Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins

L'hyperplasie intimale est la cause majeure de sténoses de pontages veineux. Différents médicaments tels que les statines permettent de prévenir les sténoses mais leur administration systémique n'a que peu d'effet. Nous avons développé une matrice d'hydrogel d'acide hyaluronique qui permet d'avoir un relargage contrôlé d'atorvastatine sur un site désiré. L'enjeu de ce projet de recherche est de démontrer que l'atorvastatine relarguée par l'hydrogel a un effet similaire sur les cellules musculaires lisses de veines saphènes humaines comparé à l'atorvastatine directement diluée dans le milieu de culture. La recherche a été conduite conjointement par le laboratoire de médecine expérimentale du département de chirurgie thoracique et vasculaire du Centre Hospitalier Universitaire Vaudois et de l'Ecole de sciences pharmaceutiques des Universités de Lausanne et de Genève. On a incorporé de l'atorvastatine calcium (Chemos GmbH, Regenstauf Allemagne) dans des gels d'acide hyaluronique (Fortelis extra) à des concentrations déterminées afin de pouvoir analyser le relargage de l'Atovastatine dans le milieu de culture cellulaire par rapport aux concentrations d'atorvastatine directement ajoutées dans le milieu. Des cellules musculaires lisses primaires ont été cultivées à partir d'expiants de veines saphènes humaines. Elles ont été identifiées grâce à l'immunohistochimie par des anticorps contre la desmine et l'alpha-smooth muscle actine. La prolifération et la viabilité de ces cellules ont été analysées à l'aide du test MTT, leur transmigration avec le test de la chambre de Boyden et leur migration avec le principe de cicatrisation de plaies (wound healing assey). L'expression de gènes connus pour participer au développement de l'hyperplasie intimale, tels que la gap junction protein Connexin43 (Cx43), l'inhibiteur du plasminogène PAI-1, Thème oxygénase HO-1, la métalloproteinase-9 et l'inhibiteur de l'activateur du plasminogène tissulaire tPA, a été déterminée par niveau de mRNA exprimé en PCR. Leur expression en protéines a été analysée en utilisant la méthode par Western blots ainsi que l'immunohistochimie. Les expériences ont été effectuées à triple reprise en duplicats en parallèles avec de l'atorvastatine calcium directement ajoutée dans le milieu de culture et avec l'atorvastatine relarguée par l'hydrogel d'acide hyaluronique. Conclusions L'atorvastatine est relarguée par l'hydrogel de façon contrôlée. L'hydrogel contenant l'atorvastatine diminue la viabilité et la transmigration des cellules musculaires lisses de veines saphènes humaines de façon similaire à l'atorvastatine directement introduite dans le milieu de culture. L'hydrogel contenant l'atorvastatine module de façon sélective l'expression de marqueurs de la différentiation cellulaire de cellules musculaires lisses de veines saphènes humaines avec un retard de 24 heures comparé avec les effets de l'atorvastatine directement ajoutée au milieu de culture, sans néanmoins changer la distribution intra-cellulaire des protéines Cx43, HO-1 et PAI-1. Perspectives Il s'agit d'un projet d'importance clinique majeure permettant de réaliser des améliorations du traitement des artériopathies occlusives, ainsi que de relevance pharmacologique permettant de réaliser des dépôts de molécules avec un relargage stable et contrôlé à un site spécifique

Surface engineering of biomedical devices with biocompatibility and controlled release

Modern medicine practice requires advanced medical devices with better biocompatibility, longer durability, and more complexity. Challenges arise for traditional techniques to apply a conformal modification on the complicate surfaces of modern implants in micro-scale to achieve better performance. Tailoring implant surface with hydrophilic coating was proven as an efficient strategy towards better biocompatibility. Precise modification of surface chemistry to accommodate the biological environment of the implants using initial chemical vapor deposition (iCVD) produced conformal nanocoating with excellent biocompatibility. In this study, highly crosslinked nanocoating was deposited on stainless steel surface and grafted with mixed charged polyionic using a one-pot three-step iCVD. Coated surface showed enhanced wettability with no adsorption of BSA after a seven-day incubation. Significant reduction of laminin adsorption and microglia attachment was observed, indicating excellent resistance against foreign body reaction for neural microelectrodes application. Secondly, with high density grafting, dual-charged antifouling grafting with a grafting thickness under 10 nm was synthesized with higher hydrophilicity. No BSA adhesion was shown on grafted surface from pH 7 to pH 9 and at body temperature, indicating significant enhancement of biocompatibility for implant applications that can withstand high pH. Thirdly, engineering of controlled release greatly improves the implant performance and avoids side effects. Charged nanocoating showed low permeability for opposite charged medication, making an effective diffusion barrier for controlled release of the medicine. Polyionic nanocoating provided three months of stable release, significantly suppressed smooth muscle cell growth. Adhesion of platelet on the coated surface was significantly reduced due to enhanced blood compatibility, indicating potential application in tissue reconstruction. Fourthly, further study into release control mechanism made it possible to synthesize nanocoatings with stable controlled release for non-charged medicine. Ultrathin simvastatin incorporated hydrogel with an 11-week stable release was synthesized using iCVD method. Biocompatible hydrogel coated sample provided controlled release of medicine in effective dosage without burst release. Coated sample significantly promoted preosteoblasts activity in vitro. In summary, application of vapor deposition of ultrathin coatings from commercially available reagents on different medical devices effectively improved substrate biocompatibility and drug release functionality, showing great potential in future implant application

Macroporous click-elastin-like hydrogels for tissue engineering applications

Producción CientíficaElastin is a key extracellular matrix (ECM) protein that imparts functional elasticity to tissues and therefore an attractive candidate for bioengineering materials. Genetically engineered elastin-like recombinamers (ELRs) maintain inherent properties of the natural elastin (e.g. elastic behavior, bioactivity, low thrombogenicity, inverse temperature transition) while featuring precisely controlled composition, the possibility for biofunctionalization and non-animal origin. Recently the chemical modification of ELRs to enable their crosslinking via a catalyst-free click chemistry reaction, has further widened their applicability for tissue engineering. Despite these outstanding properties, the generation of macroporous click-ELR scaffolds with controlled, interconnected porosity has remained elusive so far. This significantly limits the potential of these materials as the porosity has a crucial role on cell infiltration, proliferation and ECM formation. In this study we propose a strategy to overcome this issue by adapting the salt leaching/gas foaming technique to click-ELRs. As result, macroporous hydrogels with tuned pore size and mechanical properties in the range of many native tissues were reproducibly obtained as demonstrated by rheological measurements and quantitative analysis of fluorescence, scanning electron and two-photon microscopy images. Additionally, the appropriate size and interconnectivity of the pores enabled smooth muscle cells to migrate into the click-ELR scaffolds and deposit extracellular matrix. The macroporous structure together with the elastic performance and bioactive character of ELRs, the specificity and non-toxic character of the catalyst-free click-chemistry reaction, make these scaffolds promising candidates for applications in tissue regeneration. This work expands the potential use of ELRs and click chemistry systems in general in different biomedical fields.Ministerio de Economía, Industria y Competitividad (Projects MAT2013-42473-R, MAT2015-68901-R, MAT2016- 78903-R)Junta de Castilla y León (programa de apoyo a proyectos de investigación - Ref. VA313U14, VA015U16 y PCIN-2015-010)gobierno federal y estatal de Alemania en el marco del Programa de Posición Rotacional i³tm (2014-R4-01) y del Programa START de la Facultad de Medicina de la Universidad de Aachen (proyecto nº 691713),el centro de imágenes del Centro Interdisciplinario de Investigación Clínica (IZKF) de la Facultad de Medicina de la Universidad de Aache

Developing a 3D tissue-engineered model to study the biology and treatment of atherosclerosis

Coronary heart disease is the primary global cause of morbidity and mortality, accounting for about 33% of global deaths in 2013. Atherosclerosis is the principal cause of coronary heart disease and is caused by inflammation of the arterial wall. This begins with the accumulation of foam cells in the subendothelial space of an inflamed segment of the endothelium to create the fatty streak. The accumulation of these cells, and their apoptosis creates a proinflammatory necrotic core. This triggers smooth muscle cells migration into the subendothelial space, where these cells form a fibrous cap that mechanically strengthens the plaque. The ongoing inflammatory condition infit smooth muscle cell apoptosis which leads to cap thinning and eventual rupture of the plaque, triggering thrombus formation. Recreating this complex multi-step pathogenesis has principally relied on animal studies. However, key differences have been observed between the human and animal plaques. This has triggered attempts to develop a humanised in vitro models, however none of these have been demonstrated to reach later stages of plaque development, where plaque rupture trigger atherothrombosis. Previouslyour lab has used a layer-by-layer fabrication method to create a healthy tissue-engineered arterial construct. In this project, the aim was to develop and validate a simple 3D cell cultured neointimal model that can be inserted into this arterial construct to provide a novel tissue-engineered atherosclerotic plaque model. A protocol was developed to generate a 3D neointimal culture model in which the THP-1 monocytic cell line can be differentiated into THP-1-derived foam cells within a compressed collagen hydrogel. The cells were demonstrated to remain viable and secrete greater quantities of proinflammatory cytokines (such as TNF-α and IL-6) than macrophages. The neointimal construct was found to possess significant tissue factor activity and could be observed to induce a slow platelet aggregation. These prothrombotic effects were reduced when the 3D neointimal model was treated with atorvastatin during the ox LDL loading period of the culture. These results provided the first demonstration that a tissue-engineered atherosclerotic plaque model could replicate the prothrombotic properties of the native neointima. A co-culturing method was successfully developed that allowed reversible attachment of the neointimal model to the previously developed tissue-engineered medial layer using a fibrin hydrogel. Through treatment with plasmin containing solutions, the different layers of the co-culture could be shown to detach from one another, providing a basis for creating the first plaque rupture model in an in vitro atherosclerosis model. Additionally, it was possible to observe the migration of human coronary artery smooth muscle cells from the medial layer into the neointima. This provides the first evidence that tissue-engineered atherosclerosis models can elicit this key event in the development of the advanced stage of fibroatheroma. Overall, this thesis demonstrates the power of using a layer-by-layer fabrication method to develop a 3D human neointimal model that can replicate the early events in fibroatheroma. This ability to replicate both early and more advanced stage events highlight the potential for this construct to be further developed into an effective model of atherosclerotic plaque rupture to allow us to study human atherothrombosis more effectively in an ex vivo environment, and as a replacement to current in vivo animal models

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment

The establishment and use of tissue engineered vascular models to investigate the pleiotropic effects of statins

Cardiovascular disease (CVD) has been identified as the leading cause of mortality in westernised society. The triggering factor for the majority of cardiovascular diseases is atherosclerosis, defined as an accumulation of fatty materials in the vascular sub-endothelial space. This results in the initiation and propagation of inflammatory responses that result in the narrowing of the vascular lumen, as well as thickening and hardening of arteries. The initiating stimulus in atherosclerosis is elevated levels of low-density lipoprotein (LDL) in circulation, and the cells involved in this process are primarily endothelial cells-which interact with blood-, smooth muscle cells-which facilitate the contraction and relaxation of large diameter blood vessels-, and macrophages, which are immune cells that are able to take up and store lipids. As inflammation progresses, the endothelium becomes dysfunctional and expresses adhesion molecules that facilitate the entry of monocytes into the sub-endothelial space, where they differentiate into macrophages, take up LDL and become foam cells. These lipid rich foam cells are a key component of atherosclerotic plaques, together with dead cells that make up the necrotic core. Statins have been established as the gold standard for the treatment of atherosclerosis, and have been useful in decreasing morbidity and mortality in CVD patients. They function by preventing cholesterol synthesis through inhibition of HMG-CoA, thus lowering amounts of circulating cholesterol. In addition to this function, a number of pleiotropic effects have been associated with statin treatment including, increasing numbers of circulating endothelial progenitor cells (EPCs), reduce inflammation, improve atherosclerotic plaque stability and improve engraftment of MSCs into sites of vascular injury. To investigate these pleiotropic effects of this ubiquitous drug used in the treatment of the most prevalent disease, we developed tissue engineered blood vessel models that incorporated endothelial cells (Human umbilical vein endothelial cells (HUVECs)) and smooth muscle cells (Human cardiac artery smooth muscle cells (HCASMCs)) to represent the intimal and medial layers of the vasculature and could be used individually (Tissue engineered intimal layer-TEIL and tissue engineered medial layer-TEML) or in concert as a full blood vessel (tissue engineered blood vessel-TEBV). These vessel models/constructs were subjected to shear stress and used to evaluate the effect atorvastatin has on the homing of endothelial progenitor cells, the production of SDF-1 and expression of its receptor CXCR4. Further to this, the effect of atorvastatin on initiating cholesterol efflux was also investigated with considerations made to examine the role of HUVECs and smooth muscle cells in this process. The experiments conducted for this thesis were able to determine that atorvastatin increases the density of cells attached onto the surface of a lesioned construct. This was observed for the partial blood vessel models (TEIL and TEML) as well as the TEBV. This effect was noted for human mesenchymal stem cells (hMSCs) as well as EPCs. Observations in EPCs were consistent under both high (22.16 dyne/cm2) and low (2.2 dyne/cm2) shear stress. We were also able to determine that atorvastatin is more functional when used in conditions of oxidative stress through examination of different lesioning techniques. FeCl3 induced oxidative damage resulted in the recruitment of more cells to the surfaces of the lesioned constructs as well as higher levels of SDF-1, compared to the mechanical lesion which generated a mild surface abrasion. It was also possible to demonstrate that atorvastatin increases secretion of SDF-1 and expression of CXCR4, which are the main cytokine and receptor associated with cell homing and migration. This effect was determined to be both time and dose dependent. Through the use of different blood vessel models, it was determined that the cells in each layer have differing responses to the composite tissue model i.e., observations of cell attachment and SDF-1 production on TEBV were an amalgam of TEIL and TEML responses. Through the use of nanofiber inserts to create a novel HUVEC RAW264 co-culture system, we were able to demonstrate that atorvastatin triggers consistent cholesterol efflux from cultured foam cells compared to drug free controls, resulting in up to a 13% reduction in amounts off internalised cholesterol, a phenomenon that is affected by HUVEC integrity i.e., lesioned HUVECs promoted cholesterol efflux, especially in the presence of atorvastatin and IFN-γ. Atorvastatin was also able to restrict nitric oxide (NO) production in macrophages and may reverse the effects of the inflammatory cytokine IFN γ. The models used here proved a useful tool for investigating the effects of atorvastatin, and could prove useful in evaluating cellular responses to a wider array of pharmacologic, or other stimuli

Progress in Stimuli-Responsive Biomaterials for Treating Cardiovascular and Cerebrovascular Diseases

Cardiovascular and cerebrovascular diseases (CCVDs) describe abnormal vascular system conditions affecting the brain and heart. Among these, ischemic heart disease and ischemic stroke are the leading causes of death worldwide, resulting in 16% and 11% of deaths globally. Although several therapeutic approaches are presented over the years, the continuously increasing mortality rates suggest the need for more advanced strategies for their treatment. One of these strategies lies in the use of stimuli-responsive biomaterials. These "smart" biomaterials can specifically target the diseased tissue, and after "reading" the altered environmental cues, they can respond by altering their physicochemical properties and/or their morphology. In this review, the progress in the field of stimuli-responsive biomaterials for CCVDs in the last five years, aiming at highlighting their potential as early-stage therapeutics in the preclinical scenery, is described.Peer reviewe

Optimizing the Physical Properties of Vascular Targeted Carriers for Maximum Efficacy in Inflammatory Disease

Vascular targeted carriers (VTCs) increase the specificity of drug delivery while also protecting drugs from degradation in the bloodstream, and therefore, have the potential to revolutionize many clinical treatments of common diseases. It has previously been shown that rigid microparticles (MPs) are significantly more efficient than rigid nanoparticles (NPs) at adhering to target vasculature from bulk blood flow; yet, despite this increased efficiency, MP VTCs have not been successfully developed for therapeutic applications, and are routinely passed over in favor of NP systems, which better evade capillary occlusion and promote tissue transport. Here, we investigate different physical properties of VTCs, including the interplay between particle modulus, size, and targeting ligand regime, to enhance the translational potential of MP VTC therapeutics and to optimize the overall design of VTCs for a range of clinical applications. We systematically varied the physiochemical properties of particle modulus via poly(ethylene glycol) crosslinking density, targeting ligand (varied density, composition), and size (50 nm polystyrene to 2 µm hydrogels) and evaluated the impact of each property on targeted adhesion. VTC designs were evaluated in vitro using parallel plate flow chamber assays with inflamed human umbilical vein endothelial cells enabling controlled hemodynamic shear with primary human blood. VTC designs were evaluated in vivo using real time imaging of acute mouse mesentery inflammation, and full biodistribution studies following acute lung injury. The methods developed and employed here represent accurately simulated physiological conditions to encourage translatability of trends into that expected in the body. We confirmed that MPs were significantly better in targeted adhesion than NPs for all experimental conditions, with anywhere from 50% to 5,450% increase versus NPs, depending on the hemodynamic conditions. We found that both VTC modulus and targeting ligand regime could be tailored in vitro and in vivo to optimize adhesion given known hemodynamics. More deformable particles performed better at low wall shear rate (WSR), while more rigid particles adhered better at high WSRs. At high WSR, an increased ligand surface density improved the adhesion of deformable particles 27-fold, but not sufficiently to match the adhesion of rigid counterparts. While local shear rate dictated the optimal particle modulus, the local cellular protein expression dictated the adhesion kinetics required for optimized rigid NP and hydrogel MP adhesion. We found that a 25%-75% mix of ligand, skewed to the receptor which is lesser expressed, was consistently the most efficient at providing NP VTC adhesion, producing up to 9-fold more adhesion. We found that the addition of targeting ligand to MPs did not significantly decrease the circulation in vivo. Targeted, deformable MPs showed maximal retention at the target site over time versus rigid particles of any size. Finally, we showed that hydrogel MPs can greatly increase the transport of 50 nm NPs to the vascular wall, up to 5,450% versus free 50 nm NPs. This work explores and explains trends that depend on both the physiological conditions and particle properties in vitro and in vivo. Overall, we emphasize the importance of particle size, modulus, targeting ligand regime, and the local targeted tissue environment in engineering maximally efficient VTCs. We present work from VTC formulation which concludes with applications in multiple in vivo models, to provide a big picture view of how multiple particle properties cooperate to affect targeted adhesion.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144180/1/fishmarg_1.pd

Trilayer Tissue Engineered Heart Valves for Aortic Valve Replacement

Heart valve disease often progresses asymptomatically until valve damage has advanced to the point where replacement is unavoidable. Unfortunately, current valve replacements - including mechanical, bioprosthetic and autografts - have serious drawbacks, which often require replacement surgeries or lifelong anticoagulant therapy. The field of tissue engineering aims to overcome these drawbacks by combining scaffolds, stem cells, and chemical and physical stimuli to produce living tissues. The aortic heart valve has a unique structure composed of three discrete layers – fibrosa, spongiosa, and ventricularis - that work together in concert with the resident valvular interstitial cells to maintain a functioning valve. As a result, current tissue-engineered heart valves miss the mark for successful aortic valve replacement in one of two ways: either by being too weak to endure the stresses of the aortic environment or by being insufficiently recellularized and incapable of self-repair. The primary focus of this research was to create a functional heart valve replicating the unique trilayer structure developed by nature. We showed that valves can be modeled from medical imaging data, 3D printed, and used as molds to create patient-specific heart valves. The valve scaffolds supported cell attachment, growth, and proliferation. Porous, dry scaffolds were effectively glued together to form one cohesive trilayer scaffold. These scaffolds resemble the human valve’s unique histoarchitecture. A meta-analysis of literature defined maximum normal stresses and strains experienced by the native valve; providing a target set of mechanical properties to be replicated by the tissue-engineered valve. Increasing porosity and microneedle rolling treatments produced scaffolds with excellent mechanical strength that were more than strong enough to function in physiological conditions. A novel cell seeding technique was developed to rapidly seed porous and microneedle treated fibrous scaffolds; resulting in full-thickness cell seeding. Functional heart valves were made using a crush-mounting system. This system allowed for rapid and reproducible production of valves for in vitro testing. A comparison between mechanical, bioprosthetic, and trilayer valves revealed outstanding hemodynamic performance of trilayer valves. These valves functioned well for three weeks in a heart valve bioreactor. This research produced functional, tissue-engineered heart valves with excellent mechanical and hemodynamic properties

Interactions between valvular cells: implications for heart valve tissue engineering

Thesis (Ph.D.)--Boston UniversityApproximately 1 in 1000 children are born with congenital cardiovascular defects yearly in the US, including many abnormalities in heart valves. Tissue engineered heart valves (TEHVs) offer a solution for replacement or repair of affected valves. However, its therapeutic application is limited, and in ovine models, no TEHV has performed satisfactorily in vivo for longer than twenty weeks, in part due to the absence of supporting data for selection of the appropriate cell type(s) to be incorporated into the construct. This partially owes to the lack of a full understanding of the cells that inhabit the valve, which includes valve interstitial cells (VICs) and valve endothelial cells (VECs), and on the molecular mechanism underlying their interactions that maintain valve homeostasis. During embryonic valve development, the vast majority of VICs are derived from VECs via endothelial to mesenchymal transformation (EMT). EMT in postnatal valves is rare but it has been implicated in diseased valves. Yet, relatively little is known about VECs and VICs in post-natal valves in terms of specialized features, and how VECs and VICs might influence each other. This lack of knowledge has made it difficult to determine what type of cells should be used to create a TEHV. In order to achieve the optimal construction of a tissue engineered heart valve we look to the native valve as our guide for proper valve structure and function. Examination of the native valve leaflets can contribute to our understanding of the proper cellular environment and how disruption of this environment affects the valves. Many common mitral valve pathologies including mitral valve prolapse are characterized by thickening of the valve spongiosa, the presence of activated myofibroblasts, and excessive remodeling of the extracellular matrix. By examining the cell-cell interactions in healthy native valves, and comparing this with observations from pathogenic valves, a greater understanding can be achieved and then applied to the field of TEHV. In this thesis we explored the cell dynamics of the heart valve as related to natural homeostasis, disease progression, and tissue engineering. Using an in vitro co-culture model we revealed a novel two-way communication between mitral valve endothelial and interstitial cells. We propose that this communication promotes a healthy valve phenotype and function by inhibiting EndMT and suppressing VIC activation. We made a similar observation in the aortic valve, where VEC-VIC communication may prevent the process of an EndMT mediated osteogenesis in the context of calcific aortic valve disease. We have also used the VEC-VIC co-culture model to identify possible candidate cell sources for a tissue engineered heart valve. And finally, we show that cells that populated a tissue engineered pulmonary valve leaflet, created using an acellular scaffold, are phenotypically and functionally similar to native valve cells. These studies contribute to an understanding of the dynamics of the cellular interactions between VECs and VICs, and provide a new framework for identifying and testing the functionality of appropriate cell sources for building a TEHV with the ability to grow with the child, maintain homeostasis, and prevent fibrosis and calcification