Bart Sanders

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.

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

In vivo collagen remodeling in the vascular wall of decellularized stented tissue-engineered heart valves

BACKGROUND Decellularized tissue-engineered heart valves (TEHVs) are under investigation as alternative for current heart valve prostheses with the potential to rapidly repopulate with cells within the body. Ideally, these valves are stented for transapical or minimally invasive delivery. It is unclear if and how the matrix of these valves remodels under in vivo hemodynamic loading conditions and in the presence of a stent. Here, we study the evolution of collagen orientation and tissue maturation in the wall of stented decellularized TEHVs with time after implantation. METHODS AND RESULTS In a previous study, stented TEHVs based on rapidly degrading scaffolds were cultured in bioreactors, decellularized, and transapically implanted as pulmonary valve replacement in sheep. In the present study, collagen (re)orientation in the initially isotropic valvular wall was assessed using a fluorescent collagen probe combined with confocal imaging and image analysis of explanted tissue at 8, 16, and 24 weeks following implantation. Collagen tortuosity or waviness in the explants, as a measure of matrix maturity, was quantified using a Gabor wavelet method and compared with tortuosity in native sheep vascular wall tissue. Results indicate that on the luminal side of the valvular wall, fibers became aligned in circumferential direction, while tortuosity increased with implantation time, showing striking similarities with the native collagen structure after 24 weeks. On the outside of the wall, where the engineered tissue touches the stent, collagen fibers in the vicinity of the struts aligned along the struts, whereas collagen fibers in between struts were randomly oriented. Immunohistochemistry was performed to evaluate the presence of elastin and collagen type I and III. After 8 weeks, collagen types I and III were mostly present at the luminal side of the wall, whereas at 16 and 24 weeks, a homogenous distribution of collagen I and III was observed throughout the wall. Elastin was mostly expressed at the luminal side after 24 weeks. Biochemical assays showed that the amount of DNA (as a measure of cell number) increased significantly after 8 and 24 weeks, glycosaminoglycans increased significantly after 8, 16, and 24 weeks, and hydroxyproline, as a measure of collagen amount, increased significantly after 24 weeks compared to the controls. CONCLUSIONS The collagen matrix in the wall of decellularized TEHVs shows clear structural remodeling and maturation with time. While collagen orientation rapidly remodels toward a native anisotropic architecture on the luminal side of the engineered valvular wall, it is dominated and guided by stent geometry on the outer side of the wall. Collagen tortuosity was increased with implantation time and was accompanied by an increase in elastin, especially on the luminal side of the vessel.

Computationally Designed 3D Printed Self-Expandable Polymer Stents with Biodegradation Capacity for Minimally Invasive Heart Valve Implantation: A Proof-of-Concept Study

Abstract The evolution of minimally invasive implantation procedures and the in vivo remodeling potential of decellularized tissue-engineered heart valves require stents with growth capacity to make these techniques available for pediatric patients. By means of computational tools and 3D printing technology, this proof-of-concept study demonstrates the design and manufacture of a polymer stent with a mechanical performance comparable to that of conventional nitinol stents used for heart valve implantation in animal trials. A commercially available 3D printing polymer was selected, and crush and crimping tests were conducted to validate the results predicted by the computational model. Finally, the degradability of the polymer was assessed via accelerated hydrolysis.

Computational modeling guides tissue-engineered heart valve design for long-term in vivo performance in a translational sheep model

Computational modeling–inspired heart valve designs guide tissue remodeling and ensure long-term functionality in tissue-engineered heart valves in sheep. Modeling remodeling Patients with valvular heart disease such as aortic stenosis (narrowing of the aortic valve in the heart) receive artificial or bioprosthetic valve replacements, but these have limited longevity and cannot grow with younger patients. Emmert et al. used computational modeling to design tissue-engineered heart valves from polymer scaffolds seeded with vascular cells. After 4 weeks of bioreactor culture, the grafts were decellularized before transcatheter implantation in sheep as pulmonary valve replacements. Nine of the 11 grafts remained functional up to 1 year later. Computational modeling predicted that valve leaflets would shorten in vivo during dynamic remodeling before reaching equilibrium, which was confirmed in the sheep. This work suggests that tissue engineering strategies should incorporate computational simulation to lead to more successful outcomes and more predictable clinical translation. Valvular heart disease is a major cause of morbidity and mortality worldwide. Current heart valve prostheses have considerable clinical limitations due to their artificial, nonliving nature without regenerative capacity. To overcome these limitations, heart valve tissue engineering (TE) aiming to develop living, native-like heart valves with self-repair, remodeling, and regeneration capacity has been suggested as next-generation technology. A major roadblock to clinically relevant, safe, and robust TE solutions has been the high complexity and variability inherent to bioengineering approaches that rely on cell-driven tissue remodeling. For heart valve TE, this has limited long-term performance in vivo because of uncontrolled tissue remodeling phenomena, such as valve leaflet shortening, which often translates into valve failure regardless of the bioengineering methodology used to develop the implant. We tested the hypothesis that integration of a computationally inspired heart valve design into our TE methodologies could guide tissue remodeling toward long-term functionality in tissue-engineered heart valves (TEHVs). In a clinically and regulatory relevant sheep model, TEHVs implanted as pulmonary valve replacements using minimally invasive techniques were monitored for 1 year via multimodal in vivo imaging and comprehensive tissue remodeling assessments. TEHVs exhibited good preserved long-term in vivo performance and remodeling comparable to native heart valves, as predicted by and consistent with computational modeling. TEHV failure could be predicted for nonphysiological pressure loading. Beyond previous studies, this work suggests the relevance of an integrated in silico, in vitro, and in vivo bioengineering approach as a basis for the safe and efficient clinical translation of TEHVs.

Are adipose-derived stem cells cultivated in human platelet lysate suitable for heart valve tissue engineering?

Tissue‐engineered heart valves represent a promising strategy for the growing need for valve replacements in cardiovascular medicine. Recent studies have shown that adipose‐derived stem cells (ADSC) are a viable cell source, as they are readily available in both the young and the elderly, show diverse differentiation potential and adapt their extracellular matrix (ECM) to a varying mechanical load. In vitro culture medium is usually enriched with fetal calf serum (FCS). However, a promising substitute has recently been found in human platelet lysate (HPL), which is superior in terms of proliferation speed and allogenicity. This study sought to elucidate the suitability of ADSC and HPL for heart valve tissue engineering (TE). ADSC harvested from five healthy individuals were cultured in both FCS and HPL. The cells were observed for differentiation potential, proliferation speed and immunophenotype, using immunohistochemistry and FACS analysis. Neotissue was assessed for ECM composition, human collagen I (hColl1) formation, histomorphology and mechanical stiffness under uniaxial tensile stress. Neotissue cultured in HPL was found to be significantly inferior in mechanical rigidity; it showed a three‐fold higher proliferation rate and a more dense ECM, but also a more heterogeneous hColl1 distribution. ECM analysis showed significantly higher amounts of DNA and glycosaminoglycans (GAG) in HPL‐cultured tissue. No significant differences were observed for differentiation potential and immunophenotype, apart from a lower CD166 expression in HPL. The mechanical inferiority of neotissue cultured in HPL represents a limitation to the use of HPL‐enriched media for heart valve TE with ADSC. This result concurs with data published about HPL and myofibroblasts derived from the venous wall. Similarly, the mechanical inferiority is not rooted in a difference in ECM composition, but rather in hColl1 architecture. Stem cell properties, as documented in the literature, are retained with HPL. A possible connection between the mechanical inferiority and the observed decrease in CD166 needs further investigation. Copyright

Adipose Derived Tissue Engineered Heart Valve

Abstract Introduction: A major challenge associated with heart valve tissue engineering is the in vitro creation of mature tissue structures compliant with native valve functionality. Various cell types have been investigated for heart valve tissue engineering. In addition to prenatal, umbilical cord- and vascular-derived cells, mesenchymal stem cells (MSCs) have gained large interest for tissue engineering purposes, because of their broad differentiation potential. However, bone marrow derived MSCs require a highly invasive harvesting procedure and decline in both cell number and differentiation potential proportionally with the donor’s age. In contrast, adipose derived stem cells (ADSCs) represent an interesting alternative. The ease of repeated access to subcutaneous adipose tissue as well as the less invasive donation procedures provide clear advantages. Therefore, this study investigated the suitability of ADSCs as alternative cell source for tissue engineered heart valves (TEHVs). Methods: Human ADSCs were seeded on TEHV-scaffolds (n=11) made of nonwoven polyglycolic acid coated with poly-4-hydroxybutyrate. TEHVs were cultivated in diastolic-pulse-duplicator-bioreactor systems and subsequently seeded with a superficial layer of ADSC-derived endothelial cells. Quantitative assessment of extracellular matrix composition of the TEHV-leaflets was performed with biochemical analyses for sulphated glycosaminoglycans, hydroxyproline and DNA content. Microstructural evaluation was performed on representative samples of the TEHVleaflets by (immuno-)histochemistry and scanning electron microscopy. The mechanical properties of the ADSC derived TEHV-leaflets were characterized by biaxial tensile tests. Results: ADSC-derived TEHV-leaflets showed a homogenous vital cell distribution throughout the whole leaflet structure that consisted of large amounts of glycosaminoglycans and collagen and was endothelialized. Furthermore, the mechanically stable matrix of the ADSC-derived TEHVs showed a stiffness range in the right order of magnitude for heart valve applications. Conclusion: Human ADSCs represent a promising alternative autologous mesenchymal cell source for TEHVs that is of large clinical relevance due to their easy accessibility, efficient proliferation and excellent tissue formation capacities.