In Situ Myocardial Regeneration With Tissue Engineered Cardiac Patch Using Spheroid-Based 3-Dimensional Tissue.

BACKGROUND: We have developed a tissue engineered cardiac patch derived from a 3-dimensional (3D) myocardial tissue reinforced with extracellular matrix in an effort to enhance in situ myocardial regeneration. The feasibility of the patch was evaluated in a porcine model by various modalities to assess both the constructive and functional aspects of regeneration. METHODS: A spheroid-based 3D multicellular tissue was created using a 3D net mold system that incorporated cardiomyocytes and embryonic fibroblast cells. The 3D multicellular tissue was incorporated with extracellular matrix sheets and surgically implanted into the right ventricle of a healthy porcine model (n = 4). After 60 days, the implanted patches were evaluated by cardiac magnetic resonance imaging and electroanatomic mapping studies as well as by post-euthanasia analyses, including measurements of mechanical viscoelasticity. RESULTS: Cardiac magnetic resonance imaging revealed improved regional tissue perfusion in the patch area. Electroanatomic mapping exhibited regenerated electrical conductivity in the patch, as evidenced by relatively preserved voltage regions (1.11 ± 0.8 mV) in comparison to the normal right ventricle (4.7 ± 2.8 mV). Histologic and tissue analyses confirmed repopulation of site-specific host cells, including premature cardiomyocytes and active vasculogenesis. These findings were supported by quantitative reverse transcription-polymerase chain reaction. CONCLUSIONS: The tissue engineered cardiac patch effectively facilitated in situ constructive and functional myocardial regeneration, characterized by increased regional tissue perfusion and positive electrical activity in the porcine model.

Integrins and extracellular matrix proteins modulate adipocyte thermogenic capacity.

Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and ß1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.

Cell-Laden Gradient Hydrogel Scaffolds for Neovascularization of Engineered Tissues.

Gradients in mechanical properties, physical architecture and biochemical composition exist in a variety of complex tissues, yet 3D in vitro models that enable investigation of these cues on cellular processes, especially those contributing to vascularization of engineered tissues are limited. Here, a photopolymerization approach to create cell-laden hydrogel biomaterials with decoupled and combined gradients in modulus, immobilized cell adhesive peptide (RGD) concentration, and proteolytic degradation enabling spatial encapsulation of vascular spheroids is reported to elucidate their impact on vascular sprouting in 3D culture. Vascular spheroids encapsulated in these gradient scaffolds exhibit spatial variations in total sprout length. Scaffolds presenting an immobilized RGD gradient promote biased vascular sprouting toward increasing RGD concentration. Importantly, biased sprouting is found to be dependent on immobilized RGD gradient characteristics, including magnitude and slope, with increases in these factors contributing to significant enhancements in biased sprouting responses. Conversely, reduction in biased sprouting responses is observed in combined gradient scaffolds possessing opposing gradients in RGD and modulus. The presented work is the first to demonstrate the use of a cell-laden biomaterial platform to systematically investigate the role of multiple scaffold gradients as well as gradient slope, magnitude and orientation on vascular sprouting responses in 3D culture.

Induced Pluripotent Stem Cell-Derived Endothelial Networks Accelerate Vascularization But Not Bone Regeneration.

Vascularization is critical for engineering mineralized tissues. It has been previously shown that biomaterials containing preformed endothelial networks anastomose to host vasculature following implantation. However, the networks alone may not increase regeneration. In addition, a clinically applicable source of cells for vascularization is needed. In this study, vascular networks were generated from endothelial cells (ECs) derived from human induced pluripotent stem cells (iPSCs). Network formation by iPSC-ECs within fibrin gels was investigated in a mesenchymal stem cells (MSCs) coculture spheroid model. Statistical design of experiments technique was evaluated for its predicting capability during the optimization of experimental parameters. The prevascularized units were combined with hydroxyapatite nanoparticles to develop a vascularized composite hydrogel that was implanted in a rodent critical-sized cranial defect model. Immunohistological staining for human-specific CD31 at week 1 indicated the presence and maintenance of the implanted vessels. At 8 weeks, the prevascularized systems resulted in higher vessel density over MSC-only scaffolds. The implanted vessels appeared to establish flow with host vasculature. While there was a slight increase in bone volume in the prevascularized bone construct compared to MSC-only bone constructs, there was not a profound increase in bone regeneration. These results show that scaffolds with network structures can be generated from ECs derived from iPSC and that the networks survive and inosculate with the host postimplantation in a bone model. Impact statement Vascularization is critical for engineering bone. Prevascularized scaffolds have been shown to improve postimplantation vascularization. Herein, vascularized networks were generated from induced pluripotent cells derived from endothelial cells. These vascularized units were combined with a fibrin/hydroxyapatite scaffold to develop a prevascularized construct for bone regeneration. Implantation of these scaffolds in a small animal cranial defect model resulted in network inosculation and increased vascularization, but exhibited only a limited effect on bone formation. This study provides insight into the challenges of generating vascularized bone.

Immobilized RGD concentration and proteolytic degradation synergistically enhance vascular sprouting within hydrogel scaffolds of varying modulus.

Insufficient vascularization limits the volume and complexity of engineered tissue. The formation of new blood vessels (neovascularization) is regulated by a complex interplay of cellular interactions with biochemical and biophysical signals provided by the extracellular matrix (ECM) necessitating the development of biomaterial approaches that enable systematic modulation in matrix properties. To address this need poly(ethylene) glycol-based hydrogel scaffolds were engineered with a range of decoupled and combined variations in integrin-binding peptide (RGD) ligand concentration, elastic modulus and proteolytic degradation rate using free-radical polymerization chemistry. The modularity of this system enabled a full factorial experimental design to simultaneously investigate the individual and interaction effects of these matrix cues on vascular sprout formation in 3 D culture. Enhancements in scaffold proteolytic degradation rate promoted significant increases in vascular sprout length and junction number while increases in modulus significantly and negatively impacted vascular sprouting. We also observed that individual variations in immobilized RGD concentration did not significantly impact 3 D vascular sprouting. Our findings revealed a previously unidentified and optimized combination whereby increases in both immobilized RGD concentration and proteolytic degradation rate resulted in significant and synergistic enhancements in 3 D vascular spouting. The above-mentioned findings would have been challenging to uncover using one-factor-at-time experimental analyses.

The Absence of Laminin α4 in Male Mice Results in Enhanced Energy Expenditure and Increased Beige Subcutaneous Adipose Tissue.

Laminin α4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4-/-) mice exhibit significantly higher energy expenditure (EE) relative to wild-type (WT) mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4-/- mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 is higher in subcutaneous white adipose tissue of Lama4-/- mice relative to WT animals on either a chow diet or a high-fat diet. In contrast, uncoupling protein 1 expression was not increased in brown adipose tissue. Lama4-/- mice exhibit significantly improved insulin sensitivity compared with WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in EE, weight gain, and systemic insulin sensitivity.

Synthesis and evaluation of dual crosslinked alginate microbeads.

Alginate hydrogels have been investigated for a broad variety of medical applications. The ability to assemble hydrogels at neutral pH and mild temperatures makes alginate a popular choice for the encapsulation and delivery of cells and proteins. Alginate has been studied extensively for the delivery of islets as a treatment for type 1 diabetes. However, poor stability of the encapsulation systems after implantation remains a challenge. In this paper, alginate was modified with 2-aminoethyl methacrylate hydrochloride (AEMA) to introduce groups that can be photoactivated to generate covalent bonds. This enabled formation of dual crosslinked structure upon exposure to ultraviolet light following initial ionic crosslinking into bead structures. The degree of methacrylation was varied and in vitro stability, long term swelling, and cell viability examined. At low levels of the methacrylation, the beads could be formed by first ionic crosslinks followed by exposure to ultraviolet light to generate covalent bonds. The methacrylated alginate resulted in more stable beads and cells were viable following encapsulation. Alginate microbeads, ionic (unmodified) and dual crosslinked, were implanted into a rat omentum pouch model. Implantation was performed with a local injection of 100 µl of 50 µg/ml of Lipopolysaccharide (LPS) to stimulate a robust inflammatory challenge in vivo. Implants were retrieved at 1 and 3 weeks for analysis. The unmodified alginate microbeads had all failed by week 1, whereas the dual-crosslinked alginate microbeads remained stable up through 3 weeks. The modified alginate microbeads may provide a more stable alternative to current alginate-based systems for cell encapsulation. STATEMENT OF SIGNIFICANCE: Alginate, a naturally occurring polysaccharide, has been used for cell encapsulation to prevent graft rejection of cell transplants for people with type I diabetes. Although some success has been observed in clinical trials, the lack of reproducibility and failure to reach insulin dependence for longer periods of time indicates the need for improvements in the procedure. A major requirement for the long-term function of alginate encapsulated cells is the mechanical stability of microcapsules. Insufficient mechanical integrity of the capsules can lead to immunological reactions in the recipients. In this work, alginate was modified to allow photoactivatable groups in order to allow formation of covalent crosslinks in addition to ionic crosslinking. The dual crosslinking design prevents capsule breakdown following implantation in vivo.

Laminin α4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain.

Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. In this manuscript we describe the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4-/-) and compared to wild-type (Lama4+/+) control animals. Lama4-/- mice exhibited reduced weight gain in response to both age and high fat diet. Interestingly, the mice had decreased adipose tissue mass and altered lipogenesis in a depot-specific manner. In particular, epididymal adipose tissue mass was specifically decreased in knock-out mice, and there was also a defect in lipogenesis in this depot as well. In contrast, no such differences were observed in subcutaneous adipose tissue at 14 weeks. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.

Influence of crosslinking on the stiffness and degradation of dermis-derived hydrogels.

Natural hydrogels have been investigated for three-dimensional tissue reconstruction and regeneration given their ability to emulate the structural complexity of multi-component extracellular matrices (ECM). Hydrogels rich in ECM can be extracted and assembled from soft tissues, retain a composition specific to the tissue source, and stimulate vascularized tissue formation. However, poor mechanical properties and rapid degradation hinder their performance in regenerative applications. This study investigates the effect of glutaraldehyde (GA) crosslinking on the mechanical properties, biological activity, and degradation of dermis-isolated ECM-rich hydrogels. Compression tests indicated that hydrogel elastic moduli and yield stress values increased significantly with GA exposure time. Lyophilization was shown to decrease yield stress values with respect to non-lyophilized gels. Crosslinked ECM, unlike non-crosslinked gels, was resistant to pepsin degradation in vitro. In a rodent subcutaneous implant model, crosslinking for 0.5 hours or longer drastically slowed degradation relative to controls. Inflammation was low and mature vascularized granulation tissue was observed in all gels, with an increase in vessel density at 1 week in crosslinked gels relative to controls. These results support the potential use of dermis-derived hydrogels as materials for tissue engineering applications and suggest that crosslinking can enhance mechanical properties and prolong hydrogel lifetime while promoting vascularized tissue formation.