Multiscale analysis of human tissue engineered matrices for next generation heart valve 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 a 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 characteristics. 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.

Cardiovascular tissue engineering: From basic science to clinical application.

Valvular heart disease is an increasing population health problem and, especially in the elderly, a significant cause of morbidity and mortality. The current treatment options, such as mechanical and bioprosthetic heart valve replacements, have significant restrictions and limitations. Considering the increased life expectancy of our aging population, there is an urgent need for novel heart valve concepts that remain functional throughout life to prevent the need for reoperation. Heart valve tissue engineering aims to overcome these constraints by creating regenerative, self-repairing valve substitutes with life-long durability. In this review, we give an overview of advances in the development of tissue engineered heart valves, and describe the steps required to design and validate a novel valve prosthesis before reaching first-in-men clinical trials. In-silico and in-vitro models are proposed as tools for the assessment of valve design, functionality and compatibility, while in-vivo preclinical models are required to confirm the remodeling and growth potential of the tissue engineered heart valves. An overview of the tissue engineered heart valve studies that have reached clinical translation is also presented. Final remarks highlight the possibilities as well as the obstacles to overcome in translating heart valve prostheses into clinical application.

Development of a Novel Human Cell-Derived Tissue-Engineered Heart Valve for Transcatheter Aortic Valve Replacement: an In Vitro and In Vivo Feasibility Study.

Transcatheter aortic valve replacement (TAVR) is being extended to younger patients. However, TAVR-compatible bioprostheses are based on xenogeneic materials with limited durability. Off-the-shelf tissue-engineered heart valves (TEHVs) with remodeling capacity may overcome the shortcomings of current TAVR devices. Here, we develop for the first time a TEHV for TAVR, based on human cell-derived extracellular matrix and integrated into a state-of-the-art stent for TAVR. The TEHVs, characterized by a dense acellular collagenous matrix, demonstrated in vitro functionality under aortic pressure conditions (n = 4). Next, transapical TAVR feasibility and in vivo TEHV functionality were assessed in acute studies (n = 5) in sheep. The valves successfully coped with the aortic environment, showing normal leaflet motion, free coronary flow, and absence of stenosis or paravalvular leak. At explantation, TEHVs presented full structural integrity and initial cell infiltration. Its long-term performance proven, such TEHV could fulfill the need for next-generation lifelong TAVR prostheses.

Organ Chips: Quality Assurance Systems in Regenerative Medicine.

A class of novel therapies leverages regenerative cell types in disease microenvironments. This complex interplay challenges established good manufacturing practices, as standards and analytical tools to measure regenerative potency are missing. That is, we can build the product right, but we do not know if we are building the right product. Here, we suggest that organ-chips, biomimetic in vitro phenotyping platforms, can serve as key quality assurance systems in regenerative medicine.

Mechanical analysis of ovine and pediatric pulmonary artery for heart valve stent design.

Transcatheter heart valve replacement is an attractive and promising technique for congenital as well as acquired heart valve disease. In this procedure, the replacement valve is mounted in a stent that is expanded at the aimed valve position and fixated by clamping. However, for this technique to be appropriate for pediatric patients, the material properties of the host tissue need to be determined to design stents that can be optimized for this particular application. In this study we performed equibiaxial tensile tests on four adult ovine pulmonary artery walls and compared the outcomes with one pediatric pulmonary artery. Results show that the pediatric pulmonary artery was significantly thinner (1.06 ± 0.36 mm (mean ± SD)) than ovine tissue (2.85 ± 0.40 mm), considerably stiffer for strain values that exceed the physiological conditions (beyond 50% strain in the circumferential and 60% in the longitudinal direction), more anisotropic (with a significant difference in stiffness between the longitudinal and circumferential directions beyond 60% strain) and presented stronger non-linear stress-strain behavior at equivalent strains (beyond 26% strain) compared to ovine tissue. These discrepancies suggest that stents validated and optimized using the ovine pre-clinical model might not perform satisfactorily in pediatric patients. The material parameters derived from this study may be used to develop stent designs for both applications using computational models.

Prenatally harvested cells for cardiovascular tissue engineering: fabrication of autologous implants prior to birth.

Using the principal of tissue engineering, several groups have demonstrated the feasibility of creating heart valves, blood vessels, and myocardial structures using autologous cells and biodegradable scaffold materials. In the current cardiovascular clinical scenario, the main medical need for a tissue engineering solution is in the field of pediatric applications treating congenital heart disease. In these young patients, the introduction of autologous viable and growing replacement structures, such as tissue engineered heart valves and vessels, would substantially reduce today's severe therapeutic limitations, which are mainly due to the need for repeat reoperations to adapt the current artificial prostheses to somatic growth. Based on high resolution imaging techniques, an increasing number of defects are diagnosed already prior to birth around week 20. For interventions, cells should be obtained already during pregnancy to provide tissue engineered implants either at birth or even prenatally. In our recent studies human fetal mesenchymal stem cells were isolated from routinely sampled prenatal amniotic fluid or chorionic villus specimens and expanded in vitro. Fresh and cryopreserved samples were used. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable scaffolds and conditioned in a bioreactor. Leaflets were endothelialized with either amniotic fluid- or umbilical cord blood-derived endothelial progenitor cells and conditioned. 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. Genotyping confirmed their fetal origin, and fresh versus cryopreserved cells showed comparable myofibroblast-like phenotypes. 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 morphologically cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. These in vitro studies demonstrated the principal feasibility of using various human cell types isolated from fetal sources for cardiovascular tissue engineering. Umbilical cord blood-, amniotic fluid- and chorionic villi-derived cells have shown promising potential for the clinical realization of this congenital tissue engineering approach. Based on these results, future research must aim at further investigation as well as preclinical evaluation of prenatally harvested stem- or progenitor cells with regard to their potential for clinical use.

Delayed two-step free wall rupture of the right and left ventricular wall after myocardial infarction.

We present a 68-year-old female who suffered extensive complications after severe myocardial infarction (MI) in the circumflex (CX) territory. At 24 hours after the initial event, the patient presented with a covered right ventricular free wall rupture (FWR) which was followed by a rupture of the left posterior wall ten days later. We report here on a rare case of delayed two-step biventricular FWR after severe MI in the CX territory.

Plasma levels of endothelin-1 after a pulmonary embolism of bone marrow fat.

BACKGROUND: During orthopedic surgery, embolization of bone marrow fat can lead to potentially fatal, intra-operative cardiovascular deterioration. Vasoactive mediators may also be released from the bone marrow and contribute to these changes. Increased plasma levels of endothelin-1 (ET-1) have been observed after pulmonary air and thrombo-embolism. The role of ET-1 in the development of acute cardiovascular deterioration as a result of bone marrow fat embolization during vertebroplasty was therefore investigated. METHODS: Bone cement was injected into three lumbar vertebrae of six sheep in order to force bone marrow fat into the circulation. Invasive blood pressures and heart rate were recorded continuously until 60 min after the last injection. Cardiac output, arterial and mixed venous blood gas parameters and plasma ET-1 concentrations were measured at selected time points. Post-mortem, lung biopsies were taken for analysis of intravascular fat. RESULTS: Cement injections resulted in a sudden (within 1 min) and severe increase in pulmonary arterial pressure (>100%). Plasma concentrations of ET-1 started to increase after the second injection, but no significant changes were observed. Intravascular fat and bone marrow cells were present in all lung lobes. CONCLUSION: Cement injections into vertebral bodies elicited fat embolism resulting in subsequent cardiovascular changes that were characterized by an increase in pulmonary arterial pressure. Cardiovascular complications as a result of bone marrow fat embolism should thus be considered in patients undergoing vertebroplasty. No significant changes in ET-1 plasma values were observed. Thus, ET-1 did not contribute to the acute cardiovascular changes after fat embolism.

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 incomplete.

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 defects.

Tissue engineering of vascular conduits: fabrication of custom-made scaffolds using rapid prototyping techniques.

BACKGROUND: The technique of stereolithography, which automatically fabricates models from X-ray computed tomography or magnetic resonance imaging (MRI) data linked to computer-aided design programs, has been applied to the fabrication of scaffolds for tissue engineering. We previously reported on the application of stereolithography in scaffold fabrication of a trileaflet heart valve. In our current experiment we demonstrate a new technique for the fabrication of custom-made conduits for the potential replacement of a coarcted aortic segment. METHODS AND RESULTS: In this experiment the image data derived from a 12-year-old male patient with aortic coarctation scanned by MRI were processed by a computer-aided design program to reconstruct the aortic arch with isthmus stenosis three dimensionally. By defining the stenotic section and the adjacent normal vessel a custom-made nonstenotic descending aorta was reconstructed to replace the stenosed part. The rapid prototyping technique was used to establish stereolithographic models for fabricating biocompatible and biodegradable vascular scaffolds with the anatomic structure of the recalculated human descending aorta through a thermal processing technique. CONCLUSION: Our results suggest that the re-creation and reproduction of complex vascular structures by computer-aided design techniques may be useful to fabricate custom-made polymeric scaffolds for the tissue engineering of living vascular prostheses.

Effect of low molecular weight heparin (dalteparin) and fondaparinux (Arixtra) on human osteoblasts in vitro.

BACKGROUND: The prolonged administration of heparin for prevention and treatment of venous thromboembolism has been associated with a risk of heparin-induced osteoporosis. Fondaparinux is a new antithrombotic drug that specifically inhibits factor Xa. Because of the known interactions of other antithrombotic agents with bone remodelling, the effects of fondaparinux on human osteoblasts were analysed in vitro. METHODS: Primary human osteoblast cell cultures were incubated with either the low molecular weight heparin dalteparin at concentrations of 30, 300 and 900 microg/ml or with fondaparinux at concentrations of 25, 50, 100, 150, 200 and 250 microg/ml. Cellular proliferation rate and protein synthesis were measured. Expression of genes encoding osteocalcin, collagen type I and alkaline phosphatase was examined by reverse transcriptase-polymerase chain reaction. RESULTS: Incubation with dalteparin led to a significant, dose-dependent inhibition of osteoblast proliferation, inhibition of protein synthesis, and inhibited expression of phenotype markers (osteocalcin and alkaline phosphatase genes) after 3 and 7 days. No inhibitory effects were observed in the fondaparinux-treated cells. CONCLUSION: Fondaparinux did not inhibit osteoblast proliferation in vitro and may reduce the risk of heparin-induced osteoporosis associated with long-term heparin administration.

The relevance of large strains in functional tissue engineering of heart valves.

BACKGROUND: Exposing the developing tissue to flow and pressure in a bioreactor has been shown to enhance tissue formation in tissue-engineered heart valves. Animal studies showed excellent functionality in these valves in the pulmonary position. However, they lack the mechanical strength for implantation in the high-pressure aortic position. Improving the in vitro conditioning protocol is an important step towards the use of these valves as aortic heart valve replacements. In this study, the relevance of large strains to improve the mechanical conditioning protocol was investigated. METHODS: Using a newly developed device, engineered heart valve tissue was exposed to increasing cyclic strain in vitro. Tissue formation and mechanical properties were analyzed and compared to unstrained controls. RESULTS: Straining resulted in more pronounced and organized tissue formation with superior mechanical properties over unstrained controls. Overall tissue properties improved with increasing strain levels. CONCLUSIONS: The results demonstrate the significance of large strains in promoting tissue formation. This study may provide a methodological basis for tissue engineering of heart valves appropriate for systemic pressure applications.

Endoscopic saphenous vein harvesting for CABG -- a randomized, prospective trial.

BACKGROUND: The saphenous vein is an established conduit for coronary revascularization. Disadvantages of traditional harvest technique are significant pain and morbidity. We compared the endoscopic harvest technique with the traditional method. METHOD: 140 coronary artery bypass graft (CABG) patients were randomized into 2 groups: endoscopic vein harvesting (EVH; n = 80) and traditional open vein harvesting (OVH; n = 60). Analysis included preoperative risk factors for wound complication, harvesting time, graft injury, and intraoperative and postoperative complications. Patient follow-up lasted 3 months. RESULTS: The preoperative risk profiles of the groups were comparable. In the EVH group, 5 patients (7.1 %) had to be switched to the open technique. EVH time was 45 +/- 6.2 min vs. 31.1 +/- 6.5 min. Two patients (2.5 %) had to be revised because of bleeding complication vs. 6 (10 %) in the OVH group. No local infections or wound complications were observed in the EVH group vs. 11 (18 %) cases in the OVH group. Two OVH cases (3.6 %) were readmitted for wound debridement. All EVH patients reported less pain and were completely satisfied by the cosmetic results. CONCLUSION: EVH is a safe and efficient technique for CABG. Morbidity was significantly lower, with reduced pain and better cosmetic results. EVH time was significantly longer compared to the traditional harvesting technique.

Preoperative administration of steroids: influence on adhesion molecules and cytokines after cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with tissue damage mediated by adhesion molecules and cytokines. Prebypass steroid administration may modulate the inflammatory response, resulting in improved postoperative recovery. METHODS: Fifty patients undergoing elective coronary operations under normothermic CPB were randomized into two groups: group A (n = 24) received intravenous methylprednisolone (10 mg/kg) 4 hours preoperatively, and group B (n = 26) served as controls. Cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin-2R [IL-2R], IL-6, IL-8), soluble adhesion molecules (sE-selectin, sICAM-1), C-reactive protein, and leukocytes were measured before steroid application, then 24 and 48 hours, and 6 days postoperatively. Adhesion molecules were measured by enzyme-linked immunosorbent assay, cytokines by chemiluminescent immunoassay. Postoperatively, hemodynamic measurements, inotropic agent requirements, blood loss, duration of mechanical ventilation, and intensive care unit stay were compared. RESULTS: Aortic cross-clamp and CPB time was similar in both groups. Prednisolone administration reduced postoperative levels of IL-6 (611 versus 92.7 pg/mL; p = 0.003), TNF-alpha (24.4 versus 11.0 pg/L, p = 0.02), and E-selectin (327 versus 107 ng/mL, p = 0.02). Postoperative recovery did not differ between groups. CONCLUSIONS: Preoperative administration of methylprednisolone blunted the increase of IL-6, TNF-alpha, and E-selectin levels after CPB but had no measurable effect on postoperative recovery.

Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.

Arterial vascular cells have been successfully utilized for tissue engineering in human cardiovascular structures, such as heart valves. The present study evaluates saphenous vein-derived myofibroblasts as an alternative, easy-to-access cell source for human cardiovascular tissue engineering. Biodegradable polyurethane scaffolds were seeded with human vascular myofibroblasts. Group A consisted of scaffolds seeded with cells from ascending aortic tissue; in group B, saphenous vein-derived cells were used. Analysis included histology, electron microscopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content was comparable in both groups. Collagen and stress at maximum load was significantly higher in group B. Morphology showed viable, layered cellular tissue in all samples, with collagen fibrils most pronounced in group B. In conclusion, saphenous vein myofibroblasts cultured on biodegradable scaffolds showed excellent in vitro tissue generation. Collagen formation and mechanical properties were superior to aortic tissue derived constructs. Therefore, the easy-to-access vein cells represent a promising alternative cell source for cardiovascular tissue engineering.

Genetic predisposition in patients undergoing cardiopulmonary bypass surgery is associated with an increase of inflammatory cytokines.

OBJECTIVE: Cardiopulmonary bypass (CPB) surgery induces a transient rise in pro-inflammatory cytokines typically released by activated monocytes. The E4 variant of apolipoprotein E is a recognized risk factor for atherosclerosis. It has recently been shown that apolipoprotein E affects monocyte functions in vitro and leads to higher levels of median lipoprotein (a) in humans. The aim of the study is to investigate if the E4 genetic variant of apolipoprotein E affects cytokine release after CPB surgery. METHODS: 22 patients were operated on with standard coronary artery bypass grafting. Concentrations of interleukin 8 (IL-8) and tumor necrosis factor (TNF-alpha) were measured by automated Immulite immunoassay at regular intervals within 48 h after surgery. Total apparent cytokine outputs were calculated as area under the curve. Results are expressed as mean+/-standard deviation and compared by unpaired t-test. RESULTS: In the presented patient population 6 (27%) carried the E4 allele. Sixteen (63%) showed no E4 allele. Mean cross clamp time (CCT) was 56.2+/-13.5 min versus 55.7+/-12.1 min and CPB time was 91.8+/-17.5 versus 93.5+/-15.7 min. No statistical difference between E4-carriers and E4 non-carriers regarding CCT and CPB was observed. The total amount of IL-8 and TNF-alpha was higher in patients carrying the E4 genetic variant of apolipoprotein E in comparison to E4 non-carriers (P<0.08, P<0.039). CONCLUSION: The presence of the E4 allele is associated with increased release of IL-8 and TNF-alpha after CBP surgery. The preoperative determination of E4 in patients undergoing cardiac surgery may lead to additional perioperative measures for the treatment of an increased systemic inflammatory response.

Tissue engineering of small caliber vascular grafts.

OBJECTIVE: Previous tissue engineering approaches to create small caliber vascular grafts have been limited by the structural and mechanical immaturity of the constructs. This study uses a novel in vitro pulse duplicator system providing a 'biomimetic' environment during tissue formation to yield more mature, implantable vascular grafts. METHODS: Vascular grafts (I.D. 0.5 cm) were fabricated from novel bioabsorbable polymers (polyglycolic-acid/poly-4-hydroxybutyrate) and sequentially seeded with ovine vascular myofibroblasts and endothelial cells. After 4 days static culture, the grafts (n=24) were grown in vitro in a pulse duplicator system (bioreactor) for 4, 7, 14, 21, and 28 days. Controls (n=24) were grown in static culture conditions. Analysis of the neo-tissue included histology, scanning electron microscopy (SEM), and biochemical assays (DNA for cell content, 5-hydroxyproline for collagen). Mechanical testing was performed measuring the burst pressure and the suture retention strength. RESULTS: Histology showed viable, dense tissue in all samples. SEM demonstrated confluent smooth inner surfaces of the grafts exposed to pulsatile flow after 14 days. Biochemical analysis revealed a continuous increase of cell mass and collagen to 21 days compared to significantly lower values in the static controls. The mechanical properties of the pulsed vascular grafts comprised supra-physiological burst strength and suture retention strength appropriate for surgical implantation. CONCLUSIONS: This study demonstrates the feasibility of tissue engineering of viable, surgically implantable small caliber vascular grafts and the important effect of a 'biomimetic' in vitro environment on tissue maturation and extracellular matrix formation.

Surgical management of retro-aortic left renal vein in combined abdominal aortic and coronary surgery.

Although generally retro-aortic left renal vein is a rare anatomic finding, it occurs in 0.8% of the patients admitted for abdominal aortic aneurysm surgery. Surgeons fear fatal bleeding during clamping of the aorta, caused by a more caudal insertion of the retro-aortic left renal vein and a greater vulnerability of the anomalous tissue. Once such a complication occurs, a reconstruction of the retro-aortic left renal vein using a synthetic graft should be performed to obtain adequate renal venous flow and maintain renal function.