scholarly journals Delivery of targeted gene therapies using a hybrid cryogel-coated prosthetic vascular graft

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7377
Author(s):  
Cindy Huynh ◽  
Ting-Yu Shih ◽  
Alexander Mammoo ◽  
Amruta Samant ◽  
Saif Pathan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Shape Recovery ◽  
Neointimal Hyperplasia ◽  
Prosthetic Graft ◽  
Arterial Disease ◽  
Biologic Agents ◽  
Human Aortic Endothelial Cell ◽  
Swelling Ratio ◽  
Pore Connectivity ◽  
Rgd Peptides

ObjectivesThe success of prosthetic vascular grafts in the management of peripheral arterial disease is frequently limited by the development of anastomotic neointimal hyperplasia (ANIH), with the host response to prosthetic grafts beginning soon after implantation. To address this, we combine a platform of polyethylene terephthalate (PET) fabric with an applied cryogel layer containing biologic agents to create a bioactive prosthetic graft system, with the ability to deliver therapeutics targeting modulators of the ANIH-associated transcriptome response, along with antithrombotic agents.MethodsHybrid graft materials were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun (ePET), knitted PET (kPET), or woven PET (wPET). Arg-Gly-Asp (RGD) peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Physical properties such as swelling ratio, pore connectivity, shape recovery, and stiffness were evaluated. Human aortic endothelial cell (HAoEC) adherence was visualized using confocal microscopy after 24 hours and proliferation was evaluated with a resazurin-based assay for 7 days. Confocal microscopy was used to assess delivery of adeno-associated virus (AAV-GFP) after incubation of hybrid grafts with HAoECs. Heparin activity of the materials was measured using an anti-Xa assay.ResultsSEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. Hybrid materials showed a trend towards increased shape recovery, increased stiffness, decreased swelling ratio, and no difference in pore connectivity. HAoECs incorporated, adhered, and proliferated over 7 days on all materials. HAoECs were successfully transduced with AAV-GFP from the hybrid graft materials. Anti-Xa assay confirmed continued activity of heparin from all materials for over 7 days.ConclusionsWe have developed a bioactive prosthetic graft system with a cryogel coating capable of delivering biologic agents with antithrombotic activity. By applying the cryogel and selected agents onto PET prior to graft implantation, this study sets the stage for the system to be individualized and tailored to the patient, with bioengineering and targeted gene therapy strategies dovetailing to create an improved prosthetic graft adaptable to emerging knowledge and technologies.

Abstract 117: Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Cindy Huynh ◽  
Ting-Yu Shih ◽  
Amruta Samant ◽  
Saif G Pathan ◽  
David W Nelson ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Prosthetic Graft ◽  
Cell Interaction ◽  
Biologic Agents ◽  
Prosthetic Material ◽  
Limiting Factor ◽  
Human Aortic Endothelial Cell ◽  
Graft Material ◽  
Buffer Solution ◽  
Pet Fabric

Objectives: Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS). Methods: Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay. Results: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days. Conclusion: We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.

Drug eluting grafts for hemodialysis access

2017 ◽  
Vol 18 (1_suppl) ◽  
pp. S53-S55 ◽  
Author(s):  
Marc Glickman
Keyword(s):  
Neointimal Hyperplasia ◽  
Graft Function ◽  
Prosthetic Graft ◽  
Great Promise ◽  
Graft Material ◽  
Eptfe Graft ◽  
Gene Therapies ◽  
New Methods ◽  
Drug Elution ◽  
Drug Eluting

The development of new methods for drug elution of graft material, biofiber films and resurfacing of prosthetic graft surfaces offers new opportunities for improvement of graft function in arteriovenous (AV) access. Three areas of research include developing grafts that reduce the development of neointimal hyperplasia, reducing infection and reducing thrombogenicity. The only drug eluting graft presently being used, is the heparin coated expanded polytetrafluoroethylene (ePTFE) graft, which has been shown to decrease the incidence of early thrombosis. New drug eluting grafts include those with paclitaxel and those with antibiotics. The development of a hybrid coated prosthetic graft that can deliver targeted gene therapies holds great promise in the field.

scholarly journals Assessment of Mortality and Factors Affecting Outcome of Use of Paclitaxel-Coated Stents and Bare Metal Stents in Femoropopliteal PAD

2020 ◽  
Vol 9 (7) ◽  
pp. 2221
Author(s):  
Aleksander Falkowski ◽  
Hubert Bogacki ◽  
Marcin Szemitko
Keyword(s):  
Clinical Benefit ◽  
Neointimal Hyperplasia ◽  
Randomized Study ◽  
Bare Metal Stents ◽  
Total Mortality ◽  
Arterial Disease ◽  
Metal Stents ◽  
Bare Metal ◽  
Factors Affecting ◽  
Increased Risk

The use of drug-coated devices in intravascular therapy is aimed at preventing neointimal hyperplasia caused by excessive proliferation of vascular smooth muscle and thereby restenosis. Although its use seemed initially promising, a recent publication has shown an increased risk of mortality with paclitaxel-coated devices, and there is an urgent need to reaffirm assessments of drug-eluting stents (DES). Objective: The aim of the study was to compare mortality and effectiveness of paclitaxel-coated stents and bare-metal stents (BMS) in the treatment of peripheral arterial disease (PAD) with long-term follow-up. Materials and methods: In a single center randomized study, 256 patients with PAD were treated intravascularly with stent implantation. Patients were randomized into two groups: the first (n = 126) were treated with DES, and the second (n = 130) were treated with BMS. The study included evaluation after the procedure, after about 6 months and 36 months. Co-morbidities, with risks for atherosclerosis, were analyzed in all patients. Patients were evaluated for clinical outcome, restenosis frequency, and safety (complications and total mortality). Results: Clinical benefit at the end of the investigation was statistically significantly better in the DES group compared with the BMS group: 85.7% versus 66.2% (p = 0.0003), respectively. Restenosis occurred significantly less frequently in patients with DES: 16.0% versus BMS: 35.0%, p = 0.012. There was no significant effect of comorbidities on the frequency of restenoses. There were no differences in all-cause mortality over the three years with paclitaxel and no-paclitaxel stents cohorts (8.7% versus 7.1%; long-rank p = 0.575). No association was found with mortality and treatment with DES or BMS. Conclusions: The use of paclitaxel-coated stents gave good clinical benefit and caused a significantly lower frequency of restenosis compared to bare-metal stents. The use of paclitaxel-coated stents did not increase mortality.

scholarly journals Level of Patency for a One Year Period of Infra-inguinal Arterial Bypass in Patients with Peripheral Artery Disease

2019 ◽  
Vol 1 (1) ◽  
pp. 26-29
Author(s):  
Nyityasmono Tri Nugroho ◽  
Raden Suhartono
Keyword(s):  
Risk Factors ◽  
Smoking Cessation ◽  
Peripheral Arterial Disease ◽  
Patency Rate ◽  
Artery Bypass ◽  
Prosthetic Graft ◽  
Arterial Disease ◽  
Arterial Bypass ◽  
One Year ◽  
Peripheral Arterial

Introduction: Peripheral arterial disease (PAD) is the most common macroangiopathic complication in type II diabetes mellitus, arising from inadequate blood sugar control. In the presence of PAD, the risk of limb loss will also increase, and arterial bypass is one method to reduce the risk of amputation. In Indonesia, the level of patency for the arterial bypass has not yet been published. On bypass with venous grafts, the patency rates at the location of infrapopliteal reach at 70-80%, while the prosthetic graft is 30-50%. Method: From 2009 to 2012, patients with arterial bypasses were analyzed. The level of patency was described by ultrasound examination and pulsation on clinical examination in the distal anastomosis, reduced pain, and other examinations that support adequate revascularization. Identification of risk factors that affect patency, particularly protective risk factors, were also taken into account. Results: From 2009 to 2012, 29 patients with infra-inguinal arterial bypass were collected. The ratio of men to women was 5:1, with a one-year patency rate of 88% in men, and 75% in women, for an overall of 86.2%. The irreversible risk factor affecting patency was male (p = 0.117). Modifiable risk factors that decreasing patency level were smoking (p = 0.042) and more advanced stage of PAD (p = 0.067). Smoking cessation (p = 0.041) and the use of drugs after bypass procedure (p = 0.072) were known to increase the level of patency. Conclusion: The one-year patency rate for infra-inguinal artery bypass was 37-89%. Smoking cessation was known to increase the level of patency. Keywords: patency level, arterial bypass, infra-inguinal, peripheral arterial disease, diabetes

scholarly journals Roles of MicroRNAs in Peripheral Artery In-Stent Restenosis after Endovascular Treatment

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Mo Wang ◽  
Weichang Zhang ◽  
Lei Zhang ◽  
Lunchang Wang ◽  
Jiehua Li ◽  
...  
Keyword(s):  
Endovascular Repair ◽  
Neointimal Hyperplasia ◽  
Cell Types ◽  
Underlying Mechanism ◽  
Arterial Disease ◽  
Transluminal Angioplasty ◽  
Stent Restenosis ◽  
Peripheral Arterial ◽  
Different Cell Types ◽  
In Stent Restenosis

Endovascular repair including percutaneous transluminal angioplasty (PTA) and stent implantation has become the standard approach for the treatment of peripheral arterial disease; however, restenosis is still the main limited complication for the long-term success of the endovascular repair. Endothelial denudation and regeneration, inflammatory response, and neointimal hyperplasia are major pathological processes occurring during in-stent restenosis (ISR). MicroRNAs exhibit great potential in regulating several vascular biological events in different cell types and have been identified as novel therapeutic targets as well as biomarkers for ISR prevention. This review summarized recent experimental and clinical studies on the role of miRNAs in ISR modification, with the aim of unraveling the underlying mechanism and potential therapeutic strategy of ISR.

scholarly journals Pathogenesis and Clinical Significance of In-Stent Restenosis in Patients with Diabetes

2021 ◽  
Vol 18 (22) ◽  
pp. 11970
Author(s):  
Grzegorz K. Jakubiak ◽  
Natalia Pawlas ◽  
Grzegorz Cieślar ◽  
Agata Stanek
Keyword(s):  
Risk Factor ◽  
Cardiovascular Diseases ◽  
Clinical Significance ◽  
Neointimal Hyperplasia ◽  
Arterial Disease ◽  
Limb Amputation ◽  
Below The Knee ◽  
Stent Restenosis ◽  
Percutaneous Balloon ◽  
In Stent Restenosis

Diabetes mellitus (DM) is a strong risk factor for the development of cardiovascular diseases such as coronary heart disease, cerebrovascular disease, and peripheral arterial disease (PAD). In the population of people living with DM, PAD is characterised by multi-level atherosclerotic lesions as well as greater involvement of the arteries below the knee. DM is also a factor that significantly increases the risk of lower limb amputation. Percutaneous balloon angioplasty with or without stent implantation is an important method of the treatment for atherosclerotic cardiovascular diseases, but restenosis is a factor limiting its long-term effectiveness. The pathogenesis of atherosclerosis in the course of DM differs slightly from that in the general population. In the population of people living with DM, more attention is drawn to such factors as inflammation, endothelial dysfunction, platelet dysfunction, blood rheological properties, hypercoagulability, and additional factors stimulating vascular smooth muscle cell proliferation. DM is a risk factor for restenosis. The purpose of this paper is to provide a review of the literature and to present the most important information on the current state of knowledge on mechanisms and the clinical significance of restenosis and in-stent restenosis in patients with DM, especially in association with the endovascular treatment of PAD. The role of such processes as inflammation, neointimal hyperplasia and neoatherosclerosis, allergy, resistance to antimitotic drugs used for coating stents and balloons, genetic factors, and technical and mechanical factors are discussed. The information on restenosis collected in this publication may be helpful in planning further research in this field, which may contribute to the formulation of more and more precise recommendations for the clinical practice.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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