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The potential of genetically modified cardiomyoblasts in treating damaged myocardium is well known. However, efficient delivery of these cells is of major concern during treatment. The limiting factors are the massive cell death that occurs soon after their intramyocardial transplantation into the beating heart. To address these problems, we generated recombinant baculoviruses (BacMam viruses) which efficiently transduced cardiomyoblast cells under optimized conditions. These genetically modified cells were then protected in a new polymeric microcapsule using poly-ethylene-glycol (PEG), alginate, and poly-L-lysine (PLL) polymers for efficient delivery. Results showed that microcapsules maintain cell viability and support cell proliferation for at least 30 days. The capsules exhibit strong immunoprotective potential and have high mechanical and osmotic stability with more than 70% intact capsules. The encased transduced cells showed a rapid transgene expression inside the capsule for at least 15 days. However, preclinical studies are needed to further explore its long-term functional benefits. [ABSTRACT FROM AUTHOR]

Internal hemorrhaging is a leading cause of death after traumatic injury on the battlefield. Although several surgical approaches such as the use of fibrin glue and tissue adhesive have been commercialized to achieve hemostasis, these approaches are difficult to employ on the battlefield and cannot be used for incompressible wounds. Here, we present shear-thinning nanocomposite hydrogels composed of synthetic silicate nanoplatelets and gelatin as injectable hemostatic agents. These materials are demonstrated to decrease in vitro blood clotting times by 77%, and to form stable clot-gel systems. In vivo tests indicated that the nanocomposites are biocompatible and capable of promoting hemostasis in an otherwise lethal liver laceration. The combination of injectability, rapid mechanical recovery, physiological stability, and the ability to promote coagulation result in a hemostat for treating incompressible wounds in out-of-hospital, emergency conditions.

Stem cell therapy and tissue engineering represent a forefront of current research in the treatment of heart disease. With these technologies, advancements are being made into therapies for acute ischemic myocardial injury and chronic, otherwise nonreversible, myocardial failure. The current clinical management of cardiac ischemia deals with reestablishing perfusion to the heart but not dealing with the irreversible damage caused by the occlusion or stenosis of the supplying vessels. The applications of these new technologies are not yet fully established as part of the management of cardiac diseases but will become so in the near future. The discussion presented here reviews some of the pioneering works at this new frontier. Key results of allogeneic and autologous stem cell trials are presented, including the use of embryonic, bone marrow-derived, adipose-derived, and resident cardiac stem cells. [ABSTRACT FROM AUTHOR]

Human adipose-derived stem cells (hASCs) are cryopreserved traditionally using dimethyl sulfoxide (DMSO) as the cryoprotectant agent. DMSO penetrates cell membranes and prevents cellular damage during cryopreservation. However, DMSO is not inert to cells, inducing cytotoxic effects by causing mitochondrial dysfunction, reduced cell proliferation, and impaired hASCs transplantation. Additionally, large-scale production of DMSO and contamination can adversely impact the environment. A sustainable, green alternative to DMSO is trehalose, a natural disaccharide cryoprotectant agent that does not pose any risk of cytotoxicity. However, the cellular permeability of trehalose is less compared to DMSO. Here, a microfluidic chip is developed for the intracellular delivery of trehalose in hASCs. The chip is designed for mechanoporation, which creates transient pores in cell membranes by mechanical deformation. Mechanoporation allows the sparingly permeable trehalose to be internalized within the cell cytosol. The amount of trehalose delivered intracellularly is quantified and optimized based on cellular compatibility and functionality. Furthermore, whole-transcriptome sequencing confirms that less than 1% of all target genes display at least a twofold change in expression when cells are passed through the chip compared to untreated cells. Overall, the results confirm the feasibility and effectiveness of using this microfluidic chip for DMSO-free cryopreservation of hASCs., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

DNA nanostructures have been widely researched in recent years as emerging biomedical materials for drug delivery, biosensing, and cancer therapy, in addition to their hereditary function. Multiple precisely designed single-strand DNAs can be fabricated into complex, three-dimensional DNA nanostructures through a simple self-assembly process. Among all of the synthetic DNA nanostructures, tetrahedral DNA nanostructures (TDNs) stand out as the most promising biomedical nanomaterial. TDNs possess the merits of structural stability, cell membrane permeability, and natural biocompatibility due to their compact structures and DNA origin. In addition to their inherent advantages, TDNs were shown to have great potential in delivering therapeutic agents through multiple functional modifications. As a multifunctional material, TDNs have enabled innovative pharmaceutical applications, including antimicrobial therapy, anticancer treatment, immune modulation, and cartilage regeneration. Given the rapid development of TDNs in the biomedical field, it is critical to understand how to successfully produce and fine-tune the properties of TDNs for specific therapeutic needs and clinical translation. This article provides insights into the synthesis and functionalization of TDNs and summarizes the approaches for TDN-based therapeutics delivery as well as their broad applications in the field of pharmaceutics and nanomedicine, challenges, and future directions., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Purpose: Conventional methods of seeding decellularized heart valves for heart valve tissue engineering have led to inconsistent results in interstitial cellular repopulation, particularly of the distal valve leaflet, and notably distinct from documented re-endothelialization. The use of bioreactor conditioning mimicking physiologic parameters has been well explored but cellular infiltration remains challenging. Non-characteristic, non-physiologic conditioning parameters within a bioreactor, such as hypoxia and cyclic chamber pressure, may be used to increase the cellular infiltration leading to increased recellularization., Methods: To investigate the effects of novel and perhaps non-intuitive bioreactor conditioning parameters, ovine aortic heart valves were seeded with mesenchymal stem cells and cultured in one of four environments: hypoxia and high cyclic pressures (120 mmHg), normoxia and high cyclic pressures, hypoxia and negative cyclic pressures (- 20 mmHg), and normoxia and negative cyclic pressures. Analysis included measurements of cellular density, cell phenotype, and biochemical concentrations., Results: The results revealed that the bioreactor conditioning parameters influenced the degree of recellularization. Groups that implemented hypoxic conditioning exhibited increased cellular infiltration into the valve leaflet tissue compared to normoxic conditioning, while pressure conditioning did not have a significant effect of recellularization. Protein expression across all groups was similar, exhibiting a stem cell and valve interstitial cell phenotype. Biochemical analysis of the extracellular matrix was similar between all groups., Conclusion: These results suggest the use of non-physiologic bioreactor conditioning parameters can increase in vitro recellularization of tissue engineered heart valve leaflets. Particularly, hypoxic culture was found to increase the cellular infiltration. Therefore, bioreactor conditioning of tissue engineered constructs need not always mimic physiologic conditions, and it is worth investigating novel or uncharacteristic culture conditions as they may benefit aspects of tissue culture.

Introduction: Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair., Methods: In this study, a biomimetic approach was used to mimic the natural mechanical stimulation of the myocardium tissue. Specifically, human adipose-derived stem cells (hASCs) were cultured on a thin gelatin methacrylamide (GelMA) hydrogel disc and placed on top of a beating cardiomyocyte layer. qPCR studies and metatranscriptomic analysis of hASCs gene expression were investigated to confirm the correlation between mechanical stimuli and cardiomyogenic differentiation. In vivo intramyocardial delivery of pre-conditioned hASCs was carried out to evaluate their efficacy to restore cardiac function in mice hearts post-myocardial infarction., Results: The cyclic strain generated by cardiomyocytes significantly upregulated the expression of both mechanotransduction and cardiomyogenic genes in hASCs as compared to the static control group. The inherent angiogenic secretion profile of hASCs was not hindered by the mechanical stimulation provided by the designed biomimetic system. Finally, in vivo analysis confirmed the regenerative potential of the pre-conditioned hASCs by displaying a significant improvement in cardiac function and enhanced angiogenesis in the peri-infarct region., Conclusion: Overall, these findings indicate that cyclic strain provided by the designed biomimetic system is an essential stimulant for hASCs cardiomyogenic differentiation, and therefore can be a potential solution to improve stem-cell based efficacy for cardiovascular repair., Competing Interests: CONFLICT OF INTEREST Aparna R. Chakravarti, Settimio Pacelli, Perwez Alam, Samik Bagchi, Saman Modaresi, Andras Czirok, Rafeeq P.H. Ahmed, and Arghya Paul declare no conflict of interests.

Introduction: Physical and mechanical properties of ceramic-based scaffolds can be modulated by introducing hydrogel coatings on their surface. For instance, hydrogels can be used as elastic layers to overcome the brittleness of synthetic ceramic materials or to control the delivery of essential osteogenic factors. In this work, we aimed to achieve both goals by fabricating a novel cytocompatible hydrogel made of gelatin-alginate as a coating for beta-tricalcium phosphate (β-TCP) scaffolds., Methods: The hydrogel synthesis was optimized by varying the concentration of the crosslinkers N-hydroxysuccinimide and N-Ethyl-N'-(3-dimethyl aminopropyl) carbodiimide (NHS/EDC). Swelling, degradability and mechanical studies were carried out to identify the suitable hydrogel coating formulation for the β-TCP scaffolds. The cytocompatibility of the coated ceramic was assessed in vitro by testing the proliferation and the osteogenic differentiation of human adipose stem cell (hASCs) for two weeks., Results: The designed hydrogel layer could withstand cyclic compression and protected the brittle internal core of the ceramic. The hydrogel coating modulated the diffusion of the model protein BSA according to the degree of crosslinking of the hydrogel layer. Additionally, the polymeric network was able to retain positively charged proteins such as lysozyme due to the strong electrostatic interactions with carboxylic groups of alginate. A higher expression of alkaline phosphates activity was found on hASCs seeded on the coated scaffolds compared to the hydrogels without any β-TCP., Conclusion: Overall, the hydrogel coating characterized in this study represents a valid strategy to overcome limitations of brittle ceramic-based materials used as scaffolds for bone tissue engineering applications., Competing Interests: Conflict of interests. Settimio Pacelli, Sayantani Basu, Cory Berkland, Jinxi Wang and Arghya Paul have no conflicts of interest to disclose.

Introduction: Heart valve tissue engineering may provide improved treatment for valvular heart disease, yet development of a tissue engineered heart valve (TEHV) has been limited by incomplete recellularization of the valve leaflets. In this study, we compare the leaflet recellularization potential of candidate cell populations., Methods: Four cell populations were tested: bone marrow mononuclear cells (MNC), 5 million bone marrow mesenchymal stem cells (MSC), 10 million bone marrow mesenchymal stem cells (MSC2), and 5 million valve interstitial cells (VIC). Candidate cell populations were seeded onto decellularized heart valves and underwent similar conditioning in a low-flow bioreactor for 2 weeks., Results: MSC2 valves demonstrated the best recellularization of the interstitial leaflet tissue as well as an appropriate cell phenotype, mechanical properties, and biochemical composition. MSC valves exhibited similar leaflet repopulation, yet had decreased mechanical and biochemical properties. MNC seeding resulted in minimal recellularization of the leaflet, though an additional time point group found cells present after 3 days, which seemed to disappear at 2 weeks. VIC seeding resulted in cell clumping on the leaflet surface and poor recellularization., Conclusions: The results of this study suggest mesenchymal stem cells are a preferred cell population for TEHV recellularization. Additionally, MSCs demonstrate the ability for repopulation of the distal valve leaflet, which will lead to more complete recellularization of future TEHVs., (© Biomedical Engineering Society 2018.)

An innovative approach for cardiac regeneration following injury is to induce endogenous cardiomyocyte (CM) cell cycle re-entry. In the present study, CMs from adult rat hearts were isolated and transfected with cel-miR-67 (control) and rno-miR-210. A significant increase in CM proliferation and mono-nucleation were observed in miR-210 group, in addition to a reduction in CM size, multi-nucleation, and cell death. When compared to control, β-catenin and Bcl-2 were upregulated while APC (adenomatous polyposis coli), p16, and caspase-3 were downregulated in miR-210 group. In silico analysis predicted cell cycle inhibitor, APC, as a direct target of miR-210 in rodents. Moreover, compared to control, a significant increase in CM survival and proliferation were observed with siRNA-mediated inhibition of APC. Furthermore, miR-210 overexpressing C57BL/6 mice (210-TG) were used for short-term ischemia/reperfusion study, revealing smaller cell size, increased mono-nucleation, decreased multi-nucleation, and increased CM proliferation in 210-TG hearts in contrast to wild-type (NTG). Likewise, myocardial infarction (MI) was created in adult mice, echocardiography was performed, and the hearts were harvested for immunohistochemistry and molecular studies. Compared to NTG, 210-TG hearts showed a significant increase in CM proliferation, reduced apoptosis, upregulated angiogenesis, reduced infarct size, and overall improvement in cardiac function following MI. β-catenin, Bcl-2, and VEGF (vascular endothelial growth factor) were upregulated while APC, p16, and caspase-3 were downregulated in 210-TG hearts. Overall, constitutive overexpression of miR-210 rescues heart function following cardiac injury in adult mice via promoting CM proliferation, cell survival, and angiogenesis., Key Messages: MiRNA-210 transfected adult rat CMs show proliferation and reduced cell death in vitro. Cell cycle inhibitor APC is a target of miR-210. MiR-210 overexpressing (210-TG) mouse hearts show CMs cell cycle re-entry and survival post myocardial injury. 210-TG mice show significant neovascularization and angiogenic potential post myocardial infarction. 210-TG hearts show reduced infarct size following ischemic injury.

A leading strategy in tissue engineering is the design of biomimetic scaffolds that stimulate the body's repair mechanisms through the recruitment of endogenous stem cells to sites of injury. Approaches that employ the use of chemoattractant gradients to guide tissue regeneration without external cell sources are favored over traditional cell-based therapies that have limited potential for clinical translation. Following this concept, bioactive scaffolds can be engineered to provide a temporally and spatially controlled release of biological cues, with the possibility to mimic the complex signaling patterns of endogenous tissue regeneration. Another effective way to regulate stem cell activity is to leverage the inherent chemotactic properties of extracellular matrix (ECM)-based materials to build versatile cell-instructive platforms. This review introduces the concept of endogenous stem cell recruitment, and provides a comprehensive overview of the strategies available to achieve effective cardiovascular and bone tissue regeneration., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Oxygen is essential to cell survival and tissue function. Not surprisingly, ischemia resulting from myocardial infarction induces cell death and tissue necrosis. Attempts to regenerate myocardial tissue with cell based therapies exacerbate the hypoxic stress by further increasing the metabolic burden. In consequence, implanted tissue engineered cardiac tissues suffer from hypoxia-induced cell death. Here, we report on the generation of oxygen-generating hydrogels composed of calcium peroxide (CPO) laden gelatin methacryloyl (GelMA). CPO-GelMA hydrogels released significant amounts of oxygen for over a period of 5 days under hypoxic conditions (1% O 2 ). The released oxygen proved sufficient to relieve the metabolic stress of cardiac side population cells that were encapsulated within CPO-GelMA hydrogels. In particular, incorporation of CPO in GelMA hydrogels strongly enhanced cell viability as compared to GelMA-only hydrogels. Importantly, CPO-based oxygen generation reduced cell death by limiting hypoxia-induced necrosis. The current study demonstrates that CPO based oxygen-generating hydrogels could be used to transiently provide oxygen to cardiac cells under ischemic conditions. Therefore, oxygen generating materials such as CPO-GelMA can improve cell-based therapies aimed at treatment or regeneration of infarcted myocardial tissue.

With recent advances in the field of nanomedicine, many new strategies have emerged for diagnosing and treating diseases. At the forefront of this multidisciplinary research, carbon nanomaterials have demonstrated unprecedented potential for a variety of regenerative medicine applications including novel drug delivery platforms that facilitate the localized and sustained release of therapeutics. Nanodiamonds (NDs) are a unique class of carbon nanoparticles that are gaining increasing attention for their biocompatibility, highly functional surfaces, optical properties, and robust physical properties. Their remarkable features have established NDs as an invaluable regenerative medicine platform, with a broad range of clinically relevant applications ranging from targeted delivery systems for insoluble drugs, bioactive substrates for stem cells, and fluorescent probes for long-term tracking of cells and biomolecules in vitro and in vivo. This review introduces the synthesis techniques and the various routes of surface functionalization that allow for precise control over the properties of NDs. It also provides an in-depth overview of the current progress made toward the use of NDs in the fields of drug delivery, tissue engineering, and bioimaging. Their future outlook in regenerative medicine including the current clinical significance of NDs, as well as the challenges that must be overcome to successfully translate the reviewed technologies from research platforms to clinical therapies will also be discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nanodiamonds (NDs) represent an emerging class of carbon nanomaterials that possess favorable physical and chemical properties to be used as multifunctional carriers for a variety of bioactive molecules. Here we report the synthesis and characterization of a new injectable ND-based nanocomposite hydrogel which facilitates a controlled release of therapeutic molecules for regenerative applications. In particular, we have formulated a thermosensitive hydrogel using gelatin, chitosan and NDs that provides a sustained release of exogenous human vascular endothelial growth factor (VEGF) for wound healing applications. Addition of NDs improved the mechanical properties of the injectable hydrogels without affecting its thermosensitive gelation properties. Biocompatibility of the generated hydrogel was verified by in vitro assessment of apoptotic gene expressions and anti-inflammatory interleukin productions. NDs were complexed with VEGF and the inclusion of this complex in the hydrogel network enabled the sustained release of the angiogenic growth factor. These results suggest for the first time that NDs can be used to formulate a biocompatible, thermosensitive and multifunctional hydrogel platform that can function both as a filling agent to modulate hydrogel properties, as well as a delivery platform for the controlled release of bioactive molecules and growth factors., Statement of Significance: One of the major drawbacks associated with the use of conventional hydrogels as carriers of growth factors is their inability to control the release kinetics of the loaded molecules. In fact, in most cases, a burst release is inevitable leading to diminished therapeutic effects and unsuccessful therapies. As a potential solution to this issue, we hereby propose a strategy of incorporating ND complexes within an injectable hydrogel matrix. The functional groups on the surface of the NDs can establish interactions with the model growth factor VEGF and promote a prolonged release from the polymer network, therefore, providing a longer therapeutic effect. Our strategy demonstrates the efficacy of using NDs as an essential component for the design of a novel injectable nanocomposite system with improved release capabilities., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In mammals, proliferative capacity of cardiomyocytes is lost soon after birth, while zebrafish and other lower organisms like newts are known to regenerate injured hearts even at an adult age. Here, we show that miR-1825 can induce robust proliferation of adult rat cardiomyocytes and can improve cardiac function in-vivo post myocardial infarction. Rat adult cardiomyocytes transfected with miR-1825 showed a significant increase in DNA synthesis, mitosis, cytokinesis, and an increase in cell number when compared to cel-miR-67 transfected control. We also observed a reduction in mitochondrial number and a decrease in ROS and DNA-damage. RNA-sequencing data identified NDUFA10, a key gene involved in the mitochondrial electron transport chain to be a direct target of miR-1825. SiRNA mediated silencing of NDUFA10 showed a significant increase in cardiomyocyte proliferation indicating its role downstream of miRNA-1825. In addition, microRNA microarray results identified miR-1825 to regulate expression of a known proliferation inducing miRNA, miR-199a. We also identified the direct targets of miR-199a, namely p16, Rb1, and Meis2 to be downregulated following miR-1825 transfection. However, miR-199a alone did not have similar proliferation inducing effects as miR-1825, indicating that miR-1825 works through multiple pathways and is a master regulator of cardiomyocyte proliferation. In addition, our in-vivo analysis in animal models of LAD ligation and intra-cardiac miRNA delivery showed proliferation of endogenous cardiomyocytes in the peri-infarcted region and an improvement in heart function. These findings establish miR-1825 as a potential therapeutic agent for induction of cardiomyocyte proliferation and cardiac regeneration, with a significant translational potential., Competing Interests: None.

There is an immense need for tissue engineered blood vessels. However, current tissue engineering approaches still lack the ability to build native blood vessel-like perfusable structures with multi-layered vascular walls. This paper demonstrated a new method to fabricate tri-layer biomimetic blood vessel-like structures on a microfluidic platform using photocrosslinkable gelatin hydrogel. The presented method enables fabrication of physiological blood vessel-like structures with mono-, bi- or tri-layer vascular walls. The diameter of the vessels, the total thickness of the vessel wall and the thickness of each individual layer of the wall were independently controlled. The developed fabrication process is a simple and rapid method, allowing the physical fabrication of the vascular structure in minutes, and the formation of a vascular endothelial cell layer inside the vessels in 3-5 days. The fabricated vascular constructs can potentially be used in numerous applications including drug screening, development of in vitro models for cardiovascular diseases and/or cancer metastasis, and study of vascular biology and mechanobiology.

Paclitaxel (PTX) is one of the most important drugs for breast cancer; however, the drug effects are limited by its systematic toxicity and poor water solubility. Nanoparticles have been applied for delivery of cancer drugs to overcome their limitations. Toward this goal, a novel single-walled carbon nanotube (SWNT)-based drug delivery system was developed by conjugation of human serum albumin (HSA) nanoparticles for loading of antitumor agent PTX. The nanosized macromolecular SWNT-drug carrier (SWNT-HSA) was characterized by TEM, UV-Vis-NIR spectrometry, and TGA. The SWNT-based drug carrier displayed high intracellular delivery efficiency (cell uptake rate of 80%) in breast cancer MCF-7 cells, as examined by fluorescence-labeled drug carriers, suggesting the needle-shaped SWNT-HSA drug carrier was able to transport drugs across cell membrane despite its macromolecular structure. The drug loading on SWNT-based drug carrier was through high binding affinity of PTX to HSA proteins. The PTX formulated with SWNT-HSA showed greater growth inhibition activity in MCF-7 breast cancer cells than PTX formulated with HSA nanoparticle only (cell viability of 63 vs 70% in 48 h and 53 vs 62% in 72 h). The increased drug efficacy could be driven by SWNT-mediated cell internalization. These data suggest that the developed SWNT-based antitumor agent is functional and effective. However, more studies for in vivo drug delivery efficacy and other properties are needed before this delivery system can be fully realized.

Objective: The aim of the present study was to construct a new drug delivery system for milrinone using microparticles. This novel technology enhances drug bioavailability and decreases toxicity, with future implications for the treatment of end-stage heart failure., Methods: Polylactic-co-glycolic acid microparticles (PLGA-MPs) loaded with milrinone were prepared using a double emulsion-solvent evaporation technique. In vitro release kinetics was evaluated at physiologic conditions. A total of 24 female Lewis rats underwent left coronary artery ligation. One week after ligation, all rats were randomized to 1 of 3 groups (n=8 per group). Group I received an intravenous injection of PLGA-MPs alone; group II, a bolus intravenous injection of milrinone; and group III an intravenous injection of milrinone-PLGA-MPs. All injections were administrated slowly by way of the tail vein over 10 minutes. Transthoracic echocardiography, noninvasive heart rate monitoring, and blood pressure measurements were performed at different predetermined intervals before and for 24 hours after the injection. All rats survived for 24 hours and were then killed by euthanasia. Serum plasma was taken for cytokine assays and determination of milrinone levels using high-performance liquid chromatography., Results: Group III had a significantly greater left ventricular ejection fraction at 90 minutes and 3, 6, and 12 hours after treatment compared with the other groups. The milrinone plasma level was significantly greater in group III than in the other groups (group I, 0 ng/mL; group II, 1.7±2.4 ng/mL; group III, 9.1±2.2 ng/mL; P<.05). The intercellular adhesion molecule and cytokine-induced neutrophil chemoattractant-1 levels were significantly lower in group III than in the other 2 groups (P<.05)., Conclusions: Drug encapsulation using microparticles can prolong the effects of milrinone. We propose a new strategy for future drug delivery in patients with end-stage heart failure., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA.

Development of a vascularized tissue is one of the key challenges for the successful clinical application of tissue engineered constructs. Despite the significant efforts over the last few decades, establishing a gold standard to develop three dimensional (3D) vascularized tissues has still remained far from reality. Recent advances in the application of microfluidic platforms to the field of tissue engineering have greatly accelerated the progress toward the development of viable vascularized tissue constructs. Numerous techniques have emerged to induce the formation of vascular structure within tissues which can be broadly classified into two distinct categories, namely (1) prevascularization-based techniques and (2) vasculogenesis and angiogenesis-based techniques. This review presents an overview of the recent advancements in the vascularization techniques using both approaches for generating 3D vascular structure on microfluidic platforms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Due to the increasing number of heart valve diseases, there is an urgent clinical need for off-the-shelf tissue engineered heart valves. While significant progress has been made toward improving the design and performance of both mechanical and tissue engineered heart valves (TEHVs), a human implantable, functional, and viable TEHV has remained elusive. In animal studies so far, the implanted TEHVs have failed to survive more than a few months after transplantation due to insufficient mechanical properties. Therefore, the success of future heart valve tissue engineering approaches depends on the ability of the TEHV to mimic and maintain the functional and mechanical properties of the native heart valves. However, aside from some tensile quasistatic data and flexural or bending properties, detailed mechanical properties such as dynamic fatigue, creep behavior, and viscoelastic properties of heart valves are still poorly understood. The need for better understanding and more detailed characterization of mechanical properties of tissue engineered, as well as native heart valve constructs is thus evident. In the current review we aim to present an overview of the current understanding of the mechanical properties of human and common animal model heart valves. The relevant data on both native and tissue engineered heart valve constructs have been compiled and analyzed to help in defining the target ranges for mechanical properties of TEHV constructs, particularly for the aortic and the pulmonary valves. We conclude with a summary of perspectives on the future work on better understanding of the mechanical properties of TEHV constructs., (© 2013 Published by Elsevier Ltd.)

Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Carbon nanotube (CNT) possesses excellent properties as a drug carrier. To overcome the challenge of drug functionalization with CNT, we have developed a lipid-drug approach for efficient drug loading onto CNT, in which a long chain lipid molecule is conjugated to the drug molecule so that the lipid-drug can be loaded directly onto CNT through binding of the lipid 'tail' in the drug molecule to CNT surfaces via hydrophobic interactions. In a proof-of-concept study, drug paclitaxel (PTX) was conjugated with a non-toxic lipid molecule docosanol for functionalization with CNT. Folic acid was also conjugated to CNT for targeted drug delivery. High level of drug loading onto SWNT could be achieved by lipid-drug approach. Conjugation of FA to SWNT-lipid-PTX led to an increase in cell penetration capacity, and the targeted SWNT-lipid-PTX showed much improved drug efficacy in vitro in comparison to free drug Taxol and non-targeted SWNT-lipid-PTX at 48 h (78.5% vs. 31.6% and 59.1% in cytotoxicity respectively, p < 0.01). In vivo analysis using a human breast cancer xenograft mice model also confirmed the improved drug efficacy. The targeted SWNT-lipid-PTX was found non-toxic as evaluated by biochemical analysis using blood samples, and by histological analysis of major organs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recently, preclinical studies have shown that allogeneic adipose-derived stem cells (ASCs), like bone marrow-derived mesenchymal stem cell (BMSCs) have significant clinical benefits in treating cardiovascular diseases, such as ischemic/infarcted heart. In this study, we tested whether ASCs are also immune tolerant, such that they can be used as universal donor cells for myocardial regenerative therapy. The study also focuses on investigating the potential therapeutic effects of human ASCs (hASCs) for myocardial infarction in xenotransplant model, and compares its effects with that of hBMSCs. The in vitro study confirms the superior proliferation potential and viability of hASCs under normoxic and stressed hypoxic conditions compared with hBMSCs. hASCs also show higher potential in adopting cardiomyocyte phenotype. The major findings of the in vivo study are that (1) both hASCs and hBMSCs implanted into immunocompetent rat hearts with acute myocardial infarction survived the extreme environment of xenogeneic mismatch for 6 weeks; (2) both hASCs and hBMSCs showed significant improvement in myocardial pro/anti-inflammatory cytokine levels with no detectable inflammatory reaction, despite the lack of any immunosuppressive therapy; and (3) hASCs contributed to the remarkable improvement in cardiac function and reduced infarction which was significantly better than that of hBMSC and untreated control groups. Thus, our findings suggest the feasibility of using ASCs, instead of BMSCs, as universal donor cells for xenogeneic or allogeneic cell therapy.

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gut-derived lipopolysaccharides (LPS) are critical to the development and progression of chronic low-grade inflammation and metabolic diseases. In this study, the effects of probiotics Lactobacillus and Bifidobacterium on gut-derived lipopolysaccharide and inflammatory cytokine concentrations were evaluated using a human colonic microbiota model. Lactobacillus reuteri, L. rhamnosus, L. plantarum, Bifidobacterium animalis, B. bifidum, B. longum, and B. longum subsp. infantis were identified from the literature for their anti-inflammatory potential. Each bacterial culture was administered daily to a human colonic microbiota model during 14 days. Colonic lipopolysaccharides, and Gram-positive and negative bacteria were quantified. RAW 264.7 macrophage cells were stimulated with supernatant from the human colonic microbiota model. Concentrations of TNF-alpha, IL-1beta, and IL-4 cytokines were measured. Lipopolysaccharide concentrations were significantly reduced with the administration of B. bifidum (-46.45 +/- 5.65%), L. rhamnosus (-30.40 +/- 5.08%), B. longum (-42.50 +/- 1.28%), and B. longum subsp. infantis (-68.85 +/- 5.32%) (p < 0.05). Cell counts of Gram-negative and positive bacteria were distinctly affected by the probiotic administered. There was a probiotic strain-specific effect on immunomodulatory responses of RAW 264.7 macrophage cells. B. longum subsp. infantis demonstrated higher capacities to reduce TNF-alpha concentrations (-69.41 +/- 2.78%; p < 0.05) and to increase IL-4 concentrations (+16.50 +/- 0.59%; p < 0.05). Colonic lipopolysaccharides were significantly correlated with TNF-alpha and IL-1beta concentrations (p < 0.05). These findings suggest that specific probiotic bacteria, such as B. longum subsp. infantis, might decrease colonic lipopolysaccharide concentrations, which might reduce the proinflammatory tone. This study has noteworthy applications in the field of biotherapeutics for the prevention and/or treatment of inflammatory and metabolic diseases.

Despite outstanding progress in the area of cardiovascular diseases, significant challenges remain in designing efficient delivery systems for myocardial therapy. Nanotechnology provides the tools to explore such frontiers of biomedical science at cellular level and thus offers unique features for potential application in the field of cardiac therapy. This chapter focuses on the methodology, based on the work done in our lab, to prepare and investigate two kinds of biocompatible nanoparticles (NPs) that can be useful for sustained delivery of single or multiple angiogenic growth factors to damaged sites, such as in myocardially infarcted heart to promote myocardial angiogenesis and reduce scar area.

Stem cell transplantation has been considered as a major breakthrough for treating ischemic heart disease. However, survival and retention of transplanted cells at the site of infarction remains tenuous. This chapter details a method of creating polymeric microcapsules for cell delivery, resulting in increased retention of transplanted cells at the target site, while achieving minimal mechanical trauma and cell loss. Simultaneously biocompatible and biodegradable, polymeric microcapsules have important implications in regenerative cell therapy.

This study report the development of a nanobiohybrid hydrogel based endovascular stent device capable of preventing postangioplasty in-stent restenosis (ISR) by promoting significant vascular endothelial recovery in a site-specific manner. The hydrogel is comprised of fibrin matrices, assembled layer-by-layer (LbL) on stent surface with alternate layers carrying endosomolytic Tat peptide/DNA nanoparticles (NPs) or NPs hybridized to polyacrylic acid (PAA) wrapped single-walled carbon nanotubes (NP-CNT). Here, the hydrogel works as a reservoir to carry, protect, and simultaneously deliver pro-angiogenic, vascular endothelial growth factor (Vegf) and Angiopoietin-1(Ang1), gene carrying NPs to the target site. In vitro results demonstrated that CNTs incorporated in the hydrogel layers play a major role in tuning the bioactivity of the stent. In addition, the developed stent formulation can significantly reduce the loss of therapeutics while traversing through the vessel and during deployment. In vivo experiments in balloon-injured canine femoral artery demonstrated that the NCS (+) group, carrying NP(vegf+Ang1), can significantly enhance re-endothelialization of injured artery compared to control NCS (-), carrying NP(Null), and bare metal stent (BMS) groups, attenuate stenosis (18.5±9.03% vs 39.56±13.8 vs 45.34±8.3%; n=8, p<0.05) and prevent neointima formation (1.53±0.36 mm(2) vs 2.51±0.27 mm(2) vs 2.66±0.14 mm(2); n=8, p<0.05) as analyzed angiography and histomorphometric analysis. These data collectively implicate that this new technology can be useful for stent and other biomedical devices through controlled delivery of multiple biotherapeutics., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs)., Methods: AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions., Results: PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection., Conclusion: The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.

Small interfering RNA (siRNA) molecules have great potential for developing into a future therapy for breast cancer. To overcome the issues related to rapid degradation and low transfection of naked siRNA, polyethylenimine (PEI)-coated human serum albumin (HSA) nanoparticles have been characterized and studied here for efficient siRNA delivery to the MCF-7 breast cancer cell line. The optimized nanoparticles were ~90 nm in size, carrying a surface charge of +26 mV and a polydispersity index (PDI) less than 0.25. The shape and morphology of the particles was studied using electron microscopy. A cytotoxicity assessment of the nanoparticles showed no correlation of cytotoxicity with HSA concentration, while using high molecular weight PEI (MW of 70 against 25 kDa) showed higher cytotoxicity. The optimal transfection achieved of fluorescin-tagged siRNA loaded into PEI-coated HSA nanoparticles was 61.66 ± 6.8%, prepared with 6.25 μg of PEI (25 kDa) added per mg of HSA and 20 mg/ml HSA, indicating that this nonviral vector may serve as a promising gene delivery system.

The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NP(Ang1) showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than Bac(Ang1), with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NP(Ang1) to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than Bac(Ang1)). Furthermore, the Bac-NP(Ang1) group showed significantly higher cardiac performance in echocardiography than Bac(Ang1) (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NP(Ang1) and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate-chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600 pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72 h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.

Background: Research towards the application of nanoparticles as carrier vehicles for the delivery of therapeutic agents is increasingly gaining importance. The angiogenic growth factors, human vascular endothelial growth factor (VEGF) and human angiopoietin-1 are known to prevent vascular endothelial cell apoptosis and in fact to stimulate human vascular endothelial cell (HUVEC) proliferation. This paper aims to study the combined effect of these bioactive proteins coencapsulated in human serum albumin nanoparticles on HUVECs and to evaluate the potential application of this delivery system towards therapeutic angiogenesis., Methods and Results: The angiogenic proteins, human VEGF and human angiopoietin-1, were coencapsulated in albumin nanoparticles for better controlled delivery of the proteins. The application of a nanoparticle system enabled efficient and extended-release kinetics of the proteins. The size of the nanoparticles crosslinked with glutaraldehyde was 101.0 ± 0.9 nm and the zeta potential was found to be -18 ± 2.9 mV. An optimal concentration of glutaraldehyde for the nanoparticle coating process was determined, and this provided stable and less toxic nanoparticles as protein carriers. The results of the study indicate that nanoparticles crosslinked with glutaraldehyde produced nanoparticles with tolerable toxicity which provided efficient and controlled release of the coencapsulated proteins. The nanoparticles were incubated for two weeks to determine the release profiles of the proteins. At the end of the two-week incubation period, it was observed that 49% ± 1.3% of human angiopoietin-1 and 59% ± 2.1% of human VEGF had been released from the nanoparticles. The proliferation and percent apoptosis of the HUVECs in response to released proteins was observed., Conclusion: The results indicate that the released proteins were biologically active and the combined application of both the proteins demonstrated a significant highly proliferative and antiapoptotic effect on HUVECs as compared with the effect demonstrated by the individual proteins released. These studies could serve as a basis to encourage further research into the potential in vivo application of these protein-loaded nanoparticles in the field of therapeutic angiogenesis.

Background: Acute myocardial ischemia results in scar formation with ventricular dilatation and eventually heart failure. Placental growth factor (PlGF) is reported to stimulate angiogenesis and improve cardiac function. In this study, it was hypothesized that intramyocardial injection of PlGF contained in nanoparticles can be released at the site of action for an extended time period as a sustained slow-release protective mechanism that accelerates myocardial recovery in a rat model of ischemic cardiomyopathy., Methods: PlGF-loaded chitosan-alginate nanoparticles were injected into an acute myocardial infarction model in rats (n = 10 per group). Transthoracic echocardiography was performed at different time intervals. Enzyme-linked immunosorbent assay was used to measure the serum cytokines levels at 8 weeks. Hearts were stained with Masson's trichrome for scar area analysis. Immunofluorostaining was performed to evaluate the extent of myocardial angiogenesis at the infarction border. PlGF enzyme-linked immunosorbent assay was used to measure the in vitro release kinetics of PlGF-loaded nanoparticles., Results: At 8 weeks after coronary ligation, hearts that were treated with PlGF-loaded chitosan-alginate nanoparticles had significant increases in left-ventricular function (P < 0.01), vascular density (P < 0.01), and in the serum level of the anti-inflammatory cytokine interleukin-10 (P < 0.05). There was significant decrease in scar area formation (P < 0.05) and in serum levels of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 (P < 0.01). In vitro PlGF-release kinetic studies showed a sustained release of PlGF from the particles over a 120-hour period., Conclusion: The use of nanoparticles as a vehicle for PlGF delivery, as opposed to the direct injection of the growth factor after acute myocardial infarction, can provide sustained slow-release PlGF therapy, enhancing the positive effects of the growth factor in the setting of acute myocardial ischemia.

Introduction. Bone-marrow-derived mesenchymal stem cells (MSCs) have been studied for treatment of myocardial infarction (MI). Since MSCs from older donors show quantitative and qualitative senescent changes, we hypothesized that a better outcome may be achieved if aged recipients are given MSCs obtained from young donors, rather than using their own autologous MSCs. Methods. In vitro studies compared properties of young and old MSCs. Aged rats randomized into 3 groups underwent coronary artery ligations and were then injected with either old (O) or young (Y) MSCs, or ligation alone. Echocardiography evaluated ejection fractions (EF). At 16 weeks, scar deposition was analyzed. Results. Old MSCs exhibited decreased cell viability, proliferation, and differentiation potentials. EF significantly improved early in both cell therapy groups (P < .05). However, at later stages of the study, group Y showed significantly better function which correlated with decreased scar deposition. Conclusions. The significant difference between young and old cells indicates the possible advantage for allotransplanting MSCs from young donors to elderly patients with MI.

Present therapeutic strategies for most cancers are restricted mainly to the primary tumors and are also not very effective in controlling metastatic states. Alternatively, gene therapy can be a potential option for treating such cancers. Currently mammalian viral-based cancer gene therapy is the most popular approach, but the efficacy has been shown to be quite low in clinical trials. In this study, for the first time, the insect cell-specific baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been evaluated as a vector for gene delivery to colorectal cancer cells. Experiments involving factorial design were employed to study the individual and combined effects of different parameters such as multiplicity of infection (MOI), viral incubation time and epigenetic factors on transduction efficiency. The results demonstrate that baculovirus gene delivery system holds immense potential for development of a new generation of highly effective virotherapy for colorectal, as well as other major carcinomas (breast, pancreas, and brain), and offers significant benefits to traditional animal virus-based vectors with respect to safety concerns.