living human umbilical cord-cell derived TEOMs based on composite matrices as a potential future autologous therapy of intracardiac wall defects.

Upregulation of transmembrane endothelial junction proteins in human cerebral cavernous malformations

OBJECT Cerebral cavernous malformations (CCMs) are among the most prevalent cerebrovascular malformations, and endothelial cells seem to play a major role in the disease. However, the underlying mechanisms, including endothelial intercellular communication, have not yet been fully elucidated. In this article, the authors focus on the endothelial junction proteins CD31, VE-cadherin, and occludin as important factors for functional cell-cell contacts known as vascular adhesion molecules and adherence and tight junctions. METHODS Thirteen human CCM specimens and 6 control tissue specimens were cryopreserved and examined for the presence of VE-cadherin, occludin, and CD31 by immunofluorescence staining. Protein quantification was performed by triplicate measurements using western blot analysis. RESULTS Immunofluorescent analyses of the CCM sections revealed a discontinuous pattern of dilated microvessels and capillaries as well as increased expression of occludin, VE-cadherin, and CD31 in the intima and in the enclosed parenchymal tissue compared with controls. Protein quantification confirmed these findings by showing upregulation of the levels of these proteins up to 2-6 times. CONCLUSIONS A protocol enabling the molecular and morphological examination of the intercellular contact proteins in human CCM was validated. The abnormal and discontinuous pattern in these endothelial cell-contact proteins compared with control tissue explains the loose intercellular junctions that are considered to be one of the causes of CCM-associated bleeding or transendothelial oozing of erythrocytes. Despite the small number of specimens, this study demonstrates for the first time a quantitative analysis of endothelial junction proteins in human CCM.