Specific Binding of Alzheimer's Aß Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aß42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aß42 is detectable in the plasma, a phenomenon thought to result from Aß becoming more aggregated in the brain and less Aß42 and Aß40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse "cargos" of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aß42 and Aß40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aß and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aß42 and Aß40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aß incubation with EVs overnight yielded larger amounts of bound Aß peptide that was fibrillar in structure. These findings point to a specific amyloid-EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

Trisomy 21 Alters Cell Proliferation and Migration of iPSC-Derived Cardiomyocytes on Type VI Collagen.

Purpose: Individuals with Down syndrome (DS) are 2000 times more likely to develop a congenital heart defect (CHD) than the typical population Freeman et al. in Am J Med Genet 80:213-217 (1998). The majority of CHDs in individuals with DS characteristically involve the atrioventricular (AV) canal, including the valves and the atrial or ventricular septum. Type VI collagen (COLVI) is the primary structural component in the developing septa and endocardial cushions, with two of the three genes encoding for COLVI located on human chromosome 21 and upregulated in Down syndrome (von Kaisenberg et al. in Obstet Gynecol 91:319-323, 1998; Gittenberger-De Groot et al. in Anatom Rec Part A 275:1109-1116, 2023). Methods: To investigate the effect of COLVI dosage on cardiomyocytes with trisomy 21, induced pluripotent stem cells (iPSC) from individuals with DS and age- and sex-matched controls were differentiated into cardiomyocytes (iPSC-CM) and plated on varying concentrations of COLVI. Results: Real time quantitative PCR showed decreased expression of cardiac-specific genes of DS iPSC-CM lines compared to control iPSC-CM. As expected, DS iPSC-CM had increased expression of genes on chromosome 21, including COL6A1, COL6A2, as well as genes not located on chromosome 21, namely COL6A3, HAS2 and HYAL2. We found that higher concentrations of COLVI result in decreased proliferation and migration of DS iPSC-CM, but not control iPSC-CM. Conclusions: These results suggest that the increased expression of COLVI in DS may result in lower migration-driven elongation of endocardial cushions stemming from lower cell proliferation and migration, possibly contributing to the high incidence of CHD in the DS population. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00791-x.

Vascularization of PEGylated fibrin hydrogels increases the proliferation of human iPSC-cardiomyocytes.

Studies have long sought to develop engineered heart tissue for the surgical correction of structural heart defects, as well as other applications and vascularization of this tissue has presented a challenge. Recent studies suggest that vascular cells and a vascular network may have regenerative effects on implanted cardiomyocytes (CM) and nearby heart tissue separate from perfusion of oxygen and nutrients. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived CM. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells to human dermal fibroblasts, the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1 × 106 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived CM were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. CM proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased CM proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower CM maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7, and phospholamban compared to hydrogels without vascular cells and hydrogels with vascular networks. Altogether, this study details a robust method of forming rudimentary vascular networks in a fibrin-based hydrogel and shows that a hydrogel containing endothelial cells and fibroblasts can induce proliferation in adjacent CM, and these cells do not hinder CM gene expression when organized into a vascular network.

An In Vitro Characterization of a PCL-Fibrin Scaffold for Myocardial Repair.

Each year in the United States approximately 10,000 babies are born with a complex congenital heart defect (CHD) requiring surgery in the first year of after birth. Several of these operations require the implantation of a full-thickness heart patch; however, the current patch materials available to pediatric heart surgeons are exclusively non-living and non-degradable, which do not grow with the patient and are prone to fail due to an inability to integrate with the heart. In this work, the goal was to develop a full-thickness, tissue engineered myocardial patch (TEMP) that is made from biodegradable components, strong enough to withstand the mechanical forces of the heart wall, and able to integrate with the heart and drive neotissue formation. Here, a thick and porous electrospun PCL scaffold filled with high-salt PEGylated fibrin was developed. The scaffold was found to be mechanically sufficient for heart wall repair. Vascular cells were able to infiltrate more than halfway through the scaffold in static culture within three weeks. The scaffold maintained pluripotent stem cells for at least four days, supports viable iPSC-derived cardiomyocytes, and fostered tissue thickening in vitro. The TEMP developed here and tested in vitro is promising for the repair of structural CHD and will next be assessed in situ.

Bioreactor Design for Culturing Vascularized Engineered Tissue in Flow Conditions.

BACKGROUND: Current treatments for congenital heart defects often require surgery and implantation of a synthetic patch or baffle that becomes a fibrous scar and leads to a high number of reoperations. Previous studies in rats have shown that a pre-vascularized scaffold can integrate into the heart and result in regions of vascularized and muscularized tissue. However, increasing the thickness of this scaffold for use in human hearts requires a method to populate the thick scaffold and mature it under physiologic flow and electrical conditions. EXPERIMENT: We developed a bioreactor system that can perfuse up to six 7-mm porous scaffolds with tunable gravity-mediated flow and chronic electrical stimulation. Three polymers which have been reported to be biocompatible were evaluated for effects on the viability of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Bioreactor flow and electrical stimulation functions were tested, and the bioreactor was operated for up to 7 days to ensure reliability and lack of leaks in a 37C, humidified incubator. Height and flow relationships were measured for perfusion through an electrospun polycaprolactone (PCL) and gelatin scaffold previously reported by our laboratory. Culture with cells was evaluated by plating human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (hDF) on top of the scaffolds in both static and flow conditions for 2,5 and 7 days. As a proof-of concept, scaffolds were cryosectioned and cell infiltration was quantified using immunofluorescence staining. RESULTS: Neither MED610 (Stratasys), Vero (Stratasys), nor FORMLAB materials affected the viability of iPSC derived cardiomyocytes, and MED610 was chosen for manufacture due to familiarity of 3D printing from this material. The generation of electrical field stimulation from 0 to 5 volts and physiological ranges of pump capacities were verified. The relationship between height and flow was calculated for scaffolds with and without cells. Finally, we demonstrated evaluation of cell depth and structure in scaffolds cultured for 2, 5, and 7 days. CONCLUSION: The gravity-mediated flow bioreactor system we developed can be used as a platform for 3D cell culture particularly designed for perfusing vascularized tissue constructs with electrical stimulation for cardiac maturation.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

Assessment of electrospun cardiac patches made with sacrificial particles and polyurethane-polycaprolactone blends.

Congenital heart defects (CHDs) are the leading cause of death in live-born infants. Currently, patches used in the repair of CHDs are exclusively inert and non-degradable, which increases the risk of arrhythmia, follow-up surgeries, and sudden cardiac death. In this preliminary study, we sought to fabricate biodegradable scaffolds that can support cardiac regeneration in the repair of CHDs. We electrospun biodegradable scaffolds using various blends of polyurethane (PU) and polycaprolactone (PCL) with and without sacrificial poly(ethylene oxide) (PEO) particles and assessed the mechanical properties, cell infiltration levels, and inflammatory response in vitro (surface cell seeding) and in vivo (subcutaneous mouse implant). We hypothesized that a blend of the two polymers would preserve the low stiffness of PU as well as the high cell infiltration observed in PCL scaffolds. The inclusion of PU in the blends, even as low as 10%, decreased cell infiltration both in vitro and in vivo. The inclusion of sacrificial PEO increased pore sizes, reduced Young's moduli, and reduced the inflammatory response in all scaffold types. Collectively, we have concluded that a PCL patch electrospun with sacrificial PEO particles is the most promising scaffold for further assessment as in our heart defect model.

Chemokine-Induced PBMC and Subsequent MSC Migration Toward Decellularized Heart Valve Tissue.

PURPOSE: Enhancing the recellularization of a decellularized heart valve in situ may lead to an improved or ideal heart valve replacement. A promising approach is leveraging the immune response for inflammation-mediated recellularization. However, this mechanism has not been previously demonstrated in vitro. METHODS: This study investigated loading the chemokine MCP-1 into decellularized porcine heart valve tissue and measured the migration of human peripheral blood mononuclear cells (PBMCs) and mesenchymal stem cells (MSCs) toward the chemokine loaded valve tissue. RESULTS: The results of this study demonstrate that MCP-1-loaded tissues increase PBMC migration compared to non-loaded tissues. Additionally, we demonstrate MCP-1-loaded tissues that have recruited PBMCs lead to increased migration of MSCs compared to decellularized tissue alone. CONCLUSION: The results of this study provide evidence for the inflammation-mediated recellularization mechanism. Furthermore, the results support the use of such an approach for enhancing the recellularization of a decellularized heart valve.

Engineering Myocardium for Heart Regeneration-Advancements, Considerations, and Future Directions.

Heart disease is the leading cause of death in the United States among both adults and infants. In adults, 5-year survival after a heart attack is <60%, and congenital heart defects are the top killer of liveborn infants. Problematically, the regenerative capacity of the heart is extremely limited, even in newborns. Furthermore, suitable donor hearts for transplant cannot meet the demand and require recipients to use immunosuppressants for life. Tissue engineered myocardium has the potential to replace dead or fibrotic heart tissue in adults and could also be used to permanently repair congenital heart defects in infants. In addition, engineering functional myocardium could facilitate the development of a whole bioartificial heart. Here, we review and compare in vitro and in situ myocardial tissue engineering strategies. In the context of this comparison, we consider three challenges that must be addressed in the engineering of myocardial tissue: recapitulation of myocardial architecture, vascularization of the tissue, and modulation of the immune system. In addition to reviewing and analyzing current progress, we recommend specific strategies for the generation of tissue engineered myocardial patches for heart regeneration and repair.

Review on Mechanical Support and Cell-Based Therapies for the Prevention and Recovery of the Failed Fontan-Kreutzer Circulation.

Though the current staged surgical strategy for palliation of single ventricle heart disease, culminating in a Fontan circulation, has increased short-term survival, mounting evidence has shown that the single ventricle, especially a morphologic right ventricle (RV), is inadequate for long-term circulatory support. In addition to high rates of ventricular failure, high central venous pressures (CVP) lead to liver fibrosis or cirrhosis, lymphatic dysfunction, kidney failure, and other comorbidities. In this review, we discuss the complications seen with Fontan physiology, including causes of ventricular and multi-organ failure. We then evaluate the clinical use, results, and limitations of long-term mechanical assist devices intended to reduce RV work and high CVP, as well as biological therapies for failed Fontan circulations. Finally, we discuss experimental tissue engineering solutions designed to prevent Fontan circulation failure and evaluate knowledge gaps and needed technology development to realize a more robust single ventricle therapy.

Embedding of Precision-Cut Lung Slices in Engineered Hydrogel Biomaterials Supports Extended Ex Vivo Culture.

Maintaining the three-dimensional architecture and cellular complexity of lung tissue ex vivo can enable elucidation of the cellular and molecular pathways underlying chronic pulmonary diseases. Precision-cut lung slices (PCLS) are one human-lung model with the potential to support critical mechanistic studies and early drug discovery. However, many studies report short culture times of 7-10 days. Here, we systematically evaluated poly(ethylene glycol)-based hydrogel platforms for the encapsulation of PCLS. We demonstrated the ability to support ex vivo culture of embedded PCLS for at least 21 days compared with control PCLS floating in media. These customized hydrogels maintained PCLS architecture (no difference), viability (4.7-fold increase, P < 0.0001), and cellular phenotype as measured by SFTPC (1.8-fold increase, P < 0.0001) and vimentin expression (no change) compared with nonencapsulated controls. Collectively, these results demonstrate that hydrogel biomaterials support the extended culture times required to study chronic pulmonary diseases ex vivo using PCLS technology.

Evaluation of a polyurethane-reinforced hydrogel patch in a rat right ventricle wall replacement model.

Congenital heart defects affect about 1% births in the United States. Many of the defects are treated with surgically implanted patches made from inactive materials or fixed pericardium that do not grow with the patients, leading to an increased risk of arrhythmia, sudden cardiac death, and heart failure. This study investigated an angiogenic poly(ethylene glycol) fibrin-based hydrogel reinforced with an electrospun biodegradable poly(ether ester urethane) urea (BPUR) mesh layer that was designed to encourage cell invasion, angiogenesis, and regenerative remodeling in the repair of an artificial defect created onto the rat right ventricle wall. Electrocardiogram signals were analyzed, heart function was measured, and fibrosis, macrophage infiltration, muscularization, vascularization, and defect size were evaluated at 4- and 8-weeks post-surgery. Compared with rats with fixed pericardium patches, rats with BPUR-reinforced hydrogel patches had fewer arrhythmias and greater right ventricular ejection fraction and cardiac output, as well as greater left ventricular ejection fraction, fractional shorting, stroke work and cardiac output. Histology and immunofluorescence staining showed less fibrosis and less patch material remaining in rats with BPUR-reinforced hydrogel patches at 4- and 8-weeks. Rats with BPUR-reinforced hydrogel patches also had a greater volume of granular tissue, a greater volume of muscularized tissue, more blood vessels, and a greater number of leukocytes, pan-macrophages, and M2 macrophages at 8 weeks. Overall, this study demonstrated that the engineered BPUR-reinforced hydrogel patch initiated greater regenerative vascular and muscular remodeling with a limited fibrotic response, resulting in fewer incidences of arrhythmia and improved heart function compared with fixed pericardium patches when applied to heal the defects created on the rat right ventricle wall. STATEMENT OF SIGNIFICANCE: The study tested a polyurethane-reinforced hydrogel patch in a rat right ventricle wall replacement model. Compared with fixed pericardium patches, these reinforced hydrogel patches initiated greater regenerative vascular and muscular remodeling with a reduced fibrotic response, resulting in fewer incidences of arrhythmia and improved heart function at 4- and 8-weeks post surgery. Overall, the new BPUR-reinforced hydrogel patches resulted in better heart function when replacing contractile myocardium than fixed pericardium patches.

Epigenetics and Mechanobiology in Heart Development and Congenital Heart Disease.

: Congenital heart disease (CHD) is the most common birth defect worldwide and the number one killer of live-born infants in the United States. Heart development occurs early in embryogenesis and involves complex interactions between multiple cell populations, limiting the understanding and consequent treatment of CHD. Furthermore, genome sequencing has largely failed to predict or yield therapeutics for CHD. In addition to the underlying genome, epigenetics and mechanobiology both drive heart development. A growing body of evidence implicates the aberrant regulation of these two extra-genomic systems in the pathogenesis of CHD. In this review, we describe the stages of human heart development and the heart defects known to manifest at each stage. Next, we discuss the distinct and overlapping roles of epigenetics and mechanobiology in normal development and in the pathogenesis of CHD. Finally, we highlight recent advances in the identification of novel epigenetic biomarkers and environmental risk factors that may be useful for improved diagnosis and further elucidation of CHD etiology.

Controlled Release of Small Molecules for Cardiac Differentiation of Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have been shown to differentiate to functional cardiomyocytes (CM) with high efficiency through temporally controlled inhibition of the GSK3/Wnt signaling pathways. In this study, we investigated the ability of temporally controlled release of GSK3/Wnt small-molecule inhibitors to drive cardiac differentiation of iPSC without manual intervention. Porous silica particles were loaded with GSK3 inhibitor CHIR99021 or Wnt inhibitor IWP2, and the particles containing IWP2 were coated with 5 wt% poly(lactic-co-glycolic acid) 50:50 to delay release by ∼72 h. iPSCs reprogrammed through mRNA transfection were cultured with these particles up to 30 days. High-performance liquid chromatography suggests a burst release of CHIR99021 within the first 24 h and a delayed release of IWP2 after 72 h. Annexin V/propidium iodide staining did not show a significant effect on apoptosis or necrosis rates. Cultured cells upregulated both early (Nkx 2.5, Isl-1) and late (cTnT, MHC, Cx43) cardiac markers, assayed with a quantitative real-time polymerase chain reaction, and began spontaneous contraction at 3.0 ± 0.6 Hz at 15-21 days after the start of differentiation. CM had clear sarcomeric striations when stained for ß-myosin heavy chain, and showed expression and punctate membrane localization of gap junction protein Connexin43. Calcium and voltage-sensitive imaging showed both action potential and calcium transients typical of immature CM. This study showed that the cardiac differentiation of pluripotent stem cells can be directed by porous silica vectors with temporally controlled release of small-molecule inhibitors. These results suggest methods for automating and eliminating variability in manual maintenance of inhibitor concentrations in the differentiation of pluripotent stem cells to CM.

Bioengineering Cardiac Tissue Constructs With Adult Rat Cardiomyocytes.

Bioengineering cardiac tissue constructs with adult cardiomyocytes may help treat adult heart defects and injury. In this study, we fabricated cardiac tissue constructs by seeding adult rat cardiomyocytes on a fibrin gel matrix and analyzed the electromechanical properties of the formed cardiac tissue constructs. Adult rat cardiomyocytes were isolated with a collagenase type II buffer using an optimized Langendorff perfusion system. Cardiac tissue constructs were fabricated using either indirect plating with cardiomyocytes that were cultured for 1 week and dedifferentiated or with freshly isolated cardiomyocytes. The current protocol generated (3.1 ± 0.5) × 10 (n = 5 hearts) fresh cardiomyocytes from a single heart. Tissue constructs obtained by both types of plating contracted up to 30 days, and electrogram (ECG) signals and contractile twitch forces were detected. The constructs bioengineered by indirect plating of dedifferentiated cardiomyocytes produced an ECG R wave amplitude of 15.1 ± 5.2 µV (n = 7 constructs), a twitch force of 70-110 µN, and a spontaneous contraction rate of about 390 bpm. The constructs bioengineered by direct plating of fresh cardiomyocytes generated an ECG R wave amplitude of 6.3 ± 2.5 µV (n = 8 constructs), a twitch force of 40-60 µN, and a spontaneous contraction rate of about 230 bpm. This study successfully bioengineered three-dimensional cardiac tissue constructs using primary adult cardiomyocytes.

Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells.

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and ß-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and ß-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

Full-Thickness Heart Repair with an Engineered Multilayered Myocardial Patch in Rat Model.

In a rat model of right free wall replacement, the transplantation of an engineered multilayered myocardial patch fabricated from a polycaprolactone membrane supporting a chitosan/heart matrix hydrogel induces significant muscular and vascular remodeling and results in a significantly higher right ventricular ejection fraction compared to use of a commercially available pericardium patch.

The Effect of Substrate Stiffness on Cardiomyocyte Action Potentials.

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs - polysiloxane-based and epoxy-based - are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 °C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.