Maximilian Y. Emmert

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.

Hyperfibrinolysis diagnosed by rotational thromboelastometry (ROTEM) is associated with higher mortality in patients with severe trauma.

BACKGROUND: We investigated whether hyperfibrinolysis and its severity was associated with outcome of traumatized and nontraumatized patients. METHODS: From April 2008 to April 2010, all emergency patients with hyperfibrinolysis were enrolled in this study. Hyperfibrinolysis patients were divided into traumatized (trauma hyperfibrinolysis group) and nontraumatized (nontrauma hyperfibrinolysis group). The trauma hyperfibrinolysis group was matched with 24 polytrauma patients without hyperfibrinolysis (matched trauma group). Data from rotational thromboelastometry measurements, blood gas analysis (metabolic state), laboratory analysis, injury severity score, and 30-day mortality were collected. RESULTS: Thirty-five patients with hyperfibrinolysis were identified (13 traumatized, 22 nontraumatized). Overall mortality for hyperfibrinolysis was 54%. Mortality in the trauma hyperfibrinolysis group (77% ± 12%) was significantly higher than in the nontrauma hyperfibrinolysis group (41% ± 10%; P = 0.001, 95% CI 5%–67%) and the matched trauma group (33% ± 10%; P = 0.009, 95% CI 13%–74%). Hyperfibrinolysis is significantly (P = 0.017) associated with mortality in trauma patients. In the blood gas analysis representing the metabolic state, only pH (P = 0.02) and potassium (P = 0.01) were significantly lower in the trauma hyperfibrinolysis group compared to the nontrauma hyperfibrinolysis group. CONCLUSIONS: Mortality from hyperfibrinolysis is significantly higher in trauma compared with nontrauma patients, and hyperfibrinolysis is an independent factor predicting mortality in trauma patients. Rotational thromboelastometry provides real-time recognition of hyperfibrinolysis allowing early treatment.

Transplantation and tracking of human-induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment, and distribution by hybrid single photon emission computed tomography/computed tomography of sodium iodide symporter transgene expression

Background— Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. Methods and Results— Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NISpos-hiPSCs) were established. Iodide uptake, efflux, and viability of NISpos-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NISpos-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of 123I to follow donor cell survival and distribution and with the use of 99mTC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NISpos-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NISpos-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. Conclusions— This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NISpos-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.Background— Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. Methods and Results— Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NISpos-hiPSCs) were established. Iodide uptake, efflux, and viability of NISpos-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NISpos-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of 123I to follow donor cell survival and distribution and with the use of 99mTC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NISpos-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NISpos-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. Conclusions— This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NISpos-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models. # Clinical Perspective {#article-title-36}

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.

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.

Relative concentrations of haemostatic factors and cytokines in solvent/detergent-treated and fresh-frozen plasma

BACKGROUND Indications, efficacy, and safety of plasma products are highly debated. We compared the concentrations of haemostatic proteins and cytokines in solvent/detergent-treated plasma (SDP) and fresh-frozen plasma (FFP). METHODS Concentrations of the following parameters were measured in 25 SDP and FFP samples: fibrinogen (FBG), factor (F) II, F V, F VII, F VIII, F IX, F X, F XIII, von Willebrand factor (vWF), D-Dimers, ADAMTS-13 protease, tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10. RESULTS Mean FBG concentrations in SDP and FFP were similar, but in FFP, the range was larger than in SDP (P<0.01). Mean F II, F VII, F VIII, F IX, and F XIII levels did not differ significantly. Higher concentrations of F V (P<0.01), F X (P<0.05), vWF (P<0.01), and ADAMTS-13 (P<0.01) were found in FFP. With the exception of F VIII and F IX, the range of concentrations for all of these factors was smaller (P<0.05) in SDP than in FFP. Concentrations of TNF-α, IL-8, and IL-10 (all P<0.01) were higher in FFP than in SDP, again with a higher variability and thus larger ranges (P<0.01). CONCLUSIONS Coagulation factor content is similar for SDP and FFP, with notable exceptions of less F V, vWF, and ADAMTS-13 in SDP. Cytokine concentrations (TNFα, IL-8, and IL-10) were significantly higher in FFP. The clinical relevance of these findings needs to be established in outcome studies.

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.

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 Influence of Laboratory Coagulation Tests and Clotting Factor Levels on Rotation Thromboelastometry (rotem®) During Major Surgery with Hemorrhage

BACKGROUND: The aim of this study was to determine the association between standard laboratory tests, coagulation factor concentrations, and Rotation Thromboelastometry (ROTEM® delta, TEM® International GmbH, Munich, Germany) in patients undergoing major surgery with hemorrhage. METHODS: In 45 patient’s fibrinogen, factor VIII, factor XIII, International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), thrombin time, hemoglobin, leukocytes, and platelet count were simultaneously measured intraoperatively with ROTEM (EXTEM, INTEM, FIBTEM, APTEM) measurements. ROTEM parameters were: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), and &agr;-angle. Demographic and laboratory data were expressed as mean ± SD and median [range]; nonparametric Spearman rank correlations and multiple linear regressions were performed; P-values ⩽0.003 were considered significant. RESULTS: Significant correlations (P ⩽ 0.003) were found for CFT, &agr;-angle, and MCF, in EXTEM, INTEM, and APTEM with platelets, INR, and fibrinogen. Factor VIII (18 measurements) showed a strong correlation (r ≥ 0.7 or r ⩽ −0.7; all P ⩽ 0.003) with MCF, CFT, and &agr;-angle of EXTEM, INTEM, MCF of FIBTEM excluding CT of EXTEM, INTEM, FIBTEM and strong significant correlation for &agr;-angle of APTEM and moderate for CFT and MCF of APTEM. A significant moderate to strong correlation of factor XIII with MCF of EXTEM, INTEM, FIBTEM, and APTEM was found. Hemoglobin was moderately correlated (r = 0.3–0.7 or r = −0.3 to −0.7) with MCF in APTEM (P = 0.003). A moderate to strong correlation of the standard coagulation tests with all ROTEM parameters was found, in particular the CT. The aPTT correlated significantly moderate to strong with CT, CFT, &agr;-angle, and MCF of INTEM. However, multiple linear regressions were not able to show an influence of INR on ROTEM parameters except for APTEM-MCF. A significant impact of the aPTT on INTEM-CT was found. EXTEM, INTEM, and APTEM are significantly influenced by fibrinogen and platelets. CONCLUSIONS: The results confirm the clinical assumption that EXTEM, INTEM, and APTEM are associated with fibrinogen and platelets levels; INTEM-CT significantly to aPTT; and FIBTEM significantly to fibrinogen. Factor VIII showed a significant correlation with all ROTEM parameters except CT of EXTEM, INTEM, FIBTEM, and CFT and MCF of APTEM.