Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis.

Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

Extracellular Matrix-Based Biomaterials for Cardiovascular Tissue Engineering.

Regenerative medicine and tissue engineering strategies have made remarkable progress in remodeling, replacing, and regenerating damaged cardiovascular tissues. The design of three-dimensional (3D) scaffolds with appropriate biochemical and mechanical characteristics is critical for engineering tissue-engineered replacements. The extracellular matrix (ECM) is a dynamic scaffolding structure characterized by tissue-specific biochemical, biophysical, and mechanical properties that modulates cellular behavior and activates highly regulated signaling pathways. In light of technological advancements, biomaterial-based scaffolds have been developed that better mimic physiological ECM properties, provide signaling cues that modulate cellular behavior, and form functional tissues and organs. In this review, we summarize the in vitro, pre-clinical, and clinical research models that have been employed in the design of ECM-based biomaterials for cardiovascular regenerative medicine. We highlight the research advancements in the incorporation of ECM components into biomaterial-based scaffolds, the engineering of increasingly complex structures using biofabrication and spatial patterning techniques, the regulation of ECMs on vascular differentiation and function, and the translation of ECM-based scaffolds for vascular graft applications. Finally, we discuss the challenges, future perspectives, and directions in the design of next-generation ECM-based biomaterials for cardiovascular tissue engineering and clinical translation.

Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease.

Background: Chemical modification of mRNA (mmRNA) substantially improves their stability and translational efficiency within cells. Nanofibrillar collagen scaffolds were previously shown to enable the spatially localized delivery and temporally controlled release of mmRNA encoding HGF both in vitro and in vivo. Materials & methods: Herein we developed an improved slow-releasing HGF mmRNA scaffold and tested its therapeutic efficacy in a porcine model of peripheral arterial disease. Results & conclusion: The HGF mmRNA was released from scaffolds in a temporally controlled fashion in vitro with preserved transfection activity. The mmRNA scaffolds improved vascular regeneration when sutured to the ligated porcine femoral artery. These studies validate the therapeutic potential of HGF mmRNA delivery from nanofibrillar scaffolds for treatment of peripheral arterial disease.

Endothelial Cell Mechanotransduction in the Dynamic Vascular Environment.

The vascular endothelial cells (ECs) that line the inner layer of blood vessels are responsible for maintaining vascular homeostasis under physiological conditions. In the presence of disease or injury, ECs can become dysfunctional and contribute to a progressive decline in vascular health. ECs are constantly exposed to a variety of dynamic mechanical stimuli, including hemodynamic shear stress, pulsatile stretch, and passive signaling cues derived from the extracellular matrix. This review describes the molecular mechanisms by which ECs perceive and interpret these mechanical signals. The translational applications of mechanosensing are then discussed in the context of endothelial-to-mesenchymal transition and engineering of vascular grafts.

Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA.

RNA-based vector delivery is a promising gene therapy approach. Recent advances in chemical modification of mRNA structure to form modified mRNA (mmRNA or cmRNA or modRNA) have substantially improved their stability and translational efficiency within cells. However, mmRNA conventionally delivered in solution can be taken up nonspecifically or become cleared away prematurely, which markedly limits the potential benefit of mmRNA therapy. To address this limitation, we developed mmRNA-incorporated nanofibrillar scaffolds that could target spatially localized delivery and temporally controlled release of the mmRNA both in vitro and in vivo. To establish the efficacy of mmRNA therapy, mmRNA encoding reporter proteins such as green fluorescence protein or firefly luciferase (Fluc) was loaded into aligned nanofibrillar collagen scaffolds. The mmRNA was released from mmRNA-loaded scaffolds in a transient and temporally controlled manner and induced transfection of human fibroblasts in a dose-dependent manner. In vitro transfection was further verified using mmRNA encoding the angiogenic growth factor, hepatocyte growth factor (HGF). Finally, scaffold-based delivery of HGF mmRNA to the site of surgically induced muscle injury in mice resulted in significantly higher vascular regeneration after 14 days, compared to implantation of Fluc mmRNA-releasing scaffolds. After transfection with Fluc mmRNA-releasing scaffold in vivo, Fluc activity was detectable and localized to the muscle region, based on noninvasive bioluminescence imaging. Scaffold-based local mmRNA delivery as an off-the-shelf form of gene therapy has broad translatability for treating a wide range of diseases or injuries.

Multicellular Interactions in 3D Engineered Myocardial Tissue.

Cardiovascular disease is a leading cause of death in the US and many countries worldwide. Current cell-based clinical trials to restore cardiomyocyte (CM) health by local delivery of cells have shown only moderate benefit in improving cardiac pumping capacity. CMs have highly organized physiological structure and interact dynamically with non-CM populations, including endothelial cells and fibroblasts. Within engineered myocardial tissue, non-CM populations play an important role in CM survival and function, in part by secreting paracrine factors and cell-cell interactions. In this review, we summarize the progress of engineering myocardial tissue with pre-formed physiological multicellular organization, and present the challenges toward clinical translation.

Electrosprayed Montelukast/poly (lactic-co-glycolic acid) particle based coating: A new therapeutic approach towards the prevention of in-stent restenosis.

UNLABELLED: Drug-eluting stents (DESs), have shown promising results in prevention of in-stent restenosis after percutaneous coronary intervention (PCI). The elevated level of leukotrienes (LTs) detected in injured arteries after PCI, together with the potential role of LTs in inflammatory cascades and structural alterations in arterial wall provides the rationale for development of therapeutic strategies for prevention of in-stent restenosis using LTs receptor antagonists. Montelukast (MK) is a selective cysLT1 receptor antagonist, with anti-inflammatory and anti-proliferative properties, which has been used for treatment of various diseases. Here, we report on the fabrication of MK/PLGA particles by electrospraying, aiming towards the development of particle based coating of DESs. The electrosprayed particles incorporated with 3% and 6% w/w MK exhibited fairly spherical shape with smooth surfaces and narrow size distribution. Sustained release of MK for up to 40days was obtained for both formulations, with higher initial burst release and drug release rate for the particles with higher drug loading. The LTD4 induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) by 35% and 85%, respectively, which was substantially antagonized using MK incorporated particles. Nevertheless, MK antagonism preserved the normal proliferation and migration of human coronary artery endothelial cells (HCAECs). Moreover, MK antagonism inhibited the LTD4 induced phenotypic transition of HCASMCs from contractile to synthetic type. The electrosprayed MK-PLGA particles can be employed as a coating for DESs to inhibit the formation of neointimal hyperplasia responsible for in-stent restenosis, yet preserve the healing rate of the stented vessel. STATEMENT OF SIGNIFICANT: Montelukast (MK) is a selective cysLT1 receptor antagonist, with anti-inflammatory and anti-proliferative properties. The LTD4 induced proliferation and migration of human coronary artery smooth muscle cells by 35% and 85%, respectively, which was substantially antagonized using MK incorporated particles. MK antagonism preserved the normal proliferation and migration of human coronary artery endothelial cells. The MK antagonism inhibited the phenotypic transition of human coronary artery smooth muscle cells from contractile to synthetic one induced by LTD4. The electrosprayed MK-PLGA particles can be employed as coating for DESs to inhibit formation of neointimal hyperplasia, responsible for in-stent restenosis.

Electrospraying technique for the fabrication of metronidazole contained PLGA particles and their release profile.

Advanced engineering of materials for the development of drug delivery devices provides scope for novel and versatile strategies for treatment of various diseases. 'Electrospraying' was used to prepare PLGA microparticles and further encapsulate the drug, metronidazole (Met) within the particles to function as a drug delivery system. Two different solvents were utilized for the preparation of drug loaded PLGA particles, whereby the polymeric solution in dichloromethane (DCM) produced particles of bigger sizes than using trifluoroethanol (TFE). Scanning electron microscopy showed the spherical morphology of the particles, with sizes of 3946±407nm and 1774±167nm, respectively for PLGA-Met(DCM) and PLGA-Met(TFE). The FTIR spectroscopy proved the incorporation of metronidazole in the polymer, but without any specific drug-polymer interaction. The release of the drug from the particles was studied in phosphate buffered saline, where a sustained drug release was obtained for at least 41days. Cytotoxicity evaluation of the drug extract using mesenchymal stem cells (MSCs) showed not hindering the proliferation of MSCs, and the cell phenotype was retained after incubation in the drug containing media. Electrospraying is suggested as a cost-effective and single step process for the preparation of polymeric microparticles for prolonged and controlled release of drug.

Controlled delivery of stromal derived factor-1α from poly lactic-co-glycolic acid core-shell particles to recruit mesenchymal stem cells for cardiac regeneration.

Stromal derived factor-1α (SDF-1α) has shown promising results in treatment of myocardial infarction (MI), via recruitment of endogenous stem cells into the injured myocardium. However, the bioactivity of this susceptible signalling chemokine is reduced significantly during the common fabrication processes of drug delivery systems, due to the exposure to organic-aqueous interfaces or elevated temperature. In this study, we developed a novel SDF-1α delivery system using coaxial electrospraying, the technique which enables fabrication of core-shell particles with minimized contact of organic-aqueous phases. The SDF-1α incorporated PLGA particles exhibited distinct core-shell structure, confirmed by transmission electron microscopy (TEM). Controlled release of SDF-1α was obtained for at least 40days, and the release rate was tailored by co-encapsulation of bovine serum albumin (BSA) into the core of the particles. The SDF-1α released from PLGA/SDF-1α and PLGA/BSA-SDF-1α particles retained its chemotactic activity, and enhanced the number of migrated mesenchymal stem cells (MSCs) by 38% and 54%, respectively, compared to basal medium used as the control. Moreover, both SDF-1α and BSA supported the proliferation of MSCs within 3days of cell culture. The SDF-1α incorporated core-shell particles developed by electrospraying technique, can be effectively employed as injectable drug delivery system for in situ cardiac regeneration.

Protein encapsulated core-shell structured particles prepared by coaxial electrospraying: investigation on material and processing variables.

Biodegradable polymeric particles have been extensively investigated for controlled drug delivery of various therapeutic agents. 'Coaxial' electrospraying was successfully employed in this study, to fabricate core-shell PLGA particles containing bovine serum albumin (BSA) as the model protein, and the results were also compared to particles prepared by 'emulsion' electrospraying. Two different molecular weights of PLGA were employed to encapsulate the protein. Solution properties and processing parameters were found to influence the morphology of the core-shell particles. Depending on the type of solvent used to dissolve the polymer as well as the polymer concentration and molecular weight, the mean diameter of the particles varied between 3.0 to 5.5 µm. Fluorescence microscopic analysis of the electrosprayed particles using FITC-conjugated BSA demonstrated the core-shell structure of the developed particles. The encapsulation efficiency and release behavior of BSA was influenced by shell:core feeding ratio, protein concentration, and the electrospraying method. The encapsulation efficiency of BSA within the core-shell particles of high and low molecular weight PLGA was found 15.7% and 25.1% higher than the emulsion electrosprayed particles, respectively. Moreover, the total amount of BSA released from low molecular weight PLGA particles was significantly higher than high molecular weight PLGA particles within 43 days of release studies, with negligible effect on encapsulation efficiency. The technique of coaxial electrospraying has high potential for encapsulation of susceptible protein-based therapeutic agents such as growth factors for multiple drug delivery applications.

Phenotypic modulation of smooth muscle cells by chemical and mechanical cues of electrospun tecophilic/gelatin nanofibers.

The ability of mature smooth muscle cells (SMCs) to modulate their phenotype in response to environmental cues is a critical issue related to vascular diseases. A tissue engineered vascular graft shall promote the contractile phenotype of vascular SMCs. To this aim, Tecophilic/gelatin (TP/gel) was electrospun at different weight ratios of TP/gelatin (100:0, 70:30, 50:50, 30:70), leading to differences in biochemical and mechanical properties of the nanofibers which in turn influenced the phenotype of SMCs. Results indicated that both the substrate with higher ligand density and lower stiffness could enhance SMC contractility and reduce cell proliferation. However, observing the highest SMCs contractility on electrospun TP(70)/gel(30) among the composite scaffolds demonstrated stiffness as the most critical parameter. Due to conflicting effects of softness versus minor fraction of gelatin (reduced ligand density) within TP(70)/gel(30) fibers, a relatively high proliferation of SMCs was still observed on TP(70)/gel(30) scaffold. The surface of TP(70)/gel(30) scaffold was further modified through physical adsorption of gelatin molecules so as to increase the ligand density on its surface, whereby a functional vascular construct that promotes the contractile behavior of SMCs with low cell proliferation was developed.

Advances in drug delivery via electrospun and electrosprayed nanomaterials.

Electrohydrodynamic (EHD) techniques refer to procedures that utilize electrostatic forces to fabricate fibers or particles of different shapes with sizes in the nano-range to a few microns through electrically charged fluid jet. Employing different techniques, such as blending, surface modification, and coaxial process, there is a great possibility of incorporating bioactive such molecules as drugs, DNA, and growth factors into the nanostructures fabricated via EHD techniques. By careful selection of materials and processing conditions, desired encapsulation efficiency as well as preserved bioactivity of the therapeutic agents can be achieved. The drug-loaded nanostructures produced can be applied via different routes, such as implantation, injection, and topical or oral administration for a wide range of disease treatment. Taking advantage of the recent developments in EHD techniques like the coaxial process or multilayered structures, individually controlled delivery of multiple drugs is achievable, which is of great demand in cancer therapy and growth-factor delivery. This review summarizes the most recent techniques and postmodification methods to fabricate electrospun nanofibers and electrosprayed particles for drug-delivery applications.

Composite poly(vinyl alcohol)/poly(vinyl acetate) electrospun nanofibrous mats as a novel wound dressing matrix for controlled release of drugs.

The aim of this study was to develop novel biomedicated nanofiber electrospun mats for controlled drug release, especially drug release directly to an injury site to accelerate wound healing. Nanofibers of poly(vinyl alcohol) (PVA), poly(vinyl acetate) (PVAc), and a 50:50 composite blend, loaded with ciprofloxacin HCl (CipHCl), were successfully prepared by an electrospinning technique for the first time. The morphology and average diameter of the electrospun nanofibers were investigated by scanning electron microscopy. X-ray diffraction studies indicated an amorphous distribution of the drug inside the nanofiber blend. Introducing the drug into polymeric solutions significantly decreased solution viscosities as well as nanofiber diameter. In vitro drug release evaluations showed that both the kind of polymer and the amount of drug loaded greatly affected the degree of swelling, weight loss, and initial burst and rate of drug release. Blending PVA and PVAc exhibited a useful and convenient method for electrospinning in order to control the rate and period of drug release in wound healing applications. Also, the thickness of the blend nanofiber mats strongly influenced the initial release and rate of drug release.

Controlled release of metronidazole benzoate from poly epsilon-caprolactone electrospun nanofibers for periodontal diseases.

Poly epsilon-caprolactone (PCL) nanofibers containing metronidazole benzoate (MET) were successfully electrospun and evaluated for periodontal diseases. Solutions of 10.5% w/v PCL and 5-15%w/w MET in mixtures of dichloromethane (DCM)/N,N-dimethylformamide (DMF) with ratios of 90:10, 80:20 and 70:30 v/v were prepared, and the nanofibers were produced by electrospinning technique. Scanning electron microscopy (SEM) was used to investigate the morphology and average diameter of the electrospun nanofibers. DSC results indicated a molecular dispersion of MET in the PCL nanofibers and showed a decrease in crystallinity of PCL nanofibers by adding MET. Results showed that an increase in the DCM:DMF ratio led to a decrease in the solution conductivity and an increase in the solution viscosity as well as in the nanofibers diameter. Also increasing metronidazole benzoate concentration caused an increase in the solution conductivity and a decrease in the solution viscosity as well as in the nanofibers diameter. In vitro drug release studies in phosphate buffer solution (pH 7.4) showed that the drug release rate was affected by the solvents ratio and the drug concentration. Moreover, the burst release was low, and sustained drug release was prolonged to at least 19 days.