Structural and biomechanical characterizations of acellular porcine mitral valve scaffolds: anterior leaflets, posterior leaflets, and chordae tendineae.

Mitral valve (MV) tissue engineering is still in its early stage, and one major challenge in MV tissue engineering is to identify appropriate scaffold materials. With the potential of acellular MV scaffolds being demonstrated recently, it is important to have a full understanding of the biomechanics of the native MV components and their acellular scaffolds. In this study, we have successfully characterized the structural and mechanical properties of porcine MV components, including anterior leaflet (AL), posterior leaflet (PL), strut chordae, and basal chordae, before and after decellularization. Quantitative DNA assay showed more than 90% reduction in DNA content, and Griffonia simplicifolia (GS) lectin immunohistochemistry confirmed the complete lack of porcine α-Gal antigen in the acellular MV components. In the acellular AL and PL, the atrialis, spongiosa, and fibrosa trilayered structure, along with its ECM constitutes, i.e., collagen fibers, elastin fibers, and portion of GAGs, were preserved. Nevertheless, the ECM of both AL and PL experienced a certain degree of disruption, exhibiting a less dense, porous ECM morphology. The overall anatomical morphology of the strut and basal chordae were also maintained after decellularization, with longitudinal morphology experiencing minimum disruption, but the cross-sectional morphology exhibiting evenly-distributed porous structure. In the acellular AL and PL, the nonlinear anisotropic biaxial mechanical behavior was overall preserved; however, uniaxial tensile tests showed that the removal of cellular content and the disruption of structural ECM did result in small decreases in maximum tensile modulus, tissue extensibility, failure stress, and failure strain for both MV leaflets and chordae.

Challenges in Perioperative Animal Care for Orthotopic Implantation of Tissue-Engineered Pulmonary Valves in the Ovine Model.

BACKGROUND: Development of valvular substitutes meeting the performance criteria for surgical correction of congenital heart malformations is a major research challenge. The sheep is probably the most widely used animal model in heart valves regenerative medicine. Although the standard cardiopulmonary bypass (CPB) technique and various anesthetic and surgical protocols are reported to be feasible and safe, they are associated with significant morbidity and mortality rates. The premise of this paper is that the surgical technique itself, especially the perioperative animal care and management protocol, is essential for successful outcomes and survival. METHODS: Ten juvenile and adult female sheep aged 7.8-37.5 months and weighing 32.0-58.0 kg underwent orthotopic implantation of tissue-engineered pulmonary valve conduits on beating heart under normothermic CPB. The animals were followed-up for 6 months before scheduled euthanasia. RESULTS: Based on our observations, we established a guide for perioperative care, follow-up, and treatment containing information regarding the appropriate clinical, biological, and ultrasound examinations and recommendations for feasible and safe anesthetic, surgical, and euthanasia protocols. Specific recommendations were also included for perioperative care of juvenile versus adult sheep. CONCLUSION: The described surgical technique was feasible, with a low mortality rate and minimal surgical complications. The proposed anesthetic protocol was safe and effective, ensuring both adequate sedation and analgesia as well as rapid recovery from anesthesia without significant complications. The established guide for postoperative care, follow-up and treatment in sheep after open-heart surgery may help other research teams working in the field of heart valves tissue regeneration.

Pentagalloyl Glucose and Its Functional Role in Vascular Health: Biomechanics and Drug-Delivery Characteristics.

Pentagalloyl glucose (PGG) is an elastin-stabilizing polyphenolic compound that has significant biomedical benefits, such as being a free radical sink, an anti-inflammatory agent, anti-diabetic agent, enzymatic resistant properties, etc. This review article focuses on the important benefits of PGG on vascular health, including its role in tissue mechanics, the different modes of pharmacological administration (e.g., oral, intravenous and endovascular route, intraperitoneal route, subcutaneous route, and nanoparticle based delivery and microbubble-based delivery), and its potential therapeutic role in vascular diseases such as abdominal aortic aneurysms (AAA). In particular, the use of PGG for AAA suppression and prevention has been demonstrated to be effective only in the calcium chloride rat AAA model. Therefore, in this critical review we address the challenges that lie ahead for the clinical translation of PGG as an AAA growth suppressor.

Detergent-based decellularization strategy preserves macro- and microstructure of heart valves.

OBJECTIVES: Biological tissue has great potential to function as bioprostheses in patients for heart valve replacement. As these matrices are mainly xenogenic, the immunogenicity needs to be reduced by decellularization steps. Reseeding of bioscaffolds has tremendous potential to prevent calcification upon implantation, so intact microstructure of the material is mandatory. An optimal decellularization protocol of heart valves resulting in adequate preservation of the extracellular architecture has still not been developed. Biological scaffolds must be decellularized to remove the antigenic potential while preserving the complex mixture of structural and functional proteins that constitute the extracellular matrix. METHODS: Here, we compared 3 different decellularization strategies for their efficiency to remove cells completely while preserving the porcine heart valve ultrastructure. Porcine pulmonary heart valves were treated either with trypsin-ethylenediaminetetraacetic acid (TRP), a protocol using detergents in combination with nucleases (DET + ENZ), or with Accutase® solution followed by nuclease treatment (ACC + ENZ). The treated heart valves then were subjected to histological, DNA and scanning electron microscopic analyses. RESULTS: All DNA fragments were removed after ACC + ENZ treatment, whereas cellular removal was incomplete in the TRP group. TRP and ACC + ENZ-treated valves were enlarged and showed a disrupted architecture and degraded ultrastructure. In contrast, fully acellular heart valves with intact architecture, layer composition and surface topography were achieved with DET + ENZ treatment. DET + ENZ treatment yielded excellent results in terms of preservation of material architecture and removal of DNA content. CONCLUSIONS: Compared to TRP and ACC + ENZ procedures, DET + ENZ-treated porcine pulmonary heart valves demonstrated well-preserved macroscopic structures and microscopic matrix components and represent an excellent scaffold for further application in tissue engineering.

Interobserver variability in physician-modified endograft planning by comparison with a three-dimensional printed aortic model.

BACKGROUND: With the increasing application of fenestrated and physician-modified endografting for aneurysm repair, there is increasing concern about the accuracy of vessel position measurements based on computed tomography scans. Inaccuracies in measurements may result in a "window-shutter" or "eclipsing" phenomenon whereby the fenestration may not overlie the vessel ostium completely. We hypothesized that vessel position measurements from reconstructed imaging do not represent the true vessel position as obtained from a three-dimensional (3D) printed physical model of the visceral aortic segment. METHODS: Medical 3D modeling software was used to develop the 3D reconstructions, which were then exported to the 3D printing software. This allowed 3D models to be physically generated. The distances to the top and bottom and the angle of each of the celiac, superior mesenteric, right renal, and left renal arteries were recorded. These same measurements were obtained by each of the blinded reviewers in addition to the aortic diameter at the midpoint of each of these vessels. Measurements were compared with intraclass correlation coefficient, nonparametric Spearman rank correlation test, and one-sample t-test to assess accuracy and precision. Statistical significance was set at P < .05 for all tests. RESULTS: Both the individual measurements and the average of the measurements were statistically accurate (significant) for the bottom of the superior mesenteric artery and the top and bottom of both the right and left renal arteries. There was variability and inaccuracy in all visceral vessel angles and in the bottom of the celiac artery (the top and the angle of the celiac artery were the arbitrary referents). CONCLUSIONS: Whereas the visceral vessel orifices are largely accurately assessed and measured, the vessel angles are not. This may lead to an eclipsing phenomenon, which may contribute to branch or fenestrated vessel failure and therefore reintervention. Further efforts should assess the clinical significance of the eclipsing phenomenon and should target accurate and appropriate fenestration construction to prevent long-term morbidity.

Longitudinal Stretching for Maturation of Vascular Tissues Using Magnetic Forces.

Cellular spheroids were studied to determine their use as "bioinks" in the biofabrication of tissue engineered constructs. Specifically, magnetic forces were used to mediate the cyclic longitudinal stretching of tissues composed of Janus magnetic cellular spheroids (JMCSs), as part of a post-processing method for enhancing the deposition and mechanical properties of an extracellular matrix (ECM). The purpose was to accelerate the conventional tissue maturation process via novel post-processing techniques that accelerate the functional, structural, and mechanical mimicking of native tissues. The results of a forty-day study of JMCSs indicated an expression of collagen I, collagen IV, elastin, and fibronectin, which are important vascular ECM proteins. Most notably, the subsequent exposure of fused tissue sheets composed of JMCSs to magnetic forces did not hinder the production of these key proteins. Quantitative results demonstrate that cyclic longitudinal stretching of the tissue sheets mediated by these magnetic forces increased the Young's modulus and induced collagen fiber alignment over a seven day period, when compared to statically conditioned controls. Specifically, the elastin and collagen content of these dynamically-conditioned sheets were 35- and three-fold greater, respectively, at seven days compared to the statically-conditioned controls at three days. These findings indicate the potential of using magnetic forces in tissue maturation, specifically through the cyclic longitudinal stretching of tissues.

Immunomodulatory effects of adipose tissue-derived stem cells on elastin scaffold remodeling in diabetes.

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.

Platform technologies for decellularization, tunic-specific cell seeding, and in vitro conditioning of extended length, small diameter vascular grafts.

The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.

Janus magnetic cellular spheroids for vascular tissue engineering.

Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly.

Biological magnetic cellular spheroids as building blocks for tissue engineering.

Magnetic nanoparticles (MNPs), primarily iron oxide nanoparticles, have been incorporated into cellular spheroids to allow for magnetic manipulation into desired shapes, patterns and 3-D tissue constructs using magnetic forces. However, the direct and long-term interaction of iron oxide nanoparticles with cells and biological systems can induce adverse effects on cell viability, phenotype and function, and remain a critical concern. Here we report the preparation of biological magnetic cellular spheroids containing magnetoferritin, a biological MNP, capable of serving as a biological alternative to iron oxide magnetic cellular spheroids as tissue engineered building blocks. Magnetoferritin NPs were incorporated into 3-D cellular spheroids with no adverse effects on cell viability up to 1 week. Additionally, cellular spheroids containing magnetoferritin NPs were magnetically patterned and fused into a tissue ring to demonstrate its potential for tissue engineering applications. These results present a biological approach that can serve as an alternative to the commonly used iron oxide magnetic cellular spheroids, which often require complex surface modifications of iron oxide NPs to reduce the adverse effects on cells.

Mitigation of diabetes-related complications in implanted collagen and elastin scaffolds using matrix-binding polyphenol.

There is a major need for scaffold-based tissue engineered vascular grafts and heart valves with long-term patency and durability to be used in diabetic cardiovascular patients. We hypothesized that diabetes, by virtue of glycoxidation reactions, can directly crosslink implanted scaffolds, drastically altering their properties. In order to investigate the fate of tissue engineered scaffolds in diabetic conditions, we prepared valvular collagen scaffolds and arterial elastin scaffolds by decellularization and implanted them subdermally in diabetic rats. Both types of scaffolds exhibited significant levels of advanced glycation end products (AGEs), chemical crosslinking and stiffening -alterations which are not favorable for cardiovascular tissue engineering. Pre-implantation treatment of collagen and elastin scaffolds with penta-galloyl glucose (PGG), an antioxidant and matrix-binding polyphenol, chemically stabilized the scaffolds, reduced their enzymatic degradation, and protected them from diabetes-related complications by reduction of scaffold-bound AGE levels. PGG-treated scaffolds resisted diabetes-induced crosslinking and stiffening, were protected from calcification, and exhibited controlled remodeling in vivo, thereby supporting future use of diabetes-resistant scaffolds for cardiovascular tissue engineering in patients with diabetes.

The acellular myocardial flap: a novel extracellular matrix scaffold enriched with patent microvascular networks and biocompatible cell niches.

There is a great need for acellular, fully vascularized, and biocompatible myocardial scaffolds that provide agreeable biological, nutritional, and biomechanical niches for reseeded cells for in vitro and in vivo applications. We generated myocardial flap scaffolds comprising porcine left-anterior ventricular myocardium and its associated coronary arteries and veins and investigated the combinatorial effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) perfusion on both the myocardial extracellular matrix (ECM) and the vascular ECM. Results showed that all scaffolds displayed a fully intact and patent vasculature, with arterial burst pressures indistinguishable from native coronary arteries and perfusion to the level of capillaries. Scaffolds were free of cellular proteins and retained collagen and elastin ECM components, exhibited excellent mechanical properties, and were cytocompatible toward relevant seeded cells. SDS perfusion preserved collagen IV, laminin, and fibronectin well, but only reduced DNA content by 33%; however, this was further improved by post-SDS nuclease treatments. By comparison, NaOH was very effective in removing cells and eliminated more than 95% of tissue DNA, but also significantly reduced levels of laminin and fibronectin. Such constructs can be readily trimmed to match the size of the infarct and might be able to functionally integrate within host myocardium and be nourished by direct anastomotic connection with the host's own vasculature; they might also be useful as physiologically accurate models for in vitro studies of cardiac physiology and pathology.

Regenerative potential of decellularized porcine nucleus pulposus hydrogel scaffolds: stem cell differentiation, matrix remodeling, and biocompatibility studies.

Nucleus pulposus (NP) tissue regeneration has been proposed as an early stage interventional therapy to combat intervertebral disc degeneration. We have previously reported on the development and characterization of a novel biomimetic acellular porcine NP (APNP) hydrogel. Herein, we aimed to evaluate this material for use as a suitable scaffold for NP tissue regeneration. Human-adipose-derived stem cells (hADSCs) were cultured for 14 days on APNP hydrogels in chemically defined differentiation media and were analyzed for an NP-cell-like mRNA expression profile, evidence of hydrogel remodeling including hydrogel contraction measurements, extracellular matrix production, and compressive dynamic mechanical properties. The innate capacity of the hydrogel itself to induce stem cell differentiation was also examined via culture in media lacking soluble differentiation factors. Additionally, the in vivo biocompatibility of non-crosslinked and ethyldimethylaminopropyl carbodiimide/N-hydroxysuccinimide and pentagalloyl glucose crosslinked hydrogels was evaluated in a rat subdermal model. Results indicated that hADSCs expressed putative NP-cell-positive gene transcript markers when cultured on APNP hydrogels. Additionally, glycosaminoglycan and collagen content of hADSC-seeded hydrogels was significantly greater than nonseeded controls and cell-seeded hydrogels exhibited evidence of contraction and tissue inhibitors of metalloproteinase-1 production. The dynamic mechanical properties of the hADSC-seeded hydrogels increased with time in culture in comparison to noncell-seeded controls and approached values reported for native NP tissue. Immunohistochemical analysis of explants illustrated the presence of mononuclear cells, including macrophages and fibroblasts, as well as blood vessel infiltration and collagen deposition within the implant interstices after 4 weeks of implantation. Taken together, these results suggest that APNP hydrogels, in concert with autologous ADSCs, may serve as a suitable scaffold for NP tissue regeneration.

Structural and biomechanical characterizations of porcine myocardial extracellular matrix.

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

Form Follows Function: Advances in Trilayered Structure Replication for Aortic Heart Valve Tissue Engineering.

Tissue engineering the aortic heart valve is a challenging endeavor because of the particular hemodynamic and biologic conditions present in the native aortic heart valve. The backbone of an ideal valve substitute should be a scaffold that is strong enough to withstand billions of repetitive bending, flexing and stretching cycles, while also being slowly degradable to allow for remodeling. In this review we highlight three overlooked aspects that might influence the long term durability of tissue engineered valves: replication of the native valve trilayered histoarchitecture, duplication of the three-dimensional shape of the valve and cell integration efforts focused on getting the right number and type of cells to the right place within the valve structure and driving them towards homeostatic maintenance of the valve matrix. We propose that the trilayered structure in the native aortic valve that includes a middle spongiosa layer cushioning the motions of the two external fibrous layers should be our template for creation of novel scaffolds with improved mechanical durability. Furthermore, since cells adapt to micro-loads within the valve structure, we believe that interstitial cell remodeling of the valvular matrix will depend on the accurate replication of the structures and loads, resulting in successful regeneration of the valve tissue and extended durability.

Inflammation in cardiovascular tissue engineering: the challenge to a promise: a minireview.

Tissue engineering employs scaffolds, cells, and stimuli brought together in such a way as to mimic the functional architecture of the target tissue or organ. Exhilarating advances in tissue engineering and regenerative medicine allow us to envision in vitro creation or in vivo regeneration of cardiovascular tissues. Such accomplishments have the potential to revolutionize medicine and greatly improve our standard of life. However, enthusiasm has been hampered in recent years because of abnormal reactions at the implant-host interface, including cell proliferation, fibrosis, calcification and degeneration, as compared to the highly desired healing and remodeling. Animal and clinical studies have highlighted uncontrolled chronic inflammation as the main cause of these processes. In this minireview, we present three case studies highlighting the importance of inflammation in tissue engineering heart valves, vascular grafts, and myocardium and propose to focus on the endothelial barrier, the "final frontier" endowed with the natural potential and ability to regulate inflammatory signals.

Macrophage differentiation and polarization on a decellularized pericardial biomaterial.

The monocyte-derived macrophage (MDM), present at biomaterial implantations, can increase, decrease or redirect the inflammatory and subsequent wound healing process associated with the presence of a biomaterial. Understanding MDM responses to biomaterials is important for improved prediction and design of biomaterials for tissue engineering. This study analyzed the direct differentiation of monocytes on intact, native collagen. Human monocytes were differentiated on decellularized bovine pericardium (DBP), polydimethylsiloxane (PDMS) or polystyrene (TCPS) for 14 d. MDMs on all surfaces released high amounts of MMP-9 compared to MMP-2 and relatively little MMP-1. MDMs differentiated on DBP released more MMP-2, but less acid phosphatase activity. MDMs on all three surfaces released low amounts of cytokines, although substrate differences were found: MDMs on DBP released higher amounts of IL-6, IL-8, and MCP-1 but lower amounts of IL-10 and IL-1ra. This research provides evidence that MDMs on decellularized matrices may not be stimulated towards an activated, inflammatory phenotype, supporting the potential of decellularized matrices for tissue engineering. This study also demonstrated that the differentiation surface affects MDM phenotype and therefore study design of macrophage interactions with biomaterials should scrutinize the specific macrophage culture method utilized and its effects on macrophage phenotype.

Lectin and antibody-based histochemical techniques for cardiovascular tissue engineering.

Tissue engineering holds immense potential for treatment of cardiovascular diseases by creating living structures to replace diseased blood vessels, heart valves, and cardiac muscle. In a traditional approach, scaffolds are seeded with stem cells and subjected to stimuli in bioreactors that mimic physiologic conditions or are directly implanted into target sites in animal models. The expected results are significant cell changes, extensive remodeling of the scaffolds and creation of surrogate structures that would be deemed acceptable for tissue regeneration. Histochemical techniques are increasingly becoming essential tools in tissue engineering research. In our studies, we used lectin and antibody-based techniques to characterize novel collagen and elastin scaffolds and to ensure efficient removal of xenoantigens. Scaffolds were implanted in animals and infiltrated host cells were identified using antibodies to activated fibroblasts, macrophages, and lymphocytes. Stem cell-seeded scaffolds were subjected to mechanical strains and tested for differentiation into cardiovascular cells using antibody-based double immunofluorescence methods. Finally, living heart valves were constructed from scaffolds and stem cells, subjected to conditioning in a bioreactor and stem cell differentiation evaluated by immunofluorescence. Overall, these techniques have proven to be outstanding companions to biochemical, molecular biology and cell analysis methods used in tissue engineering research and development.