Bioresorbable polymer scaffolds

Abstract

A polymer scaffold includes a porous component having a network of resorbable polymer fibers, and a therapeutic dispersed in the porous component, the therapeutic component including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

Description

Atty. Dkt. No.642631-0045 (pMDA23-083) BIORESORBABLE POLYMER SCAFFOLDS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 514,513, filed July 19, 2023, the entire disclosure of which is incorporated herein for all purposes. TECHNICAL FIELD

[0002] The present disclosure relates generally to polymer scaffolds. More specifically, the present disclosure relates to bioresorbable tissue scaffolds for vascular applications, such as vascular wraps, vascular grafts, or vascular stents. BACKGROUND

[0003] Arteriovenous fistulas (AVFs) can be used to provide vascular access for patients with end stage renal disease (ESRD) in various patients and other subjects. However, AVFs often do not remain useable (e.g., patent) after relatively short periods of time, including but not limited to because of issues such as neointimal hyperplasia. In the US, there are 350,000 patients undergoing hemodialysis (HD). Despite being the preferred form of vascular access type, only approximately 40-60% of AVFs will be patent one year after formation of the AVFs. A major cause for AVF failure is neointimal hyperplasia, which can lead to fistula stenosis and thrombosis. SUMMARY

[0004] Systems and methods in accordance with the present disclosure can implement polymer scaffolds, including bioresorbable tissue scaffolds, that can reduce or mitigate the likelihood of structural changes to AVFs after formation that can result in AVFs becoming patent. For example, the polymer scaffold can couple with or otherwise support AVFs or other fistulas or vascular structures or targeted tissue, such as to mechanically support such structures. The polymer scaffold can include and / or deliver one or more cells, drug particles, nanoparticles, or various combinations thereof to the targeted tissue, such as to provide therapeutics to the targeted tissue. Various such mechanical and / or therapeutic effects from the polymer scaffold can reduce the likelihood of, such as to prevent, causes of failure including but not limited to neointimal hyperplasia, such as to reduce the likelihood of fistula stenosis and / or thrombosis. For example, scaffolds in accordance with the present disclosure can provide localized perivascular drug delivery, which can allow for high-dose treatment on the vascular wall without significantly increasing the risk for systemic toxicity. 1 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0005] At least one aspect relates to a polymer scaffold that includes a porous component and a therapeutic dispersed in the porous component. The porous component includes a network of resorbable polymer fibers. The therapeutic component includes at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

[0006] At least one aspect relates to a method of fabricating a polymer scaffold that includes dissolving a polymer in a solvent to form a polymer solution. The method includes blending a therapeutic component into the polymer solution to form a blended polymer solution, the therapeutic component including at least one of a plurality of drug particles, or a plurality of metallic nanoparticles. The method includes electrospinning the blended polymer solution to form a polymer scaffold including the therapeutic component dispersed in a porous network of polymer fibers. The method further includes incorporating a plurality of cells in the polymer scaffold.

[0007] At least one aspect relates to a method of treating a subject with a polymer scaffold that includes providing a polymer scaffold and deploying the polymer scaffold to a vascular structure of the subject. The polymer scaffold includes a porous component having a network of polymer fibers resorbable by a subject and a therapeutic component dispersed in the porous component. The therapeutic component includes at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

[0008] At least one aspect relates to a method of fabricating a polymer scaffold that includes dissolving a polymer in a solvent to form a polymer solution. The method includes electrospinning the blended polymer solution to form a polymer scaffold including the therapeutic component dispersed in a porous network of polymer fibers. The method further includes subsequently incorporating a therapeutic component in the polymer scaffold, the therapeutic component including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

[0009] At least one aspect relates to a polymer scaffold that includes a porous member and a therapeutic material dispersed in the porous component. The porous member includes a network of resorbable polymer fibers. The therapeutic material includes at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

[0010] These and other aspects and implementations are discussed in detail below. The foregoing information and the following detailed description include illustrative examples 2 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) of various aspects and implementations, and provide an overview or framework for understanding the nature and character of the claimed aspects and implementations. The drawings provide illustration and a further understanding of the various aspects and implementations, and are incorporated in and constitute a part of this specification. Aspects can be combined and it will be readily appreciated that features described in the context of one aspect of the invention can be combined with other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings are not intended to be drawn to scale. Like reference numbers and designations in the various drawings indicate like elements. For purposes of clarity, not every component can be labeled in every drawing. In the drawings:

[0012] FIG.1 is a perspective view of an example of a polymer scaffold;

[0013] FIG.2 is a flow diagram of an example of a method of forming a polymer scaffold;

[0014] FIGS.3A and 3B are schematic diagrams of examples of polymer scaffolds;

[0015] FIGS.4A and 4B are cross-section views of the polymer scaffolds of FIGS.3A and 3B;

[0016] FIG.5 is a flow diagram of an example of a method of using a polymer scaffold;

[0017] FIG.6 is a flow diagram of an example of a method of using a polymer scaffold;

[0018] FIG.7 is a schematic diagram of a process of fabricating and deploying a polymer scaffold;

[0019] FIG.8 depicts charts of example data of features of a polymer scaffold;

[0020] FIG.9 depicts examples of fluorescence microscopy images of samples of polymer scaffolds;

[0021] FIG.10 depicts examples of images and charts of features of polymer scaffolds in surgical procedures;

[0022] FIG.11 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0023] FIG.12 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds; 3 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0024] FIG.13 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0025] FIG.14 depicts examples of images and charts of fluorescence microscopy of subjects having AVFs and / or polymer scaffolds;

[0026] FIG.15 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0027] FIG.16 depicts examples of images of immunofluorescence of subjects having AVFs and / or polymer scaffolds;

[0028] FIG.17 depicts examples of scanning electron microscope images and charts of examples of polymer scaffolds;

[0029] FIG.18 depicts examples of charts of features of polymer scaffolds;

[0030] FIG.19 depicts examples of charts of features of polymer scaffolds;

[0031] FIG.20 depicts examples of images and charts of features of polymer scaffolds;

[0032] FIG.21 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0033] FIG.22 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0034] FIG.23 depicts examples of images of immunofluorescence of features of subjects having AVFs and / or polymer scaffolds;

[0035] FIG.24 depicts examples of a chart of features of subjects having AVFs and / or polymer scaffolds;

[0036] FIG.25 depicts examples of images of polymer scaffolds;

[0037] FIG.26 depicts examples of images of fluorescence microscopy of polymer scaffolds;

[0038] FIG.27 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0039] FIG.28 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0040] FIG.29 depicts examples of images and charts of features of subjects having 4 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) AVFs and / or polymer scaffolds; and

[0041] FIG.30 depicts examples of images of immunofluorescence staining of features of subjects having AVFs and / or polymer scaffolds.

[0042] FIG.31 depicts examples of a transmission electron microscope image and a corresponding chart, scanning electron microscope images, photographical images, X-ray images, and micro-computed tomography images of features of polymer scaffolds;

[0043] FIG.32 depicts examples of images and charts of features of polymer scaffolds;

[0044] FIG.33 depicts examples of images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0045] FIG.34 depicts examples of18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) / micro-computed tomography (mCT) images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0046] FIG.35 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0047] FIG.36 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0048] FIG.37 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0049] FIG.38 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0050] FIG.39 depicts examples of scanning electron microscope images, photographical images, X-ray images, and micro-computed tomography images of features of polymer scaffolds;

[0051] FIG.40 depicts examples of charts of features of subjects having AVFs and / or polymer scaffolds;

[0052] FIG.41 depicts examples of mCT images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0053] FIG.42 depicts examples of ultrasonography (US) images and charts of features 5 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) of subjects having AVFs and / or polymer scaffolds;

[0054] FIG.43 depicts examples of 18F-FDG PET images and charts of features of subjects having AVFs and / or polymer scaffolds;

[0055] FIG.44 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0056] FIG.45 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds;

[0057] FIG.46 depicts examples of images and charts of histological findings of subjects having AVFs and / or polymer scaffolds; and

[0058] FIG.47 is a schematic diagram of a sample including an example of a perivascular wrap based on a polymer scaffold implemented around a vascular structure. DETAILED DESCRIPTION

[0059] Below are detailed descriptions of various concepts related to, and implementations of, techniques, approaches, methods, apparatuses, and systems for polymeric tissue scaffolds, including but not limited to tissue scaffolds that include bioresorbable materials to be implemented for arteriovenous fistulas. The various concepts introduced above and discussed in greater detail below can be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

[0060] Approximately 5.7 million individuals worldwide suffer from end-stage renal disease. In the United States, the number of individuals with this condition has reached over 800,000. Globally, hemodialysis is still the most common method for replacing kidney function. For example, approximately 2.3 million individuals are undergoing chronic hemodialysis worldwide. In the United States, 62.6% of patients with prevalent end-stage renal disease depend on an AVF for hemodialysis. For vascular access for hemodialysis, an arteriovenous fistula (AVF) may be preferred over an intravenous catheter or an arteriovenous graft because of the lower risk of infection and fewer interventions to maintain patency. However, failure of an AVF to mature for hemodialysis use is reported to occur in up to 60% of patients, limiting its overall utility. Salvage interventions for AVF stenosis, such as balloon angioplasty and stent placement, are available, but these 6 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) interventions add to treatment cost and patient morbidity. Furthermore, the AVFs of patients requiring these interventions have a decreased cumulative overall survival rate. For example, vascular access complications continue to be a common source of morbidity and mortality, with close to half (40.9%) of primary unassisted arteriovenous fistulas (AVFs) failing by 1 year.

[0061] Creation of an AVF involves forming an anastomosis between an inflow artery and an outflow vein, either directly (i.e., autologous) or bridged through a synthetic graft. Ideally, the site responds to the increased blood flow by remodeling until it can support dialysis recanalization and flow rates. However, even successful maturations require an average of 115 days. Monitoring primarily relies on physical exam and duplex ultrasound, with further visualization limited to the radiopaque potential of endogenous tissues.

[0062] One of the major pathologic etiologies associated with AVF maturation failure is neointimal hyperplasia (NIH), a cellular process that results from the synergistic action of inflammation, hypoxia, and hemodynamic stress on the anastomotic tissue. These pathways together promote endothelial cell proliferation and inward migration of pro-inflammatory cells, smooth muscle cells, and myofibroblasts, which result in vascular stenosis and thrombosis. To suppress NIH, several systemic therapies have been studied, including aspirin, clopidogrel, dipyridamole, warfarin, sulfinpyrazone, HMG-CoA reductase inhibitors, and fish oil. However, no systemic therapy has shown a clear benefit in improving AVF maturation rates, and a major limitation of systemic agents is dose-limiting toxicity. Localized interventions, such as mechanical devices, targeted drug delivery, and cell therapy, have the potential to improve AVF maturation rates by providing a direct therapeutic effect at the anastomotic site without the risk of systemic toxicity, but may lack the ability to provide both mechanical and therapeutic support to AVFs and / or may not function sufficiently to support AVF through to maturation and / or after maturation. A most common finding accounting for AVF non-maturation and post-maturation decreased flow rates is vessel stenosis. While the pathophysiology of stenosis is not fully understood, it is believed to be primarily driven by postoperative abnormal proliferation of the innermost vessel wall (e.g., NIH). In patients with AVF failure secondary to NIH, elevated low- density lipoprotein cholesterol levels were an independent risk factor, leading to studies investigating systemic statin therapy to decrease NIH and improve AVF maturation and patency rates. The rationale for using systemic statins in this clinical setting results from their ability to regulate endothelial cell function and inhibit vascular inflammation and 7 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) thrombosis in ESRD patients. Despite the theoretical benefit of systemic statins, a favorable impact on AVF failure rates has not been proven, potentially owing to patient compliance to oral therapy given side effects from high-dose statin therapy (e.g., myopathy, hepatotoxicity, and blood glucose dysregulation). Hence, there is a compelling need to develop strategies to reduce or prevent AVF maturation failure.

[0063] Systems and methods in accordance with the present disclosure can link bioresorbable polymers to therapeutic agents such as mesenchymal stem cells (e.g., MSCs), metallic nanoparticles (e.g., gold nanoparticles, bismuth nanoparticles, etc.), and drugs (e.g., rosuvastatin, rapamycin, etc.), for purposes of extended-release elution of anti-inflammatory and / or anti-thrombotic agents via a perivascular scaffold. This can reduce or eliminate the occlusive effects of NIH which is the cause of stenoses in AVFs.

[0064] For example, a resorbable perivascular (e.g., vascular) scaffold can be loaded (e.g., coated, infused, embedded, synthesized, etc.) with one or more of MSCs, metallic nanoparticles, or anti-inflammatory or anti-thrombotic drugs. The perivascular scaffold (or “scaffold” for short) can act as a depot for elution of cellular products from MSCs or anti- inflammatory drugs for purposes of preventing the deleterious effects of NIH in the setting of AVF maturation. The scaffold can improve AVF maturation in various manners. For example, the scaffold can mechanically support the maturing AVF. AVFs are created by forming an anastomosis from a vein to an artery. The scaffold can be braided from a resorbable polymer (e.g., polycaprolactone (PCL)) laden with one or more of MSCs, metallic nanoparticles, or drugs. The composition of the polymer can allow for the gradual elution of MSC products or drugs. The placement of the device along the exterior of the maturing AVF can address the location of pathologic inflammation associated with NIH, which evidence suggests begins in the “outer” wall of the AVF before progressing towards the “inner” wall. PCL polymer resorbs within two to three years. PCL can have favorable mechanical properties and its resorption profile which would allow for prolonged release of products from MSCs or drugs. Furthermore, since permanent intravascular devices are associated with thrombosis and vessel stenosis, eventual resorption of the scaffold can minimize the potential long-term deleterious outcomes associated with permanent implantable medical devices.

[0065] The MSC-laden, metallic nanoparticles-infused, and / or drug-infused resorbable polymer scaffold can improve the success rates of AVF maturation for patients on HD in a transformative manner. In some implementations, drug-eluting stent technology can be 8 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) improved by coating stents with MSCs or drugs for sustained cellular product or drug elution.

[0066] MSCs also contain anti-inflammatory properties. In some implementations, the scaffold can be implemented as a resorbable perivascular scaffold that includes MSCs or anti-inflammatory drugs (e.g., rosuvastatin, rapamycin, etc.) capable of sustained and gradual elution of drugs and / or anti-inflammatory cellular products. In some implementations, the scaffold can further be infused with metallic nanoparticles that are radiopaque for improved radiologic visibility conducive to longitudinal monitoring using X- ray imaging techniques.

[0067] In some implementations, vascular stents coated with anti-inflammatory drugs (e.g., rosuvastatin) and / or MSCs would address the unmet clinical need of addressing NIH following endovascular intervention to maintain patency of AVFs and AV grafts.

[0068] In some implementations, the radiolucency of polymeric wraps and intravascular stents can render in-vivo monitoring challenging. In-vivo monitoring is an important component of medical device surveillance, and in its absence, the safety and efficacy of a medical device are both compromised. Although the wraps are clearly delineated from the surrounding vascular layers on histology, they may not be easily delineated on in-vivo imaging (e.g., ultrasonography (US),18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET), etc.). As a solution, systems and methods in accordance with the present disclosure can implement polymeric constructs (e.g., scaffolds, devices, wraps, etc.) synthesized with metallic nanoparticles to give them radiopacity that will enable the tracking of their placement, integrity, and breakdown over time. Among the currently available metallic nanoparticles, gold nanoparticles (AuNPs) and bismuth nanoparticles (BiNPs) have both shown excellent tunability, radiopacity, and biocompatibility. In some implementations, a polymer scaffold is provided as an electrospun polymeric perivascular wrap from AuNPs-infused PCL or BiNPs-infused PCL, which can be further seeded with MSCs, infused with drugs, or both, and can be compared with respect to AVF maturation against non-metallic NPs-containing wraps. The incorporation of AuNPs and BiNPs may confer sufficient radiopacity to an MSC-seeded and / or drug-infused polymeric wrap, as seen via micro-computed tomography (mCT), without substantially affecting the wrap’s beneficial effects on the outflow vein of the AVF.

[0069] In some implementations, metallic nanoparticles (e.g., AuNPs, BiNPs, etc.) are 9 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) incorporated in the synthesis of the PCL-based wraps. In the present disclosure, the terms “resorbable perivascular scaffold,” “resorbable polymer scaffold,” “polymer scaffold,” “tissue scaffold,” “perivascular wrap,” “vascular wrap,” “wrap,” “polymer wrap,” and “polymeric wrap” may be used interchangeably with one another. Some studies have shown that a mesenchymal stem cell (MSC)-seeded polymeric wrap is capable of reducing NIH and enhancing AVF maturation. However, the radiolucent nature of the polymeric wrap makes in-vivo monitoring of its deployment and structural integrity difficult. As such, the incorporated metallic nanoparticles can be useful in AVF surveillance since they can facilitate quick localization of the AVF for concurrent study using other imaging modalities, such as US or 18F-FDG PET. The integration of the metallic nanoparticles within the architecture of the wraps can facilitate the confirmation of device placement after surgery, and the reduction in detected intensity from the wrap may serve as a marker of its degradation over time.

[0070] In some implementations, combining resorbable polymers (e.g., PCL, poly(lactic acid), poly(lactic-co-glycolic acid), polyethylene glycol, and polydioxanone), MSCs, and / or rosuvastatin can provide a resorbable perivascular scaffold. In various implementations, such scaffolds can include one or more of a perivascular scaffold to aid AVF maturation by mitigating the occlusive effects of NIH; MSCs-coated and / or drug-coated synthetic vascular grafts formed from the perivascular scaffolds can be used in initial arteriovenous graft (AVG) formation; and MSCs-coated and / or drug-coated vascular stents formed from the perivascular scaffolds can be used in patients with stenoses located within an AVF or AVG. In addition to elution of cellular by-products or drugs, there are two additional unique characteristics specific to the perivascular scaffold disclosed herein. First, the perivascular scaffold is completely resorbable, which eliminates the risk for NIH formation posed by permanent medical devices. Second, the perivascular scaffold can track device absorption, which facilitates the assessments of device efficacy relative to the disease state.

[0071] In some implementations, through the process of electrospinning, bioresorbable synthetic polymers can be used to produce fibrous scaffolds that can mimic the three- dimensional architecture of collagen fibrils in the vascular extracellular matrix. PCL is a highly tunable and relatively affordable FDA-approved synthetic biodegradable polymer used in a wide variety of tissue-engineering applications due to its biocompatibility. Electrospun PCL has been used to generate engineered cardiac, tracheal, skeletal, integumentary, and vascular tissues. An electrospun PCL scaffold can support the 10 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) attachment and proliferation of MSCs, which have immunomodulatory properties that help restore homeostasis, repair damage, and regulate cellular proliferation following vascular injury. The periadventitial injection of human adipose tissue–derived MSCs into the outflow vein of the AVF in immunodeficient B6.Cg-Foxn1nu / J mice can significantly reduce the expression of known NIH mediators, such as monocyte chemotactic protein 1 (62%, P = 0.029) and hypoxia-inducible factor 1 alpha (62%, P = 0.0005) after 21 days. This strategy also significantly affected the outflow vein via increased mean luminal area (415%, P = 0.011), reduced mean neointimal area (77%, P = 0.013), and reduced neointimal cell density (83%, P < 0.0001).The use of an electrospun polymer scaffold, which provides mechanical support, and MSCs seeded onto the scaffold may synergistically act to reduce to inhibit NIH.

[0072] Ultrasonography (US) is used clinically to assess parameters that predict AVF maturation, such as luminal diameter and flow rate. However, with US, the anatomic and physiologic variables may not correlate with underlying pathophysiology.18F-FDG PET can be used to evaluate for sources of increased 18F-FDG uptake, such as inflammation, infection, and malignancy. In 18F-FDG PET, inflammation is detected when infiltrating inflammatory cells avidly take up the injected radiolabeled glucose, making it a widely used modality in identifying and monitoring inflammatory disorders. A recent case report showed that 18F-FDG PET was successfully used to identify high 18F-FDG uptake at a patient’s suspected AVF infection. However, the utility of 18F-FDG PET for measuring inflammation at the AVF anastomosis has not been assessed. Systems and methods in accordance with the present disclosure can use 18F-FDG PET to evaluate AVF inflammation, such as to provide for AVF patency support and monitoring.

[0073] As described further herein, in various implementations, the utilization of MSCs seeding can enhance the efficacy of perivascular scaffolds to minimize pathologic NIH. Furthermore, the use of US and 18F-FDG PET can identify early inflammatory changes following AVF formation, which can act as a non-invasive marker to assess patients at risk for pathologic inflammation and, therefore, at risk of AVF maturation failure. Further, infusion of metallic nanoparticles can allow for in-vivo monitoring via X-ray imaging techniques (e.g., mCT) of the placement and integrity over time of an MSC-seeded polymeric wrap while preserving the positive effects of the wrap on AVF maturation. MSCs can promote outward vascular remodeling in the setting of AVF maturation.

[0074] Referring generally to FIGS.1-47, various devices, systems, and methods 11 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) described herein relate to medical polymer (or polymeric) scaffolds, and methods of forming and using the same, for treating vascular structures in a subject. For example, the present implementations can include therapeutics-loaded resorbable polymer scaffolds that can be applied as tissue (e.g., perivascular) wraps near AVFs, which have been known to deteriorate over time due to NIH, for example. For implementations in which one or more therapeutic agents (e.g., cells, drug particles, metallic nanoparticles, etc.) are included, the polymer scaffold may also delay or prevent the development of abnormalities, such as NIH, near a targeted treatment area, such as an AVF, in the subject.

[0075] Referring to FIG.1, the present implementations provide a resorbable and / or bioresorbable polymer scaffold 100 that includes a porous component 110 (e.g., porous member, porous structure, etc.) and a therapeutic component 120 (e.g., therapeutic material, therapeutic agent, etc.) at least partially embedded or dispersed in the porous component 110. The porous component 110 includes a three-dimensional network of polymer fibers 112 embedded with pores 114. The porous component 110 can be defined by various parameters such as a diameter of the polymer fibers 112, porosity (i.e., volume fraction of the pores 114), and size (e.g., diameter) of the pores 114.

[0076] As described herein, a resorbable and / or resorbable material (e.g., the polymer fibers 112) generally refers to a material that can be dissolved and / or absorbed in a biological environment, such as by one or more types of bodily fluids. A medical device or structure comprised of a resorbable and / or bioresorbable material can be deployed (e.g., implanted) at a target area for treatment and gradually dissolve and / or be absorbed. In some instances, the period of dissolution and / or absorption may be adjusted based on the intended functions of the resorbable material and / or any therapeutic components (e.g., drug particles, etc.) dispersed therein.

[0077] The porous structure 110 can dissolve over time after being implanted at a targeted treatment area in a subject. In some implementations, the targeted treatment area may be a vascular structure. In some implementations, the targeted treatment area may be a vascular structure coupled to an AVF, such as an outflow vein of the AVF. In this regard, the polymer scaffold 100 can be a vascular wrap that circumferentially surrounds a portion of the outflow vein near the AVF. In this regard, the polymer scaffold 100 may be alternatively referred to as a perivascular wrap. Other applications of the polymer scaffold 100, such as a vascular graft or a vascular stent, implanted at different targeted treatment areas are also within the scope of the present disclosure. 12 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0078] The polymer fibers 112 can include at least one polymer configured to be resorbable at the targeted treatment area. As permanent intravascular medical devices (e.g., vascular wraps, grafts, or stents) have been associated with thrombosis and vessel stenosis, eventual resorption of the polymer scaffold 100 reduces, minimizes, or prevents the potential long-term deleterious outcomes associated with the placement of permanent intravascular medical devices.

[0079] In some implementations, the polymer fibers 112 include at least one of polycaprolactone (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), polydioxanone (PDO), or other resorbable polymers. In some implementations, the polymer fibers 113 consists essentially of PCL. In some implementations, the polymer fibers 112 include a homopolymer (i.e., each polymer chain having a single type of polymer described herein). In some implementations, the polymer fibers 112 include a copolymer (i.e., each polymer chain having segments of two or more types of polymers described herein). In some implementations, the polymer fibers 112 include a blended composition of two or more types of the polymers described herein. In one example implementation, the polymer fibers 112 include at least PCL. In some instances, PCL may be resorbed within two to three years, although the present implementations are not limited as such. In another example implementation, the polymer fibers 112 include PCL blended with PEG.

[0080] The composition of the polymer fibers 112 can be such that the polymer scaffold 100 exhibits a composite physiologic and / or mechanical property suitable for the intended applications of the polymer scaffold 100. The polymer scaffold 100 can have an elastic modulus (e.g., Young’s modulus) or ultimate tensile strength (UTS) that is at least comparable to that of the tissue at the targeted treatment area (e.g., the vascular structure). In an example implementation, the polymer scaffold 100 exhibits a UTS of at least 5 MPa, which is the upper limit of average tensile strength of naturally occurring blood vessels. In this regard, the polymer scaffold 100 can be used to mechanically support the AVF, which may be surgically created by creating an anastomosis from a vein to an artery in subjects undergoing HD.

[0081] In some implementations, the polymer fibers 112 may be aligned in a certain direction within the porous structure 110 to induce greater mechanical strength and stability as well as to promote proliferation of cells (e.g., the therapeutic component 120) along the direction of the vascular structure. In some implementations, the polymer fibers 112 may be 13 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) braided along a certain direction to form the porous component 110.

[0082] In some implementations, the therapeutic component 120 is dispersed or embedded throughout the porous component 110, such as within the pores 114 and / or attached to the polymer fibers 112. In this regard, the composition of the polymer fibers 112 allows for the therapeutic component 120 to be attached to the porous component 110 and then gradually released (or eluted) from the porous component 110 of the polymer scaffold 100, thereby providing treatment and / or promoting healing at the targeted treatment area.

[0083] The therapeutic component 120 can include at least one of mesenchymal stem cell(s) 126 (MSCs), drug particle(s) 124, metallic nanoparticle(s)f 122, or other suitable agents. The MSCs 126 can release cellular products that reduces (or prevents) occurrence of abnormal (e.g., inflammatory) tissue growth processes associated with the creation of AVF. In some instances, the cellular products of the MSCs 126 can promote luminal expansion of the outflow vein of the AVF.

[0084] In some implementations, the drug particles 124 include at least one of an anti- inflammatory drug, an anti-thrombosis drug, or other suitable drugs. In some implementations, the drug particles 124 are capable of treating or reducing NIH at an AVF. In one example implementation, the drug particles 124 include rosuvastatin (ROSU). In another example implementation, the drug particles 124 include rapamycin (RAPA). In yet another example implementation, the drug particles 124 include at least one of diclofenac, clopidogrel, dipyridamole, or exosomes.

[0085] In some implementations, the metallic nanoparticles 122 include any metal(s) exhibiting suitable properties such as biocompatibility and radiopacity. In some implementations, the metallic nanoparticles 122 include at least one radiopaque element. For example, the metallic nanoparticles 122 may include at least one of gold, bismuth, gadolinium, tungsten, ytterbium, barium, tantalum, or zirconium. In an example implementation, the metallic nanoparticles 122 include gold nanoparticles (AuNPs). In another example implementation, the metallic nanoparticles 122 include bismuth nanoparticles (BiNPs). The radiopacity of the metallic nanoparticles 122 allows in-vivo monitoring of the state of the polymer scaffold 100 (e.g., the degree of resorption or dissolution) and its therapeutic effect on the targeted treatment area throughout the duration of the implantation (e.g., from the creation to the maturation of an AVF).

[0086] In an example implementation, the polymer scaffold 100 includes the AuNPs 14 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) (e.g., the metallic nanoparticles 122) embedded in the porous component 110, which is further seeded (or embedded) with the MSCs 126. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110, which is further embedded with ROSU (e.g., the drug particles 124). The polymer scaffold 100 can include ROSU embedded in the porous component 110. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110. The polymer scaffold 100 can include the MSCs 126 seeded in the porous component 110. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110, which is further embedded with ROSU.

[0087] In another example implementation, the polymer scaffold 100 includes the BiNPs (e.g., the metallic nanoparticles 122)-embedded in the porous component 110, which is further seeded (or embedded) with the MSCs 126. The polymer scaffold 100 can include the BiNPs -embedded in the porous component 110, which is further embedded with ROSU (e.g., the drug particles 124). The polymer scaffold 100 can include ROSU embedded in the porous component 110. The polymer scaffold 100 can include the BiNPs embedded in the porous component 110. The polymer scaffold 100 can include the MSCs 126 seeded in the porous component 110. The polymer scaffold 100 can include the BiNPs -embedded in the porous component 110, which is further embedded with ROSU.

[0088] In an example implementation, the polymer scaffold 100 includes the AuNPs (e.g., the metallic nanoparticles 122)-embedded in the porous component 110, which is further seeded (or embedded) with the MSCs 126. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110, which is further embedded with RAPA (e.g., the drug particles 124). The polymer scaffold 100 can include RAPA embedded in the porous component 110. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110. The polymer scaffold 100 can include the MSCs 126 seeded in the porous component 110. The polymer scaffold 100 can include the AuNPs-embedded in the porous component 110, which is further embedded with RAPA.

[0089] In another example implementation, the polymer scaffold 100 includes the BiNPs (e.g., the metallic nanoparticles 122)-embedded in the porous component 110, which is further seeded (or embedded) with the MSCs 126. The polymer scaffold 100 can include the BiNPs -embedded in the porous component 110, which is further embedded with RAPA (e.g., the drug particles 124). The polymer scaffold 100 can include RAPA embedded in the porous component 110. The polymer scaffold 100 can include the BiNPs embedded in the porous component 110. The polymer scaffold 100 can include the MSCs 126 seeded in the 15 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) porous component 110. The polymer scaffold 100 can include the BiNPs -embedded in the porous component 110, which is further embedded with RAPA.

[0090] Incorporating therapeutic component 120 into the porous component 110 provides various advantages for improving the maturation of AVF. For example, by seeding the porous component 110 with the MSCs 126, the polymer scaffold 100 can more closely mimic the three-dimensional architecture of the vascular extracellular matrix and provide an untapped potential for the local delivery of cell therapies that inhibit occurrences of NIH. In various implementations, MSC-seeded polymer scaffold 100 promotes greater luminal expansion and potentially reduce inflammatory process related to NIH. The polymer scaffold 100 provided herein can be configured to support attachment of the MSCs 126 onto the porous component 110 and facilitate the gradual elution of the MSCs 126 at a target treatment area, such as at the AVF’s outflow vein. Additionally or alternatively, incorporating metallic nanoparticles 122, including AuNPs and / or BiNPs, for example, into the porous component 110 can enhance radiopacity of the polymer scaffold 100, thereby allowing in-vivo monitoring of the placement and degradation of the polymer scaffold 100 (e.g., seeded with the MSCs 126) over time using imaging techniques such as mCT. The benefits of incorporating the MSCs 126 include at least its extensive immunomodulatory and regenerative properties. The incorporation of the MSCs 126 onto the polymer scaffold 100 would improve the controlled release of the MSCs 126, which would enhance the therapeutic effect of the polymer scaffold 100 on AVF maturation. The presence of radiopaque metallic nanoparticles 122 onto the polymer scaffold 100 and the MSCs 126 is not compromised. Further still, incorporating the drug particles 124, including ROSU and / or RAPA, in the polymer scaffold 100 allows for controlled release of the drug particles 124, which can further reduce AVF failure and improve the imaging and histologic parameters of AVF maturation. Accordingly, when combined with the MSCs 126 and / or the metallic nanoparticles 122, the overall efficacy of the polymer scaffold 100 in inhibiting NIH and enhancing AVF maturation can be improved.

[0091] FIG.2 shows a flow diagram of a method 200 for forming the polymer scaffold 100. It should be appreciated that the method 200 needs not be performed in the order shown. Further, various operations can be omitted and additional operations can be included. The method 200 or one or more operations thereof can be performed in various combinations of sequenced, continuous, and / or batch processes.

[0092] At operation 210, a polymer (or a blend of polymers) is dissolved in a solvent to 16 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) form a polymer solution. Composition of the polymer has been described in detail above.

[0093] At operation 220, the therapeutic component 120 is blended or infused into the polymer solution to form a blended polymer solution. In some implementations, the drug particles 124 and the metallic nanoparticles 122 are blended into the polymer solution at operation 220.

[0094] At operation 230, the blended polymer solution is loaded into an electrospinning apparatus and spun into the polymer scaffold 100 including the therapeutic component 120 dispersed or embedded in the porous component 110, which includes a network of the polymer fibers 112. An example apparatus 710 used for implementing the electrospinning process is illustrated in FIG.7. Parameters of the electrospinning process may be adjusted to control the diameter, alignment, and morphology of the polymer fibers 112, as well as the size of the pores 114, which determines the porosity of the porous component 110. For example, a voltage applied at a needle tip of a syringe dispensing the polymer solution may be adjusted to achieve a certain alignment or directionality of the electrospun polymer fibers 112 in the polymer scaffold. For implementations in which the therapeutic component 120 includes the MSCs 126, the polymer scaffold 100 is formed (e.g., electrospun) first before performing additional operation(s) to seed the MSCs 126. Other methods of forming the three-dimensional porous component 110 (loaded with the therapeutic component 120) from a polymer solution are also within the scope of the present disclosure. For example, the polymer solution may be cast into a mold having a suitable shape.

[0095] At operation 240, the therapeutic component 120 is incorporated in the polymer scaffold 100. In some implementations, the MSCs 126 are seeded in the polymer scaffold 100 at operation 240, where the polymer scaffold 100 includes at least one of the drug particles 124 and the metallic nanoparticles 122 that has been blended into the polymer solution at operation 220 and subsequently electrospun into the polymer fibers 112 at operation 230. In some implementations, the MSCs 126 are seeded in the polymer scaffold 100 at operation 240, where the polymer scaffold 100 does not include the drug particles 124 or the metallic nanoparticles 122.

[0096] In some implementations, instead of proceeding from operation 210 to operation 220 during which the therapeutic component 120 is blended into the polymer solution, the method 200 proceeds from operation 210 to operation 230 during which the polymer solution is first electrospun to form the polymer fibers 112 constituting the porous 17 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) component 110 of the polymer scaffold 100. Thereafter, at operation 240, the therapeutic component 120 including at least one of the metallic nanoparticles 122, the drug particles 124, or the MSCs 126 is incorporated (e.g., seeded, injected, inserted, etc.) in the porous component 110 of the polymer scaffold 100.

[0097] Although the polymer scaffold 100 shown in FIG.1 has a sheet-like structure, the present implementations are not limited as such. For example, the polymer scaffold 100 may be formed (e.g., electrospun, cast, etc.) to have a tubular structure 300A, as shown in FIG.3A, or a rod structure 300B, as shown in FIG.3B. As shown in FIG.4A, the tubular structure 300A has an annular (i.e., ring-shaped) cross-sectional area when viewed along line AA’ of FIG.3A. As shown in FIG.4B, the rod structure 300B has a circular cross- sectional area when viewed along line BB’ of FIG.3B. The polymer scaffold 100 may be formed into any size, shape, or structure suitable for its intended applications.

[0098] For example, referring to FIGS.4A and 4B, the tubular structure 300A is defined to have an inner diameter D1 and an outer diameter D2 that is greater than D1, and the rod structure 300B is defined to have a diameter D3. For implementations in which the polymer scaffold 100 is applied as a vascular (e.g., perivascular) wrap, the inner diameter D1 can be greater than an outer diameter of the vascular structure around which the polymer scaffold is wrapped. For implementations in which the polymer scaffold 100 is applied as a vascular graft, which extends between two ends of the vascular structure, the outer diameter D1 can be comparable to the outer diameter of portions of a vascular structure to be grafted. For implementations in which the polymer scaffold 100 is applied as an intravascular stent, which is inserted into a lumen of the vascular structure, the diameter D3 can be less than an inner diameter of a vascular structure (i.e., a diameter of a lumen of a blood vessel surrounded by its intima).

[0099] Furthermore, in some implementations, the polymer fibers 112 are oriented along, or substantially along, a longitudinal direction L of the tubular structure 300A and the rod structure 300B, which corresponds to a lengthwise direction of the vascular structure to which the polymer scaffold 100 is coupled. For implementations in which the polymer scaffold 100 is seeded with the MSCs 126, the longitudinal direction L corresponds to the direction of the proliferation of the MSCs 126.

[0100] FIG.5 shows a flow diagram of a method 500 for using the polymer scaffold 100. It should be appreciated that the method 500 needs not be performed in the order 18 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) shown. Further, various operations can be omitted and additional operations can be included.

[0101] At operation 510, the polymer scaffold 100 is provided or formed according to implementations of the method 200 described above.

[0102] At operation 520, the polymer scaffold 100 is deployed at the targeted treatment area, such as a vascular structure, in the subject. In some implementations, the polymer scaffold 100 is sterilized before being deployed at the targeted treatment area. In some implementations, deploying the polymer scaffold 100 includes wrapping the polymer scaffold 100 around a vein of an AVF. In some implementations, deploying the polymer scaffold 100 includes coupling or grafting the polymer scaffold 100 to at least one open end of a vascular structure. In some implementations, deploying the polymer scaffold 100 includes inserting the polymer scaffold 100 as a stent into a lumen of a vascular structure.

[0103] FIG.6 shows a flow diagram of a method 600 for using the polymer scaffold 100 for treating an AVF. It should be appreciated that the method 600 needs not be performed in the order shown. Further, various operations can be omitted and additional operations can be included.

[0104] At operation 610, the polymer scaffold 100 is provided or formed according to implementations of the method 200 described above. In some implementations, the polymer scaffold 100 is sterilized before being deployed at the targeted treatment area.

[0105] At operation 620, an AVF is surgically formed in the subject by coupling an artery with a vein.

[0106] At operation 630, the (sterilized) polymer scaffold 100 is deployed as a vascular wrap (or perivascular wrap) by wrapping the polymer scaffold 100 around the vein (i.e., the outflow vein) of the AVF. In some implementations, the polymer scaffold 100 is deployed at the same surgery during which the AVF is created in the subject. For example, the (sterilized) polymer scaffold 100 may be wrapped around the outflow vein of the AVF after forming the AVF and achieving hemostasis at the surgery. In many implementations, deploying the polymer scaffold 100 loaded with the therapeutic component 120 (e.g., the MSCs 126, the drug particles 124, and / or the metallic nanoparticles 122) at the time of surgery would allow for the unmet clinical need of mitigating or preventing NIH in subjects undergoing HD and needing an AVF surgery.

[0107] FIG.7 is a schematic illustration of fabricating and deploying an example 19 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) implementation of the polymer scaffold 100 at an AVF 720 created in a chronic kidney disease (CKD) model using a rat subject 730.

[0108] The polymer scaffold 100 is deployed as a vascular wrap surrounding a vein 740 (e.g., a wrapped outflow vein) of the AVF 720, according to some implementations. The wrapped outflow vein 740 includes a lumen 742 surrounded by, in this order, an intima 744, a media 746, and an adventitia 748, where the polymer scaffold 100 including the AuNPs (i.e., the metallic nanoparticles 122), the ROSU (i.e., the drug particles 124), and / or the MSCs 126 embedded in the PCL porous structure 110 is sandwiched between the adventitia 748 and the media 746. For purposes of comparison, a control sample 740C is also illustrated. As shown, the control sample 740C demonstrates NIH and luminal stenosis with immune cells present in the media 746, while the wrapped vein 740 does not show signs of NIH and luminal stenosis.

[0109] Examples of various implementations of the present disclosure are provided below.

[0110] EXAMPLE 1

[0111] Overview

[0112] The MSC-seeded (or MSC-loaded) perivascular scaffold was tested in a CKD rat model of an AVF, which showed an increased luminal diameter and flow rate relative to control CKD rats, as evidenced on ultrasound. Decreased uptake of 18F-FDG on PET / CT imaging was indicated, which may reflect decreased rates of inflammation as well as reduced neointima-to-lumen and neointima-to-media ratios, which may reflect reduced NIH. Histology of the test and control CKD rats confirmed increased lumen size and decreased NIH formation in CKD rats with an AVF treated with an MSC-seeded perivascular scaffold. The addition of a perivascular MSC-seeded scaffold increased the luminal area of the AVF while simultaneously decreasing the intima:lumen ratio and the intima:media ratio by 88.3% and 87.8%, respectively. These results demonstrate the utility of a perivascular scaffold seeded with MSCs at the outflow vein immediately after AVF formation to support AVF maturation by minimizing NIH.

[0113] The rosuvastatin- loaded perivascular scaffold was tested in a CKD rat model of an AVF. At week 8 following AVF formation, CKD rats treated with the rosuvastatin- loaded perivascular scaffold were found to have a significantly lower wall thickness:luminal diameter ratio compared to the AVF of CKD rats with no scaffold or scaffold without 20 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) rosuvastatin (P< 0.0001).

[0114] Anti-inflammatory drug attachment to resorbable polymer(s): A solution of 8:3 toluene:methylene hydroxide was mixed with PCL. Rosuvastatin (potent anti-inflammatory drug in high concentrations) was incorporated into the mixture at 40 mg / mL. The PCL- rosuvastatin blend was electrospun at 1.0 mL / h and 15 kV. The rosuvastatin-loaded sample was then tested in vitro for drug-release. The rosuvastatin release rate exhibited an initial burst during week 1 that tapered gradually over weeks 2 to 12 for a total cumulative rosuvastatin release of 40.7% at by week 12. The rosuvastatin-loaded polymer scaffold was tested in two specific cell lines (e.g., immortalized human vascular endothelial cells (EC- RF24) and mouse vascular smooth muscle cells (MOVAS)) for purposes of toxicity. No significant difference in cellular viability was detected in control EC-RF24 and MOVAS cell lines without polymer or drug exposure and EC-RF24 and MOVAS cell lines exposed to rosuvastatin-containing PCL (P>0.05).

[0115] In some implementations, a bioresorbable electrospun perivascular polymer scaffold can provide mechanical strength to a maturing AVF and to support the attachment and proliferation of Sprague-Dawley rat MSCs. The polymer concentration and electrospinning voltage can be optimized to produce scaffolds with aligned fibers, as MSC- seeded scaffolds with highly aligned fibers have greater mechanical stability and yield higher degrees of cell proliferation compared to scaffolds with random orientation. The scaffold also featured properties, such as fiber diameter, pore size, and porosity, that are comparable to those found in the endogenous human extracellular matrix. The constructed polymer can be highly suitable medium for the delivery of cell-based therapy.

[0116] Fabrication and Characterization of a Polymer Scaffold

[0117] The polymer (e.g., perivascular polymer) scaffolds were fabricated from PCL having an average molecular weight Mn of about 80,000 g / mol (Sigma-Aldrich, St. Louis, MO) using a Spraybase electrospinning system (Avectas, Maynooth, Ireland). Concentration of the PCL polymer in the polymer solution was about 20%. The polymer was dissolved in chloroform / methanol (8:3) to form a sample polymer solution, and each sample polymer solution was pumped through a syringe at a constant rate of 1.0 mL / h to the 20-gauge blunt-end needle tip, resulting in a sample scaffold (e.g., polymer scaffold 100 of FIG.1). The distance between a needle tip of the syringe and an underlying mandrel collector was about 15 cm. Concentration of the polymer solution and a voltage applied 21 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) during electrospinning were optimized to produce aligned fibers that would facilitate cell- scaffold interaction and directional cell (e.g., MSCs) proliferation.

[0118] Fiber alignment, morphology, diameter, and pore size of each sample scaffold (e.g., scaffold, electrospun scaffold, electrospun PCL scaffold, etc.) were determined using a Nova NanoSEM scanning electron microscope (Field Electron and Ion Company, Hillsboro, OR), where the sample was observed to include a porous structure containing a network of polymer fibers (or “fibers” for short). At least 100 segments of fibers and pore areas were randomly chosen and measured per sample using ImageJ (National Institutes of Health, Bethesda, MD). The porosity of the sample scaffold was determined using a calculation method and a liquid intrusion method. A melting temperature (Tm) and glass transition temperature (Tg) of the electrospun scaffolds were determined using an STA PT 1000 thermogravimetric analyzer (Linseis, Selb, Germany). The sample scaffolds were formed into a tubular scaffold (e.g., polymer scaffold 300A of FIGS.3A and 4A) or a rod scaffold (e.g., polymer scaffold 300B of FIGS.3B and 4B). An ultimate tensile strength of the scaffolds (e.g., the tubular scaffolds) was determined using an MTESTQuattro materials testing system (ADMET, Norwood, MA) in ambient conditions. After two and four weeks of incubation in phosphate-buffered solution (PBS) at 37 °C, the scaffolds were reevaluated to determine the change in their ultimate tensile strength over time.

[0119] FIG.8 demonstrates the representative morphology, diameter, and pore size of the sample scaffold, which was produced using a 20% PCL solution at 15 kV. Specifically, chart a is an image of the fibers of the electrospun scaffold captured by scanning electron microscopy, demonstrating good alignment of the fibers (bar = 50 μm, 1500x magnification). Charts b and c are histograms plotting frequency distribution of the fiber diameters and scaffold pore sizes. Chart d is a bar graph showing the gradual reduction in the tensile strength of the scaffold over 4 weeks.

[0120] The physicochemical properties of the sample scaffold are summarized in Table 1. The mean diameter of the fibers (2.42 ± 1.31 μm) fell within the range of the typical diameter of endogenous human collagen, which is from about 1 μm to about 20 μm. The sample scaffold had adequate porosity—a property that promotes cell-surface interactions, nutrient and gas exchange, and cell proliferation—with a mean pore size of 22.51 ± 14.66 μm2, which can facilitate attachment of MSCs. Despite its porous architecture, the scaffold maintained a high level of tensile strength over four weeks, starting at 9.38 ± 2.04 MPa from the baseline to 6.98 ± 2.53 MPa after four weeks of incubation with the MSCs. 22 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) Table 1. Physicochemical properties of an electrospun polymer (e.g., PCL) scaffold. Properties PCL Scaffold Diameter (μm) 2.42 ± 1.31 Pore size (μm2) 22.51 ± 14.66 Porosity (calculated, %) 70.36 ± 4.90 Porosity (intrusion, %) 83.96 ± 1.61 Tg(°C) -65.64 ± 1.89 Tm (°C) 55.73 ± 0.77 Maximum stress (MPa) 9.38 ± 2.04

[0121] Cell Culture and MSC Attachment

[0122] Bone-marrow-derived Sprague-Dawley rat mesenchymal stem cells (MSCs) that express red fluorescent protein (RFP-MSCs) (Catalog no. CSC-C1315, Creative Bioarray, Shirley, NY) were cultured using DMEM with 10% FBS and 1% penicillin-streptomycin (Corning, Corning, NY). The scaffolds were subjected to ethylene oxide gas sterilization and then seeded with 1 x 105 RFP-MSCs at 3rd passage. After 12 hours, the samples were washed with phosphate-buffered saline (PBS) to remove any unattached cells. For in vitro visualization of attachment, the scaffolds were imaged using an Eclipse Ti2 fluorescence microscope (Nikon, Melville, NY), and the number of attached cells was quantified using NIS-Elements BR analysis software (Nikon). For in vivo efficacy studies, cells were kept in culture media for a maximum of 24 hours prior to surgical application.

[0123] It has been shown that an electrospun PCL scaffold can support the attachment of human GFP-expressing MSCs, with the number of viable cells attached to the scaffold increasing as much as seven-fold after one week of incubation. In this study, fluorescence microscopy similarly revealed that the seeded Sprague-Dawley rat RFP-MSCs attached to the electrospun PCL scaffold, as shown in FIG.9. Specifically, image a shows a control sample containing MSCs cultured on a polystyrene plate and image b shows Sprague- Dawley rat RFP-MSCs loaded onto electrospun PCL scaffold after 12 hours of incubation and washing (bars = 100 μm, 10x magnification).

[0124] It was discovered that higher degrees of uniform fibril alignment had a positive correlation with MSC attachment. In vitro, using the PCL scaffolds as an example, it was noticed that the alignment of metallic nanoparticles resorbable polymer fibers was 23 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) maximized via electrospinning at 15 kV. The PCL scaffolds were subsequently sterilized by incubation in 70% ethanol for 30 minutes followed by two sequential 15 minutes washes in PBS and seeded with 5 x 104 mCherry-expressing human MSCs per side and incubated at 37°C for 30 minutes to allow the MSCs to attach before the addition of culture media. After 48 hours of incubation, the MSC-seeded PCL scaffolds, now the PCL-MSC scaffolds, were imaged under a fluorescence microscope confirming the presence of MSC attachment. The PCL-MSC scaffold was shown to contain MSCs along a majority of the scaffold area with MSCs situated along the orientation of the PCL fibers.

[0125] Formation of an AVF and Application of the Scaffold as a Perivascular Wrap

[0126] All rat experiments were approved by The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (Protocol No: 1882-RN01). A chronic kidney disease (CKD) model was created using Sprague-Dawley rats based on a two-step 5 / 6th nephrectomy procedure known in the art. The first surgery involved the removal of both poles of a left kidney in a rat (e.g., a subject). The second surgery, performed seven days after the first surgery to ensure rat survival, involved the removal of the entire right kidney. Creatinine and blood urea nitrogen (BUN) were obtained after the second procedure to confirm the decline in kidney function. An AVF model was then created via surgical anastomosis of the external jugular vein (EJV) with the common carotid artery (CCA) in an end-to-side fashion based on a modified procedure known in the art. AVFs were created in non-CKD rats and CKD rats four weeks after subtotal nephrectomy. In subsequent experiments, CKD rats were divided into three groups: control (i.e., no PCL scaffold), PCL scaffold alone, and PCL scaffold seeded with MSCs (e.g., the PCL+MSC). In the PCL scaffold groups, following the AVF formation and hemostasis, the sterilized polymer scaffold (1 mm x 5 mm) was wrapped around the outflow vein (e.g., the vein) coupled to the AVF. The PCL+MSC group received a scaffold seeded with 1 x 105MSCs.

[0127] Ultrasonography (US),18F-fluorodeoxyglucose Positron Emission Tomography, and Histological Analysis

[0128] The successful creation of an AVF was confirmed by the presence of pulsatile arterial waveforms in the external jugular vein visualized using ultrasonography (US) with a Vevo 2100-LAZR system (VisualSonics, Toronto, ON, Canada). Four weeks after surgery, the rats were anesthetized and imaged. B-mode, color Doppler, and pulse wave Doppler were used to assess the wall thickness, luminal diameter, and flow rate of the AVFs. A 15- 24 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) MHz B-mode probe was used to obtain measurements of the wall thickness and luminal diameter across the outflow vein. Wall-to-lumen ratio was calculated by dividing the average wall thickness by the average luminal diameter. An 18-MHz Doppler probe was used to measure flow velocity. The flow rate was derived from the average flow velocity and average luminal diameter.

[0129] 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET; or “PET” for short) / computed tomography (CT) images were acquired in Sprague-Dawley rats at baseline (i.e., non-CKD) and 4 weeks following the two-stage surgery (i.e., CKD).18F- FDG PET / CT images were obtained using an m.CAM gamma camera (Siemens, Malvern, PA). The radioactive tracer—37 MBq of 18F-FDG in 200 μL of saline—was injected intravenously. Forty minutes after injection of the radioisotope, a 10-minute PET / CT scan was acquired of the AVF for each rat. The scans were reconstructed by a two-dimensional maximum-likelihood expectation-maximization algorithm. Default reconstruction parameters were applied. The maximum standard uptake values (SUVmax) of the AVFs were quantified.

[0130] Representative images of the surgical procedures (e.g., 5 / 6thnephrectomy) are shown in FIG.10. Images a show the two-stage surgery to induce CKD in Sprague-Dawley rats. In step one (left image), the upper and lower poles of the left kidney are removed. In step two (right image), the right nephrectomy was performed one week later. Chart b shows that, on a two-tailed t test, significantly increased serum creatinine and blood urea nitrogen (BUN) levels were observed in CKD rats (n = 5) versus control rats (n = 3) four weeks following the two-stage surgery. Images c and d show that Surgical creation of AVFs via end-to-side anastomosis of the external jugular vein with the common carotid artery.

[0131] Impaired kidney function in the Sprague-Dawley rats was confirmed by increased serum creatinine and BUN. Four weeks after the second step of the two-stage surgery (i.e., right nephrectomy), the mean serum creatinine level of the CKD rats (n = 6, 0.83 ± 0.09 mg dL-1) was 1.41 times higher than that of control rats (n = 3, 0.59 ± 0.13 mg dL-1, P < 0.05), and the mean BUN level of the CKD rats (n = 6, 49.28 ± 6.99 mg dL-1) was 1.54 times higher than that of the control rats (n = 3, 32.03 ± 2.67 mg dL-1, P < 0.002).

[0132] FIG.11 compares the AVFs in non-CKD and CKD rats after AVF creation. At four weeks, US measurements revealed that the mean wall thickness of the AVFs of the CKD rats (n = 6, 0.54 ± 0.14 mm) was 1.93 times higher than that of non-CKD rats (n = 6, 25 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) 0.28 ± 0.11 mm, P < 0.002), while the mean luminal diameter of the AVFs of the CKD rats (n = 6, 0.74 ± 0.07 mm) was significantly lower than that of non-CKD rats (n = 6, 0.88 ± 0.09 mm, P < 0.05). Consequently, a wall-to-lumen ratio of the AVFs of the CKD rats (n = 6, 0.75 ± 0.22 mm) was 2.27 times higher than that of non-CKD rats (n = 6, 0.33 ± 0.14 mm, P < 0.002).

[0133] Specifically, images and chart a shows B-mode and color Doppler US images of the AVFs in non-CKD (i.e., control) and CKD rats. Compared to non-CKD rats (n = 6), CKD rats (n = 6) had significantly higher mean wall thickness, lower luminal diameter, and higher wall-to-lumen ratio using a two-tailed t test (A, artery; L, lumen; V, vein; W, wall; bars = 1 mm). Images and chart b shows 18F-FDG PET images demonstrated significantly higher SUVmaxvalues at the outflow vein in CKD rats (n = 4) compared to non-CKD rats (n = 4) using a two-tailed t test. On histomorphometric analysis, referring to images and chart c, using a two-tailed t test, neointima-to-lumen and neointima-to-media ratios were higher for CKD rats than for non-CKD rats (bars = 100 μm, 20x magnification; I, intima; L, lumen; M, media).

[0134] For histological analysis, rats were euthanized by CO2 asphyxiation and thoracotomy after imaging. The outflow veins were harvested after perfusion for epifluorescence visualization and paraffin embedding. To monitor the retention of RFP- MSCs at two and four weeks against perivascular injection of MSCs, the harvested vessels were fixed in 10% formaldehyde for 15 minutes, stained with 4′,6-diamidino-2- phenylindole (DAPI), a nuclear stain, and viewed using an Eclipse Ti2 fluorescence microscope (Nikon, Melville, NY). Paraffin-embedded samples were sliced, and the sections were divided for hematoxylin and eosin (H&E) and immunofluorescence multiplex staining with the following markers: (1) CD45, highly expressed in leukocytes and a marker of inflammatory infiltration; (2) alpha-smooth muscle actin (α-SMA), highly expressed in smooth muscle cells but also expressed in fibroblasts; (3) vimentin, highly expressed in fibroblasts but also expressed in vascular smooth muscle cells and lymphocytes; and (4) DAPI. An Aperio LV1 real-time digital pathology system (Leica Biosystems, Buffalo Grove, IL) was used to capture images from the H&E slides and quantify the areas covered by the lumen, neointima, and media. Blood artifacts within the lumen from incomplete tissue processing were excluded from the analysis. Histologic ratios were derived from the measured areas (Figure S2). A Leica Versa fluorescent system (Leica Biosystems) was used to scan and capture images from the immunofluorescence slides, while the Halo image 26 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) analysis platform (Indica Labs, Albuquerque, NM) was used to perform fluorescence cell counting.

[0135] In addition, on the histomorphometric analysis, the AVFs of the CKD rats had a significantly higher neointima-to-lumen ratio, a measure of luminal stenosis, and a higher neointima-to-media ratio, a measure of wall thickening. The neointima-to-lumen ratio of the AVFs of the CKD rats (n = 3, 1.85 ± 0.08) was 2.89 times higher than that of non-CKD rats (n = 3, 0.64 ± 0.07, P < 0.0001). The neointima-to-media ratio of the AVFs of the CKD rats (n = 3, 3.08 ± 0.05) was 6.84 times higher than that of non-CKD rats (n = 3, 0.45 ± 0.07, P < 0.0002). Moreover, when the inflammatory response was measured via 18F-FDG PET imaging, the AVFs of the CKD rats had significantly higher 18F-FDG uptake as assessed by SUVmax (n = 4, 5.80 ± 0.87) compared to that of non-CKD rats (n = 4, 1.18 ± 0.51, P < 0.05). Therefore, in the succeeding experiments, CKD rats were used exclusively to determine the efficacy of the MSC-loaded scaffolds.

[0136] Characterization of the PCL-Based Scaffold as a Perivascular Wrap around the AVF

[0137] FIG.12 compares the B-mode and color Doppler ultrasonographic findings for AVFs in CKD Sprague-Dawley rats at four weeks following AVF creation and placement of no PCL perivascular wrap (i.e., control rats, n = 12), PCL wrap alone (n = 12), and PCL+MSC wrap (n = 12). The addition of a PCL wrap alone, when compared with control rats, resulted in significantly increased luminal diameter (1.25 ± 0.11 mm versus 0.82 ± 0.10 mm, P < 0.0001), reduced wall-to-lumen ratio (0.40 ± 0.05 versus 0.69 ± 0.17, P < 0.0001), and increased flow rate (2.95 ± 0.80 versus 1.32 ± 0.99 cm3 / s, P < 0.05). Likewise, compared to controls, the addition of PCL+MSC wraps resulted in significantly increased luminal diameter (1.74 ± 0.29 mm, P < 0.0001), reduced wall-to-lumen ratio (0.29 ± 0.06, P < 0.0001), and increased flow rate (5.74 ± 2.36 cm3 / s, P < 0.0001). When comparing PCL alone and PCL+MSC wraps, PCL+MSC resulted in a greater improvement in luminal diameter (P < 0.0001), wall-to-lumen ratio (P < 0.05), and flow rate (P < 0.0002) compared to PCL alone, suggesting that the addition of MSCs promotes better luminal expansion and blood flow through the outflow vein.

[0138] Specifically, images a shows ultrasound images of the AVF in CKD rats with no wrap (i.e., control), PCL alone, and PCL+MSC perivascular wrap at 4 weeks (A, artery; L, lumen; V, vein; W, wall; bars = 1 mm). Charts b compare wall thickness, luminal diameter, 27 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) wall-to-lumen ratio, and flow rate for the venous outflow in CKD rats with no wrap (i.e., control, C, n = 12), PCL wrap alone (P, n = 12), and PCL+MSC (P+M, n = 12) wrap. While no significant difference was detected among the three groups with respect to wall thickness, significant increases in luminal diameter, decreases in wall-to-lumen ratio, and increases in flow rate were found when comparing C versus P, C versus P+M, and P versus P+M using one-way analysis of variance.

[0139] FIG.13 compares the axial18F-FDG PET images at the outflow vein of AVF in CKD rats findings for the AVFs of CKD rats with no wrap (i.e., control, n = 4), PCL-alone wrap (n = 3), and PCL+MSC wrap (n = 3) at four weeks after AVF creation. The perivascular application of PCL+MSC wrap resulted in significantly reduced 18F-FDG uptake compared to rats without no wrap (SUVmaxversus 3.01 ± 0.80, P < 0.05). No significant difference was detected on 18F-FDG PET imaging when comparing PCL-alone versus control CKD rats (SUVmax5.80 ± 1.55 versus 6.49 ± 1.31, P > 0.9999).

[0140] Specifically, image a shows PET images from rats with no wrap (i.e., control), PCL-alone wrap, and PCL+MSC wrap were obtained 4 weeks following surgery. The location of the fistula is marked by the white arrows. Referring to chart b, SUVmaxmeasurements show that PCL+MSC (P+M, n = 3) significantly reduced18F-FDG uptake compared to control (n = 4) and PCL alone (n = 3) using a one-way analysis of variance.

[0141] The presence of RFP-MSCs was investigated two and four weeks following AVF surgery in representative rats with no wrap (i.e., control), rats treated with PCL wrap, rats treated with a perivascular injection of RFP-MSCs (iMSC), and rats treated with PCL+MSC wrap. An AVF treated with PCL+MSC showed improved RFP-MSC retention compared to the AVF that received a perivascular injection of MSCs after 4 weeks, as seen on fluorescence microscopy (Figure 7). The iMSC group (n = 3) demonstrated 140 ± 34 cells per mm2at two weeks and 86 ± 60 cells per mm2at four weeks, while the PCL+MSC group (n = 3) showed 669 ± 112 cells per mm2at two weeks and 445 ± 117 cells per mm2at four weeks. Although both groups revealed a steep reduction in the number of MSCs per mm2, the use of PCL scaffold caused a five-fold increase in the number of MSCs retained.

[0142] FIG.14 demonstrates results of fluorescence microscopy of the harvested AVFs of representative Sprague-Dawley CKD rats. Image a includes fluorescent microscopic images demonstrating the increased retention of RFP-MSCs at two weeks and four weeks following surgery in the AVF that received PCL+MSC scaffold compared to perivascular 28 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) injection of MSCs (iMSC) (bar = 100 μm). Referring to chart b, quantification of MSCs per mm2using a two-tailed t test shows a 5-fold increase in retention with the use of PCL scaffold (n = 3) versus injection (n = 3).

[0143] FIG.15 compares the histological findings for the AVFs of CKD rats with no wrap (control, n = 3), PCL-alone wrap (n = 3), and PCL+MSC wrap (n = 3) at four weeks after AVF creation. The application of a PCL-alone wrap resulted, when compared to control rats, in a significantly reduced neointima-to-lumen ratio (0.26 ± 0.01 versus 1.85 ± 0.08, P < 0.0001) and neointima-to-media ratio (0.83 ± 0.05 versus 3.08 ± 0.47, P < 0.0002). Similarly, the application of a PCL+MSC wrap resulted in a significantly reduced neointima-to-lumen ratio (0.06 ± 0.01 versus 1.85 ± 0.08, P < 0.0001) and neointima-to- media ratio (0.17 ± 0.02 versus 3.08 ± 0.47, P < 0.0001) compared to the controls. Compared to PCL-alone wrap, PCL+MSC wrap resulted in a greater reduction in the neointima-to-lumen ratio (P < 0.002), suggesting that the addition of MSCs promotes better attenuation of luminal stenosis through the outflow vein. There was no significant difference between PCL alone and PCL+MSC treatment in regard to the neointima-to- media ratio, but the PCL+MSC treatment demonstrated a trend towards further reduction. These findings confirm not only the beneficial effect of PCL wrap alone but also the added benefit conferred by MSC seeding on the outflow vein’s wall thickness and luminal diameter, which are concordant with the findings obtained via ultrasonography.

[0144] Specifically, images a show histological results of the outflow vein of AVF in rats with no wrap (i.e., control), PCL-alone wrap, and PCL+MSC wrap (bars = 100 μm, b: blood within the lumen; N: neointima; W: wrap L: lumen). Referring to chart b, histomorphometry using a one-way analysis of variance shows that both PCL (n = 3) and PCL+MSC (n = 3) perivascular wraps resulted in a significant reduction in both neointima- to-lumen and neointima-to-media ratios relative to control (n = 3).

[0145] The presence of cells positive for CD45, α-SMA, and vimentin was also investigated four weeks following AVF surgery in a representative rat with no wrap (i.e., control), rat treated with PCL-alone wrap, and rat treated with PCL+MSC wrap. The AVFs treated with PCL and PCL+MSC revealed fewer cells that were positive for CD45, α-SMA, and vimentin (FIG.16, Table 2). The findings suggest that the scaffold treatments can potentially reduce the neointimal infiltration of inflammatory cells, smooth muscle cells, and fibroblasts. 29 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0146] FIG.16 shows images of immunofluorescence staining of the AVFs of representative Sprague-Dawley CKD rats. H&E with corresponding immunofluorescent microscopic images that demonstrate reduced CD45, α-SMA, and vimentin staining for both PCL and PCL+MSC perivascular wraps compared to no intervention (i.e., control) 4 weeks following surgery (bars = 100 μm, b: blood within the lumen; W: wrap). Table 2. Immunofluorescence staining analysis of the neointima. Marker Control PCL PCL+MSC Cell count % Cell % Cell % count count CD45 37 24.03 1 0.83 1 0.57 α-SMA 97 62.99 30 24.79 31 17.61 Vimentin 60 38.96 47 38.84 44 25.00 DAPI (Total) 154 100.00 121 100.00 176 100.00 Abbreviations: α-SMA, alpha-smooth muscle actin; CD, cluster of differentiation; DAPI, 4′,6-diamidino-2-phenylindole; PCL, polycaprolactone; MSC, mesenchymal stem cell.

[0147] It was observed that the application of a PCL scaffold alone reduced the stenosis and thickening of the venous outflow wall of an AVF, as seen on US and histological analyses. These results indicate that mechanical support alone can improve the patency of a maturing AVF, which is concordant with prior research in humans. It should be noted that the optimal duration for perivascular wrap presence prior to complete dissolution remains a subject of further research effort. One of the major pathways that underlie NIH is hemodynamic stress. Specifically, disturbed or oscillatory wall shear stress leads to endothelial activation and, subsequently, endothelial proliferation. Moreover, mechanical loads that exceed the capacity of blood vessels eventually cause vascular wall injury and activation of synthetic smooth muscle cells and fibroblasts that proliferate and migrate centrally into the intima. Hence, the addition of the polymer scaffold of the present implementations applied as a perivascular wrap, which may reduce (or prevent) the overexpansion of the vascular wall and reduce the flow turbulence within the vascular lumen, is likely to improve the patency of a maturing AVF.

[0148] It was also observed that incorporating MSCs into the PCL scaffold (i.e., PCL+MSC wrap) yielded improvements in luminal diameter, wall-to-lumen ratio, and flow rate as seen on US, and a significant reduction in 18F-FDG uptake as seen on PET images. 30 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) Moreover, the addition of MSCs to the PCL wraps resulted in a further reduction in the neointima-to-lumen and neointima-to-media ratios on H&E staining and a further reduction in the percentages of cells that were positive for CD45, α-SMA, and vimentin on immunofluorescence staining. Taken together, these findings suggest that mechanical interventions to promote AVF maturation and patency may be improved by the perivascular addition of MSCs. Given the increased potential for washout of MSCs during perivascular injection, the use of a perivascular wrap to implant MSCs, in turn, improves the efficacy of MSCs towards mitigating pathologic inflammation over time in the developing AVF.

[0149] The observed trend in the improvement of the wall-to-lumen ratio derived via US correlated with the trend in the improvement of the neointima-to-lumen ratio as seen on histomorphometry, highlighting a potential role for anatomic imaging parameters for AVF surveillance. On the other hand, only the PCL+MSC group demonstrated a significant reduction in uptake on 18F-FDG PET imaging. This phenomenon can be expected because PCL wrap alone (i.e., without MSCs) appears to lack innate immunomodulatory properties in contrast to MSCs. Although the difference between control and PCL-alone wrap is insignificant, the slightly increased uptake seen with the PCL wrap could be explained by an increase in the number and activity of inflammatory cells that take up the scaffold’s degradation products in the adventitial layer. Nevertheless, the findings described herein suggest that both PCL alone and PCL+MSC can potentially reduce the infiltration of inflammatory cells in the neointimal layer. Further investigation is warranted to characterize the cell populations within the neointima, describe the cellular and extracellular composition of the remaining layers of the vascular wall, and identify the expression pattern of NIH pathway-associated genes and proteins. Variations in these parameters could provide insight into the discrepancy between the findings seen on 18F-FDG PET and histology as well as the mechanisms that underlie the efficacy of the scaffold treatments. Nonetheless, the possibility that there may be in vivo changes associated with eventual AVF maturation or, conversely, eventual AVF failure that occurs prior to visible changes in the composition and architecture of vascular tissue cannot be ruled out. Although 18F-FDG PET is associated with time and monetary costs, PET imaging offers a potential approach to estimating degrees of local inflammation within the AVFs in vivo.

[0150] EXAMPLE 2

[0151] Fabrication and Characterization of a Polymer Scaffold 31 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0152] Methanol (ACS reagent grade, 99.5%), toluene (ACS reagent grade, ?99.9%), gold (III) chloride trihydrate (HAuCl4·3H2O, 99.9%), tetrabutylammonium bromide (TBAB, ACS reagent grade, 98.0%), 1-octanethiol (98.5%), PCL (average Mn 80,000), and sodium borohydride (NaBH4, 99%) were obtained from Sigma-Aldrich (St. Louis, MO). Rosuvastatin (ROSU) calcium salt (98%) was purchased from Acros Organics (NJ). All chemicals were used without further purification unless otherwise noted.

[0153] AuNPs were synthesized. After creating HAuCl4·3H2O in distilled water, TBAB, 1-octanethiol, and NaBH4 solution were sequentially added. PCL was dissolved in 8:3 toluene / MeOH to prepare 15% w / v solutions. For AuNP-embedded wraps, AuNP was mixed with PCL at a 1:4 ratio. For ROSU-embedded wraps, ROSU was added at 40 mg / mL. Using a Spraybase electrospinning system (Avectas, Maynooth, Ireland), all solutions were electrospun at 1.0 mL / h and 15 kV.

[0154] Physicochemical characterization is an important component of medical device development. JEM 1010 transmission electron microscopy (JEOL USA, Inc., Peabody, MA) was used to characterize the size and morphology of the AuNPs. Additional parameters, including fiber diameter, pore size, and elemental composition of the wraps, were obtained using a Nova NanoSEM scanning electron microscope (SEM; Field Electron and Ion Company, Hillsboro, OR) with an EDAX energy-dispersive X-ray spectroscopy system (Ametek, Berwyn, PA). ImageJ software recorded mean fiber diameters and pore sizes (National Institutes of Health, Bethesda, MD). A combination of calculation and liquid intrusion methods were used to determine porosity. STA PT 1000 thermogravimetric analysis (Linseis, Selb, Germany) enabled the determination of melting and glass transition temperatures. Ultimate tensile strength was recorded using an MTESTQuattro materials testing system (ADMET, Norwood, MA).

[0155] The AuNPs synthesized were found to have an average diameter of 3.93 ± 0.68 nm. The representative scanning electron microscope, photographic, x-ray, and mCT images of PCL (PCL-alone), PCL with ROSU (PCL-ROSU), PCL with AuNP (PCL-Au), and PCL with AuNP and ROSU (PCL-Au-ROSU) polymer scaffolds or wraps are shown in FIG.17. Qualitatively, in the SEM images, PCL-ROSU and PCL-Au-ROSU fibers were more aligned than the fibers of PCL and PCL-Au. The successful integration of AuNP and ROSU into the polymeric wraps is supported by the representative energy-dispersive X-ray spectroscopy spectra (XPS). The PCL wrap only demonstrated peaks corresponding to carbon and oxygen. ROSU-containing wraps revealed peaks representing the sulfur and 32 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) calcium found in rosuvastatin calcium salt, while AuNP-containing wraps had peaks corresponding to gold. The addition of AuNP rendered the previously white wrap a dark gray color with increased X-ray and mCT imaging visibility.

[0156] Specifically, top three rows of FIG.17 show representative SEM images (1000x, bar = 100 μm; 5000x, bar = 20 μm) and energy-dispersive X-ray spectroscopy spectra of the electrospun wraps. Greater alignment of fibers can be seen in the PCL-ROSU and PCL-Au- ROSU wraps. The successful integration of ROSU and AuNP is confirmed by the presence of corresponding peaks in EDS. Bottom three rows of FIG.17 show photographic, X-ray, and mCT images of the electrospun wraps (bar = 1 cm). Incorporating AuNPs resulted in a characteristic gray color in photographs and enhanced X-ray and mCT imaging visualization.

[0157] The physicochemical properties of the polymeric wraps are summarized in Table 3. ROSU-containing wraps had thinner fibers, smaller pore sizes, and lower maximum stress than their counterparts. PCL-ROSU had significantly thinner fibers than PCL and PCL-Au (p < 0.022). PCL-Au wraps had the largest average pore size of the four wrap compositions (p < 0.022), while PCL-ROSU had the smallest (p < 0.006). PCL-ROSU and PCL-Au-ROSU had significantly lower maximum stress compared to PCL (p < 0.046) and PCL-Au (p < 0.042). No significant differences were found between the four wraps in terms of calculated porosity (p 0.726) and intrusion porosity (p ≥ 0.381). Table 3. Physiochemical properties of the electrospun wraps.33 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0158] The above results demonstrated that the addition of ROSU influenced the polymeric wrap’s fiber diameter, pore size, and mechanical strength. The thinner fibers of the wraps infused with ROSU may have been resulted from the increased conductivity of the solution following the dissolution of rosuvastatin calcium salt. The smaller average pore size of the ROSU-containing PCL wraps is associated with lower rates of particle dispersion. However, there was no significant difference in wrap porosity by the intrusion method or in vitro AuNP release, suggesting that the difference in pore diameter did not affect the rate of substrate intrusion. Though average wrap mechanical strength did decrease with the addition of both AuNPs and ROSU, it remained greater than 5 MPa, which is the upper limit of average tensile strength of naturally occurring blood vessels. Therefore, all wrap groups were found to have sufficient mechanical strength to support vascular flow. In addition, the increased fiber alignment seen in wraps loaded with both AuNPs and ROSU is an optimal structure for directing tissue growth, as the micro-patterning associated with alignment more effectively reconstitutes the form and function found in a native tissue environment.

[0159] In-Vitro Release of Drug and Nanoparticles

[0160] The polymeric wraps were incubated in PBS at 37°C with vigorous shaking for up to 12 weeks to simulate the hydrolytic degradation of the polymers. The PBS was sampled and replenished after each time point. The amount of ROSU released at each time point was calculated from the absorption of the supernatant at 244 nm using a UV-vis spectrophotometer (Cary 60, Agilent, Santa Clara, CA), while the AuNP content was quantified by inductively coupled plasma optical emission spectrometry (Varian 720-ES, Agilent, Santa Clara, CA).

[0161] In vitro AuNP and ROSU release rates for the polymeric wraps are plotted in FIG.18. In vitro, the ROSU release rate from the PCL-ROSU and PCL-Au-ROSU wraps exhibited a similar pattern (p ≥ 0.08) with initial burst during W1 (i.e., 78.59% and 73.78%, respectively) then tapered over W2-12 for a total cumulative release of 89.68% and 89.5%, respectively, at W12. There was also no difference in the in vitro cumulative AuNP release between PCL-Au and PCL-Au-ROSU wraps (p ≥ 0.509). Average AuNP release at W1 was 18.76% and 19.38% for PCL-Au and PCL-Au-ROSU, respectively, and then linearly increased until W4 for both PCL-Au (R2 = 0.9901) and PCL-Au-ROSU (R2 = 0.9882) wraps. AuNP release slowly plateaued from W4 to W12 with a cumulative release of 88.19% for PCL-Au and 92.42% for PCL-Au-ROSU. 34 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083)

[0162] Specifically, referring to FIG.18, analysis of % cumulative (a) ROSU and (b) AuNP release at various time points is depicted with overlaid linear models. ROSU demonstrated an initial release burst, followed by a gradual taper over weeks 1-12. AuNPs followed a linear release pattern from weeks one to four, which suggests the potential for predictive models of implanted device degradation via clinical imaging. R2 values are included for PCL-Au and PCL-Au-ROSU AuNP release from weeks one to four.

[0163] In-Vitro Cytotoxicity and Hemocompatibility Assay

[0164] Biocompatibility testing is commonly used to evaluate the safety and compatibility of medical devices. In this study, the vascular cytocompatibility and hemocompatibility of the wraps was tested. To test for cytotoxicity, immortalized human vascular endothelial cells (EC-RF24) and mouse vascular smooth muscle cells (MOVAS) were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2atmosphere. The gas-sterilized wraps were immersed in cell culture media for 24 hours to produce the treated media. EC-RF24 and MOVAS cells were seeded into 96-well plates at 1x104cells / well and incubated for 24 hours, after which the cell culture media were replaced with treated media at increasing concentrations (i.e., 0%, 25%, 75%, and 100%). After 24 hours, cell viability was measured using the alamarBlue assay. Fluorescence readings were taken at an excitation wavelength of 540 nm and emission wavelength of 590 nm using a Cytation5 microplate reader (Biotek Instruments, Winooski, VT). To test for hemolysis, 1 mm x 1 mm scaffolds were incubated in 500 μL of 2% heparinized blood for 1 hour at 37°C. Triton X, a non-ionic detergent, was used as the positive control, while heparinized blood with PBS was used as the negative control. The treated solutions were centrifuged at 1000 x g for five minutes to pellet the intact erythrocytes, and the absorbance of the supernatant was read at 540 nm.

[0165] FIG.19 demonstrates the impact of ROSU and AuNP loading on in vitro cell viability and hemocompatibility. No significant difference in cell viability was seen between PCL, PCL-ROSU, PCL-Au, and PCL-Au-ROSU treatment conditions in the EC- RF24 or MOVAS cell lines at any percentage of media treated (p ≥ 0.14), suggesting that the addition of ROSU and AuNPs is not cytotoxic to vascular cells. Moreover, the wraps did not cause significant hemolysis (> 5%) when incubated with heparinized blood. There was no significant difference in hemolysis between PCL, PCL-ROSU, PCL-Au, and PCL- Au-ROSU (p ≥ 0.57), and all the wraps had significantly lower hemolysis compared to 35 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) Triton X, a hemolytic detergent (p ≤ 0.001).

[0166] Specifically, referring to charts a, there was no significant difference in the viability of EC-RF24 immortalized human vascular endothelial cells and MOVAS mouse smooth muscle cells between any of the wraps at any concentration analyzed after 48 hours of incubation (p ≥ 0.14). Referring to chart b, when incubated with heparinized blood, none of the wraps produced significant hemolysis (> 5%). There was no significant difference between PCL, PCL-ROSU, PCL-Au, and PCL-Au-ROSU in terms of hemolysis (p ≥ 0.57).

[0167] It was found that the incorporation of AuNPs significantly increased wrap radiopacity on in vivo mCT at all time points, which indicates that AuNP loading confers greater radiographic visibility. Early radiodensity values of the PCL-Au (1,232 Hounsfield unit, or HU) and PCL-Au-ROSU (1,182 HU) wraps are concordant with previous analyses of AuNP radiodensities exceeding 1,000 HU known in the art. Wraps with AuNPs were not only more visible but also demonstrated linear decreases in both in vitro AuNP release and in vivo radiopacity from W1 (week one) to W4 (week four). These trends raise the possibility of a predictive model for wrap degradation based on non-invasive clinical imaging. The addition of ROSU to AuNP-loaded wraps showed no decrease in visibility and was found to increase wrap radiopacity at W8 (week 8), likely due to the calcium in the ROSU salt, which has similar radiodensity to bone of about 1,000 HU.

[0168] Formation of an AVF and Application of the Scaffold as a Perivascular Wrap

[0169] Approval and oversight from the MD Anderson Cancer Center Institutional Animal Care and Use Committee were obtained for all experiments using Sprague-Dawley rats (400 SAS SD, Charles River Laboratories, Wilmington, MA). Up-to-date institutional licensing and training were required for any personnel handling the animals. Standard protocol was followed for all procedures, sterilizing the surgical site using alternating chlorhexidine and alcohol in three repetitions. Sterile technique, including surgical drapes, sterile gloves, and instruments, was upheld during invasive procedures. Animals were positioned supine on the sterile field, which overlaid a heating blanket. Isoflurane anesthesia was utilized, and stimulation of intratarsal skin was applied to ensure adequate sedation before animal placement on the operating table. Buprenorphine was administered for pain control. Rats were euthanized via CO2asphyxiation followed by thoracotomy.

[0170] Briefly, a two-step 5 / 6th nephrectomy procedure known in the art was performed using Sprague-Dawley rats to model CKD in vivo. Following a four-week interval, AVFs 36 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) were surgically created based on a modified procedure also known in the art. Specifically, end-to-side surgical anastomosis of the external jugular vein with the common carotid artery was performed. For analysis, the CKD rats were divided into five groups—control AVF (i.e., no wrap), bare PCL wrap, PCL wrap embedded with ROSU (PCL-ROSU), PCL wrap embedded with AuNPs (PCL-Au), and PCL wrap embedded with AuNPs and ROSU (PCL- Au-ROSU). In all groups, excluding the control, a treatment-specific, gas-sterilized cylindrical wrap measuring 1 mm x 5 mm was incorporated into the AVF. The wrap in each group was fashioned around the outflow vein and secured using 10-0 nylon sutures to prevent migration and to ensure circumferential placement.

[0171] Characterization of the Scaffold as a Perivascular Wrap for the AVF

[0172] X-ray and mCT images of the wraps were performed prior to in vivo application. An MX-20 digital specimen radiography system (Faxitron Bioptics, Tucson, AZ) was used for radiographic visualization. A Skyscan preclinical in vivo scanner (Bruker, Billerica, MA) was used for in vitro and in vivo (at 2-, 4-, and 8-week (W) time points) mCT imaging of the wraps, with results expressed in Hounsfield units (HU).

[0173] Pulsatile arterial waveforms in the external jugular vein of successfully created AVFs were visualized using the Vevo 2100-LAZR system (VisualSonics, Toronto, ON, Canada). At 2-, 4-, and 8W time points, living rats were anesthetized and analyzed using B- mode ultrasound (US) with a 15-MHz probe. AVF outflow tract wall thickness (WT) and luminal diameter (LD) were assessed, with six measurements analyzed per scan.

[0174] Preselected rats in each group were euthanized at W2, W4, and W8 time points. The outflow veins were harvested after perfusion at the site of perivascular wrap placement for formalin fixation and paraffin embedding. Blocks were sectioned into 4-μm sections and then divided for hematoxylin and eosin (H&E) and immunofluorescence multiplex staining with the following markers: (1) von Willebrand factor (vWF), an endothelial cell marker; (2) CD45, a leukocyte marker; (3) alpha-smooth muscle actin (α-SMA), a smooth muscle cells marker; (4) vimentin, a fibroblasts marker; and (5) 4′,6-diamidino-2-phenylindole (DAPI), a nuclear stain. Slides were imaged using an Aperio LV1 real-time digital pathology system (Leica Biosystems, Buffalo Grove, IL). ImageJ software was used to quantify the luminal area (LA) and neointimal area (NIA) of the H&E-stained samples, while the Halo image analysis platform (Indica Labs, Albuquerque, NM) was used to 37 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) quantify fluorescently labeled cells in the neointima.

[0175] Images a of FIG.20 depict in vivo representative mCT images of control, PCL, PCL-ROSU, PCL-Au, and PCL-Au-ROSU wraps at W2, W4, and W8 (red box = AVF location; bar = 10 mm). The polymeric wraps loaded with AuNPs (PCL-Au and PCL-Au- ROSU) were found to be more radiopaque than the control, PCL, and PCL-ROSU wraps at all time points: W2 (p < 0.0001), W4 (p < 0.0001), and W8 (p < 0.0001). The PCL-ROSU wraps, which contain small amounts of calcium, had higher Hounsfield unit (HU) compared to control and PCL but significantly lower HU than PCL-Au and PCL-Au-ROSU at all time points (p < 0.0001). Nonetheless, the PCL-ROSU wraps were not readily visible on mCT. There was no difference between the AuNP-loaded wraps at W2 or W4 (p ≥ 0.3467), but PCL-Au-ROSU wraps demonstrated a significantly higher HU than PCL-Au wraps at W8 (p = 0.0042).

[0176] Chart b of FIG.20 shows that the average radiopacities of the AuNP-containing wraps decreased from W2 to W8. However, they remained significantly more visible than wraps without AuNPs throughout the study. This decrease in average HU was found to be linear to W4 for PCL-Au (R2 = 0.991) and PCL-Au-ROSU (R2 = 0.999) wraps. For example, HU values of the wraps at weeks 2, 4, and 8. AuNP loading conferred enhanced visibility of wraps on mCT and quantitatively greater radiodensity values compared to control, PCL, and PCL-ROSU at all time points. AuNP-associated radiopacity decreased over time as the in vivo wraps degraded. The addition of ROSU to AuNP-loaded wraps did not reduce wrap visibility, and at week 8, PCL-Au-ROSU was found to be significantly more radiopaque than PCL-Au alone.

[0177] As seen in the representative images of FIG.21, venous outflow tract lumens increased in size over time in the implanted-wrap groups but showed narrowing by week 8 in the control. The quantified analysis further demonstrated this trend, with decreased wall thickness and WT:LD ratio in the wrap groups. By week four, all implanted wrap groups showed significantly reduced markers of NIH and occlusion compared to the control. By week 8, ROSU-loaded wraps had significantly increased vessel patency compared to wraps without ROSU loading. Specifically, representative B-mode US images of the AVF site across each treatment group and time point are presented in Images a. The average WT:LD ratio, a marker of NIH, decreased over time in all conditions with a perivascular wrap and increased over time in the control as shown in charts b, which compares B-mode measurements of the AVF groups at weeks two, four, and eight. Decreasing LD, increasing 38 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) WT, and increasing WT:LD ratio are markers of NIH and loss of patency.

[0178] All the treatment groups were found to have significantly lower WT:LD compared to control at W4 (p < 0.0001). Wraps with ROSU were found to be equivalent to their counterparts without ROSU at W4 (p ≥ 0.5800). At W8, all the treatment groups maintained a lower WT:LD versus control (p < 0.0001), but wraps with ROSU had a significantly lower WT:LD ratio than all other cohorts (p ≤ 0.001). No differences in WT:LD ratio were seen between PCL-ROSU and PCL-Au-ROSU at all time points (p ≥ 0.4646).

[0179] Images a of FIG.22 provide representative hematoxylin and eosin–stained microscopic images of histologic slides with analysis tracings. Similar to the trends seen on US analysis, NIA:LA ratio decreased over time in the perivascular wrap groups and increased in the control group as shown in charts b, which compare histomorphometric measurements of the AVF groups at weeks 2, 4, and 8. W2 analysis found no difference in NIA:LA between any cohorts (p ≥ 0.179). The representative histologic images for the control group in FIG.22 demonstrate an increasing spread of NIH throughout the vessel well and decreased lumen patency at 8 weeks. Groups with implanted wrap had reduced NIH and increased luminal patency over time. These findings were quantified as NIH area, luminal area, and NIH-to-luminal area ratio, which showed that wrap groups demonstrated better vessel preservation by week 4 and ROSU-loaded wraps outperformed wraps without ROSU by week 8.

[0180] As on US, all the treatment groups were found to have significantly lower WT:LD compared to control at W4 (p ≤ 0.022). Wraps with ROSU were found to be equivalent to their counterparts without ROSU at W4 (p ≥ 0.179). By W8, PCL-ROSU and PCL-Au-ROSU were found to have a significantly lower NIA:LA ratio than both the control (p ≤ 0.02) and their counterpart wraps without ROSU addition (p < 0.035). No differences in WT:LD ratio were seen between PCL-ROSU and PCL-Au-ROSU at all time points (p ≥ 0.598).

[0181] FIG.23 provides representative images of immunofluorescence slides demonstrating the cells in the neointimal layer that are positive for vWF, CD45, α-SMA, and vimentin, indicating decreased infiltration of α-SMA-positive cells with all the treatment groups (bar = 100 µm). FIG.24 and Table 4 show that the control group had a lower proportion of vWF-positive cells and a higher proportion of α-SMA-positive and 39 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) vimentin-positive cells compared to all the wraps, suggesting that the control group promoted increased smooth muscle cell and fibroblast infiltration into the neointimal layer, while the wraps have reduced this phenomenon. Moreover, the ROSU-containing wraps showed further reduction in α-SMA-positive cells as well as CD45-positive cells compared to their non-ROSU-containing counterparts, suggesting that the addition of ROSU could reduce not only smooth muscle infiltration but also immune cell infiltration into the neointima. Table 4. Immunofluorescence analysis of the neointima. Au = gold; α-SMA, alpha smooth muscle actin; CD, cluster of differentiation; DAPI, 4′,6- diamidino-2-phenylindole; PCL, polycaprolactone; ROSU = rosuvastatin; vWF, von Willebrand factor.

[0182] PCL generally has an in vivo resorption time of 2-3 years, though an optimal time frame remains unknown. PCL was selected for its longevity to ensure that rosuvastatin elution would exceed the time for AVF maturation. There are alternative resorbable polymers with shorter or longer durations of resorptions that could be employed to maintain drug-elution throughout the maturation period. While the efficacy of ROSU shows promise for the intended purpose of mitigating the untoward effects of NIH in the maturing AVF, it should be noted that alternative drugs with anti-inflammatory (e.g., diclofenac) or anti- thrombotic properties (e.g., clopidogrel) could be used as an alternative or in addition to the resorbable polymer construct. Also, flow dynamics may play a pivotal role in AVF maturation.

[0183] Lastly, US and histologic analyses revealed a potential therapeutic synergism between the supportive perivascular wrap and ROSU. All the perivascular wraps were found to have a significant advantage by W4, with improved vessel patency and wall 40 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) structure in all wrap groups. This is consistent with previous reports that perivascular wraps confer a mechanical effect on the vessel wall that suppresses abnormal cell proliferation and vascular changes. The addition of ROSU further enhanced the vascular benefits of perivascular wraps by W8, as wraps with ROSU had significantly decreased markers of NIH and luminal narrowing on both US and histologic analysis. This may be explained by the ROSU-loaded wrap’s two-pronged approach to reducing NIH. The mechanical NIH attenuation that is inherently present in perivascular wraps may be bolstered by direct statin delivery for reduced low-density lipoprotein levels, decreased inflammation, and mitigated endothelial cell dysfunction in local tissues. By targeting abnormal vessel changes through two unique pathways, the ROSU-loaded wraps can confer improved luminal maintenance and wall integrity compared with wraps that provide mechanical effects only. Interestingly, the ROSU-containing wraps showed further reduction in α-SMA-positive and CD45- positive cell infiltration compared to their bare counterparts, which supports the role of this drug in mitigating leukocyte infiltration and smooth muscle cell proliferation in the vascular walls.

[0184] Overall, these results suggest that targeted delivery of ROSU can supplement the NIH-attenuating effect of perivascular wraps, leading to even greater control of vessel patency and fistula maturation.

[0185] EXAMPLE 3

[0186] Fabrication and Characterization of an AuNP-Infused Polymer Scaffold

[0187] AuNPs were synthesized following an example protocol. AuNPs and PCL with an average molecular weight Mn of about 80,000 g / mol (Sigma-Aldrich, St. Louis, MO) were mixed in toluene / MeOH to form a solution with 15% PCL and increasing concentrations of AuNPs—0%, 10%, 20%, and 30%. The resulting solutions were electrospun at 1.0 mL / h and 15 kV using a Spraybase electrospinning system (Avectas, Maynooth, Ireland).

[0188] The size and morphology of the AuNPs were determined using a JEM 1010 trans-mission electron microscope (TEM, JEOL USA, Inc., Peabody, MA). The fiber diameter and pore size of wraps were determined using a Nova NanoSEM scanning electron microscope (SEM, Field Electron and Ion Company, Hillsboro, OR) with an EDAX energy- dispersive spectroscopy system (Ametek, Berwyn, PA). SEM images were captured from random areas to determine the mean fiber diameter and mean pore size of each sample via 41 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) ImageJ software (National Institutes of Health, Bethesda, MD). Porosity was determined using the calculation method and liquid intrusion method known in the art. Melting temperature (Tm) and glass transition temperature (Tg) were determined using an STA PT 1000 thermogravimetric analyzer (Linseis, Selb, Germany). Ultimate tensile strength was determined using an MTESTQuattro materials testing system (ADMET, Norwood, MA).

[0189] FIG.25 shows a representative TEM image of the synthesized AuNPs as well as representative SEM, photographic, X-ray, and mCT images of the electrospun polymeric wraps. The synthesized AuNPs had an average diameter of 3.93 ± 0.68 nm. The physicochemical properties of the electrospun polymeric wraps are summarized in Table 5. These properties translated to adequate attachment of MSCs, as seen on fluorescence microscopy (FIG.26). Among the AuNP-infused wraps, PCL-AuNP20 (20% concentration of AuNPs) demonstrated a qualitatively more consistent signal intensity on both X-ray and mCT. Hence, this blend was chosen for the subsequent experiments, termed hereafter as “PCL-Au.” Table 5. Physicochemical properties of the electrospun polymeric wraps. Wrap type Diameter (μm)Pore sizePorosity (calculated, Porosity (intrusion, Maximum stress (μm) %)%)Tg(°C) Tm(°C)(MPa) PCL 2.07 ± 1.03 168.34 ± 177.94 93.21 ± 1.23 76.75 ± 8.06 52.53 -69.61 9.17 ± 0.31PCL-AuNP10 0.53 ± 0.26 98.34 ± 76.13 53.63 ± 20.62 74.31 ± 6.15 54.32 -68.19 13.41 ± 0.52PCL-AuNP20 0.25 ± 0.07 81.57 ± 86.11 65.52 ± 1.13% 71.13 ± 1.98 52.44 -69.71 9.01 ± 0.42PCL-AuNP30 0.27 ± 0.09 62.91 ± 53.40 64.02 ± 8.34 76.03 ± 3.69 52.37 -69.64 12.42 ± 1.40Abbreviations: AuNP, gold nanoparticles; PCL, polycaprolactone; Tg, glass transition temperature; Tm, melting temperature.

[0190] Cell Culture and MSC Attachment

[0191] Bone-marrow derived Sprague-Dawley rat mesenchymal stem cells that express red fluorescent protein (RFP-MSCs, P2, Creative Bioarray, Shirley, NY) were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin (Corning, Corning, NY). The wraps were subjected to ethylene oxide gas sterilization and then seeded with 1 x 105 RFP-MSCs. After 48 hours, the samples were imaged using an Eclipse Ti2 fluorescence microscope (Nikon, Melville, NY) to visualize the attachment of RFP-MSCs. The cells for in vivo testing were kept in culture media before surgical application.

[0192] FIG.26 shows attachment of Sprague-Dawley rat red fluorescent protein (RFP)– expressing MSCs onto the polymeric wraps. All the polymeric wraps allowed sufficient 42 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) attachment of Sprague-Dawley rat RFP-MSCs after 48 hours of incubation.

[0193] Formation of an AVF and Application of the Scaffold as a Perivascular Wrap

[0194] All rat experiments were approved by the Institutional Animal Care and Use Committee. A CKD model was created using Sprague-Dawley rats (400 SAS SD, Charles River Laboratories, Wilmington, MA) using the two-step 5 / 6th nephrectomy procedure known in the art. After four weeks, an AVF model was created via an end-to-side surgical anastomosis of the external jugular vein with the common carotid artery based on a procedure also known in the art. A total of 15 rats were randomly allocated into five groups—control AVF (i.e., no wrap), PCL wrap, PCL+MSC wrap, PCL-Au wrap, and PCL-Au+MSC wrap—with 3 rats per group. A minimum of 3 rats per group is needed to attain a statistical level of p < 0.05 with a power of 80%. In the various wrap groups, a 1 mm x 5 mm piece of the gas-sterilized cylindrical wrap was placed around the outflow vein and secured using 10-0 nylon sutures. The MSC groups received a wrap seeded with 1 x 105MSCs.

[0195] Characterization of Scaffold as a Perivascular Wrap around the AVF

[0196] Prior to in vivo application, a Skyscan 1276 CMOS mCT system (Bruker, Billerica, MA) was used to capture x-ray and mCT images of the wraps. The radiopacity of the implanted wraps was then determined using the same machine. The CTAn mCT image analyzer (Bruker) was used to measure the radiopacity of the wraps in Hounsfield units (HU) and perform volume rendering for three-dimensional visualization.

[0197] A Vevo 2100-LAZR system (FUJIFILM VisualSonics, Toronto, ON, Canada) was used to visualize pulsatile arterial waveforms in the external jugular vein of successfully created AVFs. At weeks two and eight, the rats were anesthetized, and a 15- MHz B-mode probe was used to get 6 measurements of the wall thickness and luminal diameter across the outflow vein. Wall-to-lumen ratio was calculated by dividing the average wall thickness by the average luminal diameter.

[0198] Rats were euthanized by CO2asphyxiation and thoracotomy following imaging. The outflow veins were harvested after perfusion for formalin fixation and paraffin embedding. Tissue blocks were cut into 4-μm sections and subjected to hematoxylin and eosin (H&E) staining or immunofluorescence multiplex staining with the following markers: (1) alpha-smooth muscle actin (α-SMA), which is highly expressed in smooth muscle cells but also expressed in fibroblasts; (2) CD45, which is highly ex-pressed in 43 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) leukocytes and a marker of inflammatory infiltration; (3) vimentin, which is highly expressed in fibroblasts but also expressed in vascular smooth muscle cells and lymphocytes; and (4) 4′,6-diamidino-2-phenylindole (DAPI), a nuclear stain. An Aperio LV1 real-time digital pathology system (Leica Biosystems, Buffalo Grove, IL) was used to capture images from the H&E slides and quantify the luminal and neointimal areas. Blood artifacts seen within the lumen of the vessels resulting from incomplete tissue processing were excluded from the analysis. A Leica Versa fluorescent-imaging system (Leica Biosystems) was used to capture images from the immunofluorescent slides, and the Halo image analysis platform (Indica Labs, Albuquerque, NM) was used to perform fluorescent cell counting.

[0199] FIG.27 and Table 6 compare the mCT findings for the 5 groups of rat AVFs 2 and 8 weeks after AVF creation. FIG.27 shows evaluation of radiopacity via mCT. Images a show reconstructed mCT images of the control (i.e., no perivascular wrap), PCL, PCL+MSC, PCL-Au, and PCL-Au+MSC arteriovenous fistulas (AVFs) at 2 and 8 weeks after AVF creation. Red boxes correspond to the position of the wrap. Chart b demonstrates Hounsfield unit (HU) values of the five groups of AVFs 2 and 8 weeks after AVF creation. At weeks 2 and 8, rat AVFs wrapped with AuNP-containing polymer scaffolds (PCL-Au and PCL-Au+MSC) had higher HU values com-pared to control (p<.001) and non–Au- containing wraps (PCL and PCL+MSC; both p<.001).

[0200] At week 2, higher radiopacity was seen in rat AVFs wrapped with polymeric wraps containing AuNPs (PCL-Au and PCL-Au+MSC) compared to control (p<.001) and non-Au containing wraps (PCL [p<.001] and PCL+MSC [p<.001]). There was no difference among the AVFs without AuNP, i.e., between control and PCL (p=.95), control and PCL+MSC (p=.85), and PCL and PCL+MSC (p>.99). There was also no difference between PCL-Au and PCL-Au+MSC (p=.21) AVFs.

[0201] At week 8, the radiopacity of AuNP-containing wraps was retained. PCL-Au and PCL-Au+MSC AVFs still had higher radiopacity compared to control (p<.001), PCL (p<.001), and PCL+MSC (p<.001) AVFs. Similar to week 2, there was no difference be- tween control and PCL (p=.89), control and PCL+MSC (p>.99), or PCL and PCL+MSC (p=.88) AVFs. There was also no difference between PCL-Au and PCL-Au+MSC (p=.98) AVFs. However, there was a reduction in the HU values of the PCL-Au (42.54%, p<.001) and PCL-Au+MSC (37.96%, p=.002) wraps at week 8 when compared to week 2. Nonetheless, the intensity level of PCL-Au and PCL-Au+MSC wraps remained similar 44 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) (p=.98), which suggests that the addition of MSCs did not affect wrap degradation over time. Table 6. Radiopacity, wall-to-lumen ratio, and neointima-to-lumen ratio of the AVF groups (n=3 per group). Wrap type Radiopacity (HU) Wall-to-lumen ratio Neointima-to-lumen ratio Week 2 Week 8 Week 2 Week 8 Week 2 Week 8 Control 98.07 ± 9.88 69.27 ± 23.81 0.88 ± 0.44 0.95 ± 0.03 2.05 ± 0.89 5.26 ± 0.53 PCL 152.0 ± 21.21 135.8 ± 37.19 0.53 ± 0.05 0.31 ± 0.08 0.52 ± 0.22 0.25 ± 0.06 PCL+MSC 171.9 ± 60.22 67.68 ± 5.61 0.5 ± 0.08 0.24 ± 0.04 0.28 ± 0.10 0.25 ± 0.06 PCL-Au 1375 ± 97.79 790.1 ± 164.0 0.61 ± 0.09 0.33 ± 0.08 0.34 ± 0.24 0.27 ± 0.15 PCL-Au+MSC 1212 ± 164.1 751.9 ± 107.1 0.4 ± 0.09 0.23 ± 0.05 0.20 ± 0.08 0.27 ± 0.13 Abbreviations: Au, gold; AVF, arteriovenousfistula; HU, Hounsfield unit; MSC, mesenchymal stem cell; PCL, polycaprolactone.

[0202] The wall-to-lumen ratio was assessed as a marker of vascular stenosis. FIG.28 and Table 6 above compare the US findings for the five groups of rat AVFs at two and eight weeks after AVF creation. It was discovered that PCL+MSC and PCL-Au+MSC AVFs had a reduced wall-to-lumen ratio, a marker of vascular stenosis, in comparison to AVFs without MSCs as early as two weeks after AVF creation.

[0203] FIG.28 shows evaluation of wall-to-lumen ratio via B-mode US. Specifically, images a show B-mode US images of the control (i.e., no perivascular wrap), PCL, PCL+MSC, PCL-Au, and PCL-Au+MSC AVFs at two and eight weeks after AVF creation. Chart b shows wall-to-lumen ratios of the five groups of AVFs two and eight weeks after AVF creation; at week 2, PCL+MSC (p=.045) and PCL-Au+MSC (p=.008) AVFs had a lower wall-to-lumen ratio compared to control, and at week 8, all scaffold groups had a lower wall-to-lumen ratio compared with the control rat AVFs (p<.001).

[0204] At week 2, the control rat AVFs had a higher wall-to-lumen ratio compared to PCL+MSC (p=.045) and PCL-Au+MSC (p=.008) AVFs. There was no difference between control and PCL (p=.072), control and PCL-Au (p=.24), PCL and PCL+MSC (p>.99), PCL and PCL-Au (p=.96), PCL and PCL-Au+MSC (p=.83), PCL+MSC and PCL-Au (p=.90), PCL+MSC and PCL-Au+MSC (p=.92), or PCL-Au and PCL-Au+MSC (p=.45) AVFs.

[0205] At week 8, the control rat AVFs had a higher wall-to-lumen ratio compared to all wrap groups: PCL (p<.001), PCL+MSC (p<.001), PCL-Au (p<.001), and PCL-Au+MSC (p<.001). There was no difference between PCL and PCL+MSC (p=.99), PCL and PCL-Au (p>.99), PCL and PCL-Au+MSC (p=.98), PCL+MSC and PCL-Au (p>.95), PCL+MSC and 45 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) PCL-Au+MSC (p>.99), or PCL-Au and PCL-Au+MSC (p=.94) AVFs. The similarity between the wall-to-lumen ratios of PCL+MSC and PCL-Au+MSC AVFs suggests that the incorporation of AuNPs does not affect the efficacy of MSCs in reducing AVF wall-to- lumen ratio.

[0206] The neointima-to-lumen ratio was evaluated to assess the presence of NIH. FIG. 29 and Table 6 above compare the histomorphometric analysis for the five groups of rat AVFs at two and eight weeks after AVF creation. It was found that 8 weeks after AVF creation, all treatment groups had improved neointima-to-lumen ratios compared to control rats.

[0207] FIG.29 shows evaluation of neointima-to-lumen ratio via histomorphometric analysis. Images a show hematoxylin and eosin–stained microscopic images of the control (i.e., no perivascular wrap), PCL, PCL+MSC, PCL-Au, and PCL-Au+MSC arteriovenous fistulas (AVFs) at two and eight weeks after AVF creation. Red blood cells within the lumen of the vessels resulting from incomplete tissue processing were excluded from the analysis. Chart b shows neointima-to-lumen ratio of the five groups of AVFs two and eight weeks after AVF creation, where the control rat AVFs had a higher neointima-to-lumen ratio compared to all the other groups.

[0208] At week 2, the control rat AVFs had a higher neointima-to-lumen ratio compared to all the other groups: PCL (p<.001), PCL+MSC (p<.001), PCL-Au (p<.001), and PCL- Au+MSC (p<.001). There was no difference between PCL and PCL+MSC (p=.92), PCL and PCL-Au (p=.97), PCL and PCL-Au+MSC (p=.81), PCL+MSC and PCL-Au (p>.99), PCL+MSC and PCL-Au+MSC (p>.99), or PCL-Au and PCL-Au+MSC (p=.99) AVFs.

[0209] At week 8, the control rat AVFs maintained a higher neointima-to-lumen ratio compared to all the other groups: PCL (p=.001), PCL+MSC (p=.002), PCL-Au (p=.002), and PCL-Au+MSC (p<.001). There was no difference between PCL and PCL+MSC (p=.93), PCL and PCL-Au (p=.95), PCL and PCL-Au+MSC (p>.99), PCL+MSC and PCL- Au (p>.99), PCL+MSC and PCL-Au+MSC (p=.94), or PCL-Au and PCL-Au+MSC (p=.96) AVFs. Similar to what was observed with the wall-to-lumen ratio, the addition of AuNPs did not influence the wraps' capacity to reduce the AVF neointima-to-lumen ratio.

[0210] Several markers that represent neointimal infiltration with smooth muscle cells, inflammatory cells, and fibroblasts (i.e., α-SMA, CD45, and vimentin, respectively) were assessed. Table 7 and FIG.30 compare the immunofluorescence analysis for representative 46 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) slides from each of the five groups of rat AVFs at eight weeks after AVF creation. When compared to control, PCL and PCL-Au wraps reduced the neointima-to-media ratio of total cells (i.e., DAPI) as well as CD45- and vimentin-positive cells but in-creased the neointima- to-media ratio of α-SMA–positive cells. In contrast, PCL+MSC and PCL-Au+MSC wraps reduced the neointima-to-media ratio of α-SMA–positive cells, and these MSC-seeded wraps further decreased the neointima-to-media ratio of total cells as well as CD45- and vimentin-positive cells when compared to their non-MSC counterparts. Table 7. Neointima-to-media ratio of immunofluorescence-stained cells on representative AVFs. Wrap type α-SMA CD45 Vimentin DAPI Control 1.0 0.76 0.74 0.79 PCL 1.21 0.30 0.71 0.69 PCL+MSC 0.98 0.17 0.09 0.64 PCL-Au 1.74 0.08 0.36 0.66 PCL-Au+MSC 0.92 0.07 0.22 0.40 Abbreviations: α-SMA, alpha-smooth muscle actin; Au, gold; AVF, arteriovenous fistula; CD45, cluster of differentiation 45; DAPI, 4′,6-diamidino-2-phenylindole; MSC, mesenchymal stem cell; PCL, polycaprolactone.

[0211] FIG.30 shows evaluation of neointimal immunofluorescence staining for α- SMA, CD45, and vimentin. Immunofluorescence microscopic images of the control (i.e., no perivascular wrap), PCL, PCL+MSC, PCL-Au, and PCL-Au+MSC arteriovenous fistulas (AVFs) at eight weeks after AVF creation, showing the different cell populations in the neointimal (N) and medial (M) vascular layers.

[0212] It was found that PCL+MSC and PCL-Au+MSC AVFs demonstrated an improved wall-to-lumen ratio, a marker of vascular stenosis, compared with AVFs without MSCs in as early as two weeks after AVF creation. This is consistent with the results of a prior study, wherein the addition of MSCs to polymeric wrap reduced the AVF wall-to- lumen ratio when compared to polymeric wrap alone at four weeks after AVF creation. These results highlight the role of MSCs in improving AVF maturation. The similarity between the wall-to-lumen ratios of PCL+MSC and PCL-Au+MSC AVFs (p=.92) also suggests that the incorporation of AuNPs does not substantially affect the activity of MSCs in reducing AVF wall-to-lumen ratio. By week 8, all treatment groups demonstrated an improved wall-to-lumen ratio. Nonetheless, the similarity between the wall-to-lumen ratios of AVFs with and without MSCs still supports the idea that the incorporation of AuNPs 47 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) does not interfere with the activity of the wraps in reducing an AVF’s wall-to-lumen ratio.

[0213] It was also found that all treatment groups demonstrated improved neointima-to- lumen ratios, a measure of NIH, versus control rats by eight weeks after AVF creation. The above findings show that the reduction in the neointima-to-lumen ratio by PCL and PCL+MSC treatment versus control seen at four weeks after AVF creation in a previous study can be seen as early as two weeks and can be maintained for up to eight weeks. Moreover, the incorporation of AuNPs did not substantially affect the ability of the wraps to reduce the AVF neointima-to-lumen ratio. Although it appears that the added benefit of MSC-containing wraps had decreased by week 8, there could be changes in the cellular and extracellular composition of the vascular wall attributable to MSCs that were not reflected on the results of in vivo imaging. Interestingly, it was found that representative MSC-seeded wraps performed better than control and non-MSC counterparts in reducing the neointima- to-media ratio of total cells as well as α-SMA–, CD45-, and vimentin-positive cells on immunofluorescence analysis. Moreover, an increase in the neointima-to-media ratio of α- SMA–positive cells with PCL and PCL-Au AVFs was observed, which indicates inward migration of smooth muscle cells within the neointima after longer periods of observation. Taken together, these findings indicate that the addition of MSCs may promote better outward vascular remodeling com-pared to a vascular wrap that serves purely as a physical support.

[0214] It should be noted that the scope of this study was limited to the assessment of the signal intensity of the wraps on mCT and the effect of the wraps on the AVF wall-to- lumen ratio as seen on US and the neointima-to-lumen ratio as seen on histology. Nonetheless, it represents a significant stride towards the development of an electrospun polymer scaffold as a perivascular wrap that can be monitored non-invasively while providing both mechanical and functional support to the maturing AVF. The use of cells that can potentially suppress NIH, such as endothelial cells and mesenchymal stem / stromal cells, will continue to be an emerging area of active research since cell-based therapies could release a wider array of immunomodulatory substances for AVF maturation compared to localized perivascular drug delivery.

[0215] Progress in this field can be accelerated through the development of new applications for known biocompatible materials, such as synthetic polymers and nanoparticles. Further studies are recommended to explore the relationship between the rate of wrap degradation and its impact on the ability of the wrap to provide attachment to 48 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) seeded MSCs as well as mechanical support to the maturing AVF. Moreover, there is a need for additional research on the cellular and extracellular composition of the vascular wall in order to determine the fate of the seeded MSCs, understand the effect of MSCs on the different cells of the outflow vein, and identify potential sources of differences between in vivo and histological observations in this study. These investigations will pave the way for the application of this research to larger animals and, eventually, to human patients who may benefit from novel AVF therapies.

[0216] EXAMPLE 4

[0217] Overview

[0218] In some implementations, a BiNPs-infused perivascular wrap allowing the sustained release of MSCs and longitudinal monitoring using conventional X-ray imaging is provided. The BiNPs-infused perivascular wrap combines two modifiers of AVF maturation into a single intervention—a radiopaque wrap, which provides mechanical support and enhanced radiologic visibility, and MSCs, which attenuate multiple pathways of AVF failure. Experimental results revealed that implementations of the BiNPs-infused perivascular wrap can mitigate endothelial dysfunction, hypoxia, and inflammation in rat AVFs, strengthening the foundation for the development of MSC-loaded radiopaque wraps that could potentially reduce salvage interventions, healthcare costs, morbidity, and mortality associated with AVF failure.

[0219] Prior studies have found that MSCs-loaded PCL scaffolds that were wrapped around AVFs in rats enhanced MSC retention and inhibited NIH in the AVFs after 4 weeks. However, the clinical application of radiolucent polymer scaffolds is hindered by the challenge of monitoring their integrity and placement. The scarcity of research investigating the long-term effects of polymer scaffolds as novel delivery systems for therapies targeting AVF failure can be attributed, in part, to this underlying factor.

[0220] The infusion of high-atomic-number (high-Z) nanoparticles presents an opportunity to improve the visibility of polymeric devices under conventional X-ray modalities. A comparative study evaluating several high-Z metals (iron, zirconium, barium, gadolinium, ytterbium, tantalum, tungsten, gold, and bismuth) revealed that bismuth had the most optimal differentiation from iodine, a common component of contrast agents used in the clinic, in dual-energy CT, while maintaining increased contrast in single-energy CT. Consequently, medical devices incorporating bismuth nanoparticles (BiNPs) may be 49 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) efficiently distinguished from iodine-based vascular contrast agents. The present disclosure provides a BiNPs-infused perivascular wrap (i.e., a BiNPs-infused polymer scaffold, that can be both used for the delivery of MSCs and monitored via conventional X-ray modalities for the surveillance of long-term AVF outcomes.

[0221] Molecular imaging technologies that can detect the development of pathologic pathways leading to AVF failure can potentially improve AVF outcomes by providing an opportunity for intervention before failure. As disclosed above, molecular imaging using 18F-FDG PET revealed neointimal infiltration with smooth muscle cells, fibroblasts, and immune cells, and consequently, the development of NIH in rat AVFs after 4 weeks. In particular, cellular infiltration within the AVFs’ neointimal layer at a single time point was investigated. Another aspect of the present disclosure, as described below, assess the utility of 18F-FDG PET in the monitoring of inflammation and hypoxia in all the layers of the AVFs using both short-term and long-term time points, thereby providing approaches for maintaining and evaluating the patency of AVFs.

[0222] In some implementations, the BiNPs-infused perivascular wrap includes BiNPs incorporated into polycaprolactone (PCL) to fabricate an electrospun perivascular wrap capable of MSC delivery and conducive to longitudinal monitoring using conventional imaging. Effects of example BiNPs-infused perivascular wraps on the attenuation of markers of neointimal hyperplasia (e.g., endothelial dysfunction, hypoxia, and inflammation), the leading cause of AVF failure, in rats with induced CKD (n = 3 per time point) were evaluated. Example implementations of the BiNPs-infused perivascular wrap include the following groups: control (no wrap), PCL wrap, PCL with MSCs, PCL-Bi (BiNPs-infused wrap), and PCL-Bi with MSCs.

[0223] Physicochemical characterization and in-vitro biocompatibility tests revealed that BiNP infusion did not alter the BiNPs-infused perivascular wrap’s non-cytotoxicity towards vascular cells, hemocompatibility, and capacity for MSC loading but facilitated long-term monitoring via micro-computed tomography. After 8 weeks, all treatment groups demonstrated significant improvement in wall-to-lumen ratio on ultrasonography (P < 0.001), neointima-to-lumen ratio on histomorphometry (P < 0.001), and attenuation of neointimal hypoxia on immunohistochemistry (P < 0.05). Compared to non-MSC wraps, MSC-loaded wraps not only attenuated endothelial dysfunction and neointimal 50 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) inflammation but also reduced hypoxia and inflammation across all vascular layers.

[0224] The following quantitative data were presented as means ± standard deviations and analyzed using a two-tailed t test or analysis of variance with GraphPad Prism software, version 10.0.3 (GraphPad, San Diego, CA). All animals were included in the analysis. Statistical significance was defined as P < 0.05.

[0225] Fabrication and Characterization of an BiNPs-Infused Polymer Scaffold

[0226] BiNPs were synthesized via one-pot thermal decomposition following a published protocol.15 PCL (average Mn 80,000, Sigma-Aldrich, St. Louis, MO) was dissolved in chloroform / methanol (8:3) to yield 12% w / v solutions. For BiNPs-infused wraps, BiNPs were mixed with PCL at a 1:4 ratio. The solutions were electrospun at 1.0 mL / hour and 15 kV using a Spraybase electrospinning system (Avectas, Maynooth, Ireland).

[0227] The average BiNP size was determined using a JEM1010 transmission electron microscope (JEOL USA, Peabody, MA). The fiber diameter and pore size of the perivascular wraps were determined using a Nova NanoSEM scanning electron microscope (Field Electron and Ion Company, Hillsboro, OR). The porosity of the wraps was determined using the liquid intrusion method. The melting temperature (Tm) and glass transition temperature (Tg) of the wraps were determined using an STA PT 1000 thermogravimetric analyzer (Linseis, Selb, Germany). The ultimate tensile strength of the wraps was determined using an MTESTQuattro materials testing system (ADMET, Norwood, MA). X-ray and mCT images of the wraps were captured using a SKYSCAN 1276 CMOS mCT system (Bruker, Billerica, MA).

[0228] FIG.31 depicts a representative TEM image (FIG.31a) of the synthesized BiNPs as well as representative SEM, photographic, X-ray, and mCT images of the BiNPs and BiNPs-loaded perivascular wraps, respectively (FIG.31b). The synthesized BiNPs were spherical and had an average diameter of 3.29 ± 0.511 nm. The synthesized wraps were composed of intertwined nanofibers, and the incorporation of BiNPs led to the production of fibers with a rougher surface and rendered the previously white wrap a dark gray color with increased visibility on X-ray and mCT imaging.

[0229] The infusion of BiNPs into the PCL wraps also produced several changes in the physicochemical properties of the wraps (Table 8). The infusion of BiNPs significantly reduced the average fiber diameter (P < 0.001) and the average pore area (P < 0.05) of the 51 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) wraps. There was no significant difference between PCL and PCL-Bi (i.e., BiNPs-infused PCL) in terms of porosity, glass transition temperature, melting temperature, and maximum stress. Despite having a porous architecture, the wraps maintained a high level of tensile strength. Table 8. Physicochemical properties of the electrospun wraps. Properties PCL PCL-Bi Diameter (μm) 0.76 ± 0.42 0.57 ± 0.27 Pore area (μm2) 8.32 ± 3.96 6.94 ± 2.98 Porosity (%) 76.61 ± 2.41 74.57 ± 4.56 Tg(°C) −65.64 ± 1.89 −63.04 ± 0.69 Tm(°C) 55.73 ± 0.77 54.91 ± 0.55 Maximum stress (MPa) 9.38 ± 2.04 7.65 ± 2.33 Abbreviations: PCL, polycaprolactone; PCL-Bi, bismuth nanoparticle-infused polycaprolactone; Tg, glass transition temperature; Tm,melting temperature.

[0230] In-Vitro Cytotoxicity and Hemocompatibility Assay

[0231] The biocompatibility of the perivascular wraps was examined by determining their cytotoxicity against selected vascular cells and by evaluating their effect on hemolysis and clotting time. The wraps were subjected to ethylene oxide (EO) gas sterilization before testing. The acute cytotoxicity of the perivascular wraps was examined against immortalized human vascular endothelial cells (EC-RF24, American Type Culture Collection (ATCC), Manassas, VA) and primary mouse vascular aortic smooth muscle cells (MOVAS, ATCC). The wraps were incubated in Dulbecco's Modified Eagle’s Medium (DMEM, Corning, Corning, NY) cell culture medium for 24 hours. The cells were then plated into a 96-well flat-bottom plate and incubated with treated media at different volume concentrations (0, 25, 50, 75, and 100%) in regular DMEM.

[0232] After 72 hours, the viability of the cells was determined using the alamarBlue assay (Invitrogen, Waltham, MA). Hemolysis and clotting time were evaluated using published protocols. For hemolysis, the wraps were incubated in an erythrocyte solution prepared from heparinized rat blood for 1 hour at 37°C. The treated solutions were 52 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) centrifuged at 1000g for 5 minutes to pellet the erythrocytes that were not lysed, and the absorbance of the supernatant was read at 540 nm. Triton X, a hemolytic detergent, was used as the positive control, and normal saline solution (NSS) was used as the negative control. For clotting time, the wraps were incubated in platelet-poor plasma derived from citrated rat blood for 30 minutes at 37°C. After this, 3% rabbit brain cephalin and 30 mM CaCl2solution were added, and the time required for the formation of a stable clot, which represents the activated partial thromboplastin time (aPTT), was recorded. Glass was used as the positive control, and NSS was used as the negative control.

[0233] Experimental results demonstrated that perivascular wraps were biocompatible and supported the attachment of MSCs. For example, FIG.32 depicts results of study of in- vitro cytotoxicity, hemocompatibility, and MSC attachment. Specifically, FIG.32a shows effect of PCL and PCL-Bi wraps vs control (no wrap) on the viability of immortalized human vascular endothelial cells (EC-RF24) and primary mouse vascular aortic smooth muscle cells (MOVAS). Different concentrations of wrap-treated medium vs untreated medium are shown. FIG.32b shows effect of the wraps on hemolysis and aPTT. Controls were Triton X, normal saline solution (NSS), glass. FIGS.32c and 32d shows seeding efficiency and attachment of mesenchymal stem cells that expressed red fluorescent protein on the electrospun wraps.

[0234] No significant difference was observed in the cell viability of EC-RF24 or MOVAS cell lines when incubated with increasing concentrations of media extracts of PCL or PCL-Bi (FIG.32a). Furthermore, the wraps did not cause substantial alterations in hemolysis and clotting time (FIG.32b). The blood compatibility of the wraps was similar to the negative control, NSS. The wraps allowed the attachment of a sufficient number of MSCs after 24 hours, with a seeding efficiency of 87.4 ± 3.87% for PCL and 85.2 ± 5.0% for PCL-Bi (FIG.32c). FIG.32d shows fluorescent microscopy images of the seeding of RFP-expressing MSCs on the wraps. The infusion of BiNPs did not affect the MSC-seeding efficiency of the wrap.

[0235] Cell Culture and Loading of MSCs in Perivascular Wraps

[0236] Bone-marrow-derived Sprague-Dawley rat MSCs that express red fluorescent protein (RFP) (catalog no. CSC-C1315, Creative Bioarray, Shirley, NY) were cultured using DMEM with 10% fetal bovine serum (Corning) and 1% penicillin-streptomycin (Corning). Wraps were subjected to EO gas sterilization and seeded with 1 x 105 MSCs at 53 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) 3rd passage. After 24 hours, MSC attachment and seeding efficiency were determined using an ECLIPSE Ti2 fluorescence microscope (Nikon, Melville, NY). For in-vivo efficacy studies, the MSC-laden wraps were kept in a culture medium for a maximum of 24 hours before surgical application.

[0237] Formation of an AVF and Application of the Scaffold as a Perivascular Wrap

[0238] All rat experiments were approved by MD Anderson Cancer Center’s Institutional Animal Care and Use Committee (protocol no.00001882-RN01). Current institutional licenses and training were mandatory for all individuals responsible for the care and handling of the rats. The rats were housed in a centralized facility under the Department of Veterinary Medicine. CKD was induced in a total of 308-week-old female Sprague- Dawley rats (400 SAS SD, Charles River Laboratories, Wilmington, MA) based on a published protocol for a two-step 5 / 6th nephrectomy.20 After 4 weeks, AVFs were created via surgical anastomosis of the external jugular vein with the common carotid artery in an end-to-side fashion in the rats with CKD.21

[0239] Based on pilot histomorphometric data, a minimum of three rats per group is needed to attain a statistical significance level of p < 0.05 and a power of 80%. In subsequent experiments, three rats were allocated to each of five groups via simple randomization—control (i.e., no perivascular wrap), PCL (polymeric wrap), PCL+MSC (wrap with MSCs), PCL-Bi (BiNPs-infused wrap), and PCL-Bi+MSC (BiNPs-infused wrap with MSCs)—per planned time point (2 weeks and 8 weeks after implantation). The group allocations were available to all the investigators. In the wrap groups, EO gas-sterilized wrap (1 mm x 5 mm) was placed around the outflow vein following AVF creation. The rats in the MSC groups received a wrap seeded with 1 x 105 MSCs. For each time point, rats were euthanized by CO2 asphyxiation and thoracotomy after imaging.

[0240] Characterization of Scaffold as a Perivascular Wrap around the AVF

[0241] The rat AVFs were imaged using US, mCT, and 18F-FDG PET / CT. A Vevo 2100-LAZR system (VisualSonics, Toronto, ON, Canada) was used to obtain B-mode US images of the AVFs for the assessment of the outflow vein’s wall-to-lumen ratio, a US marker of vascular stenosis. A Skyscan 1276 CMOS mCT system was used to capture mCT images of the wraps. CTAn mCT image analyzer software (Bruker) was used to measure the radiopacity of the wraps in Hounsfield units (HU). An Albira Preclinical PET / SPECT / CT system (Bruker) was used to acquire 18F-FDG PET / CT images. Three hours after 54 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) intravenous injection of the radioactive tracer (37 MBq of 18F-FDG in 200 μL of saline), a 10-minute PET / CT scan was acquired for each rat. The maximum standard uptake values (SUVmax) of the AVFs were determined.

[0242] Experimental results demonstrated that the radiopaque wraps facilitated long- term monitoring via micro-computed tomography. For example, FIG.33 depicts wrap implantation and in-vivo monitoring with micro-CT and US. FIG.33a shows perivascular implantation of the wrap around an outflow vein of an AVF The black arrow indicates the common carotid artery, and the white arrow indicates the perivascular wrap applied onto the external jugular vein. FIGS.33b and 33c show results of the evaluation of radiopacity of the wraps via micro-CT revealed that the infusion of BiNPs facilitated long-term wrap visualization. Red boxes represent the location of the AVFs (HU, Hounsfield units). FIG. 33d shows B-mode ultrasound images of the treatment groups with tracings that represent the lumen of the external jugular vein (yellow), the wall of the external jugular vein (cyan), and the common carotid artery (red). In FIG.33e, evaluation of wall-to-lumen ratios revealed that the mesenchymal stem cell-loaded wraps reduced outflow vein stenosis at as early as 2 weeks after implantation.

[0243] In particular, FIG.33a depicts the implantation of a BiNPs-infused perivascular wrap around the AVF’s outflow vein, and FIGS.33b and 33c depict representative in-vivo mCT images of control (no wrap) vs PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC wraps. At week 2, the average radiopacity of the control, PCL, PCL+MSC, PCL-Bi, and PCL- Bi+MSC groups were 61.07 ± 24.78, 158.7 ± 9.9, 131.9 ± 62.87, 1312 ± 173.8, and 1301 ± 217.7, respectively, while the average radiopacity of the groups at week 4 were 90.72 ± 57.58, 154.8 ± 42.15, 67.68 ± 5.61, 797.2 ± 43.29, and 800.8 ± 40.05, respectively. The polymer wraps loaded with BiNPs (i.e., PCL-Bi and PCL-Bi+MSC) were more radiopaque than the control, PCL wrap, and PCL-MSC wrap at weeks 2 (p < 0.001) and 8 (p < 0.001). There was no significant difference between the BiNPs-loaded wraps with vs without MSCs at either time point, suggesting that MSC loading did not affect the visibility of the radiopaque wraps on mCT.

[0244] The perivascular wraps improved the AVFs’ wall-to-lumen ratio. In particular, FIG.33d compares the B-mode ultrasonographic findings for the AVFs at weeks 2 and 8. At week 2, the average wall-to-lumen ratio of the control, PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC groups were 0.88 ± 0.44, 0.53 ± 0.05, 0.4 ± 0.15, 0.51 ± 0.05, and 0.35 ± 0.08, respectively, while the average wall-to-lumen ratio of the groups at week 4 were 0.95 55 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) ± 0.03, 0.31 ± 0.08, 0.24 ± 0.04, 0.31 ± 0.03, and 0.17 ± 0.08, respectively. FIG.33e shows that the MSC-loaded wraps (i.e., PCL+MSC and PCL-Bi+MSC) promoted a significant reduction in the AVFs’ wall-to-lumen ratio compared to control as early as 2 weeks (p < 0.01) until after 8 weeks (p < 0.001). At week 8, all the treatment groups were found to have a significantly lower wall-to-lumen ratio compared to control (p < 0.01), with the MSC- loaded wraps demonstrating a non-significant trend towards further reduction compared to their non-MSC-loaded counterparts. There was no significant difference between the MSC- loaded PCL vs PCL-Bi wraps at either time point.

[0245] Experimental results demonstrated that the MSC-loaded wraps reduced the 18F- FDG uptake of the AVFs. For example, FIG.34 depicts experimental results of the in-vivo monitoring with 18F-FDG PET (or “PET” for short) / CT imaging. FIG.34a shows PET / CT images of the treatment groups, where white boxes represent the location of the AVFs. In FIG.34b, evaluation of 18F-FDG uptake revealed that the mesenchymal stem cell-loaded wraps reduced the AVFs’ SUVmax at as early as 2 weeks.

[0246] In particular, FIG.34 compares the PET findings for the AVFs at weeks 2 and 8. At week 2, the average 18F-FDG uptake of the control, PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC groups were 7.43 ± 0.93, 6.73 ± 1.17, 4.93 ± 0.87, 5.58 ± 0.97, and 4.97 ± 0.86, respectively, while the average 18F-FDG uptake of the groups at week 4 were 5.76 ± 0.71, 8.11 ± 0.7, 4.07 ± 0.33, 7.42 ± 1.20, and 3.91 ± 1.17, respectively. The MSC-loaded wraps promoted a significant reduction in the AVFs’ 18F-FDG uptake compared to control at 2 weeks (p < 0.05). By week 8, the MSC-loaded wraps had the lowest uptake values and had a significantly lower uptake compared to their non-MSC-loaded counterparts (p < 0.01). At week 8, the PCL wrap had a significantly higher uptake compared to the control (p < 0.05). There was no significant difference between the MSC-loaded PCL vs PCL-Bi wraps at either time point.

[0247] Experimental results demonstrated that the perivascular wraps improved the AVFs’ neointima-to-lumen ratio. For example, FIG.35 depicts the experimental results of hematoxylin and eosin staining and histomorphometry. FIG.35a shows hematoxylin and eosin-stained microscopic images of the AVFs at 2 and 8 weeks after implantation. The dashed line delineates the neointimal layer from the medial layer. Red blood cells that were present in the lumens of the vessels due to incomplete processing of the tissue were omitted from the analysis. FIG.35b shows comparison of the average neointima-to-lumen ratios of 56 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) the treatment groups at 2 and 8 weeks.

[0248] In particular, FIG.35 compares the histological findings for the AVFs at weeks 2 and 8. At week 2, the average neointima-to-lumen ratio of the control, PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC groups were 2.05 ± 0.89, 0.52 ± 0.22, 0.28 ± 0.1, 0.76 ± 0.74, and 0.36 ± 0.11, respectively, while the average neointima-to-lumen ratio of the groups at week 4 were 5.26 ± 0.53, 0.25 ± 0.06, 0.22 ± 0.02, 0.4 ± 0.21, and 0.28 ± 0.18, respectively. All the treatment groups were found to have a significantly lower neointima-to-lumen ratio compared to control at weeks 2 (p < 0.01) and 8 (p < 0.001). At both time points, the MSC- loaded wraps demonstrated non-significant reductions in the neointima-to-lumen ratio compared to their non-MSC-loaded counterparts.

[0249] Histomorphometry and Immunohistochemistry of a Perivascular Wrap around the AVF

[0250] After euthanasia, the outflow veins were harvested, fixed, processed, and embedded in paraffin. Paraffin-embedded sections were divided for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining with the following markers: (1) von Willebrand factor (vWF), which is highly expressed in activated endothelial cells in response to hemodynamic stress or vascular injury; (2) hypoxia-inducible factor 1 alpha (HIF-1α), highly expressed in cells in hypoxic conditions; (3) ionized calcium-binding adaptor molecule 1 (Iba-1), highly expressed in macrophages, which are important mediators of inflammation; (4) Ki-67, a nuclear protein associated with cellular proliferation; and (5) RFP, an indicator of the MSCs that were loaded onto the wraps. An Aperio LV1 real-time digital pathology system (Leica Biosystems, Buffalo Grove, IL) was used to capture images from the stained slides. The areas covered by the lumen and the neointimal layer were quantified to determine each AVF’s neointima-to-lumen ratio, a histologic marker of vascular stenosis. The HALO image analysis platform (Indica Labs, Albuquerque, NM) was used to count IHC-stained cells.

[0251] Experimental results demonstrated that BiNP infusion did not affect in vivo MSC retention. For example, FIG.36 depicts in-vivo MSC retention. FIG.36a shows immunohistochemical staining of the RFP in the AVFs reveals the retention of MSCs within the MSC-loaded wraps. An MSC pellet is also shown for comparison. In FIG.36b, calculation of MSC retention showed no difference between the MSC-loaded wraps with vs 57 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) without bismuth nanoparticles.

[0252] IHC staining was utilized to ascertain the presence of RFP-expressing MSCs within the AVFs, as tissue processing can result in the loss of fluorescence of certain fluorescent proteins. FIG.36 demonstrates that MSCs were retained within the MSC-loaded wraps. The retention of the MSCs, as graphed in FIG.36, was determined by dividing the number of RFP-stained MSCs by the total number of seeded RFP-expressing MSCs. The retention of MSCs was reduced from week 2 to week 8 by around 15%. However, there was no significant difference in the retention of MSCs between the MSC-loaded wraps with vs without BiNPs at week 2 (PCL+MSC = 21.18 ± 2.25% vs. PCL-Bi+MSC = 19.53 ± 1.77%) and at week 8 (PCL+MSC = 4.57 ± 1.6% vs. PCL-Bi+MSC = 4.82 ± 0.88%), suggesting that BiNP infusion did not affect the wrap’s ability to deliver MSCs onto the outflow vein of AVFs.

[0253] Experimental results demonstrated that the MSC-loaded wraps reduced neointimal expression of vWF, HIF-1α, and Iba-1. For example, FIG.37 depicts immunohistochemical analysis of the neointima. FIG.37a shows immunohistochemically stained slides of the neointima of the AVFs at week 8. The dashed line delineates the neointimal layer from the medial layer. The NIH markers evaluated were vWF, HIF-1α, and Iba-1. Red blood cells and artifacts that were present in the lumens of the vessels due to incomplete processing of the tissue were omitted from the analysis. FIGS.37b-37d show comparison of the percentage of cells positive for vWF, HIF-1α, and Iba-1 within the neointima at 8 weeks.

[0254] After 8 weeks, the numbers of cells positive for vWF, HIF-1α, and Iba-1 within the neointima were determined to evaluate endothelial dysfunction, hypoxia, and inflammation within the layer, respectively (FIG.37). The average percentage of cells positive for vWF in the control, PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC groups were 70.96 ± 5.26, 62.71 ± 10.48, 29.93 ± 8.22, 53.93 ± 4.05, and 29.89 ± 2.08, respectively. The average percentage of cells positive for HIF-1α in the groups were 43.8 ± 5.93, 24.7 ± 4.07, 8.15 ± 8.05, 23.99 ± 6.36, and 11.53 ± 8.38, respectively. The average percentage of cells positive for Iba-1 in the groups were 9.04 ± 3.23, 22.82 ± 8.21, 4.11 ± 3.42, 23.14 ± 6.46, and 4.8 ± 4.85, respectively.

[0255] All the treatment groups had a significantly lower percentage of cells expressing HIF-1α compared to the control AVF (p < 0.05). The MSC-loaded wraps had significantly 58 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) lower vWF and Iba-1 staining compared to the non-MSC wraps (p < 0.05). Furthermore, the MSC-loaded wraps demonstrated a non-significant trend toward reduction in HIF-1α staining compared to the non-MSC wraps. There was no difference between the MSC- loaded PCL vs PCL-Bi wraps for any of the examined markers. These results suggest that the addition of MSCs promotes better attenuation of endothelial dysfunction and inflammation and could also potentially improve the inhibition of hypoxia by perivascular wraps.

[0256] Experimental results demonstrated that the MSC-loaded wraps reduced overall expression of HIF-1 α, Iba-1, and Ki-67 in AVFs. For example, FIG.38 depicts immunohistochemical analysis of the whole outflow vein. FIG.38a shows immunohistochemically stained slides of the AVFs at week 8. The markers evaluated were NIH markers HIF-1α and Iba-1 and cellular proliferation marker Ki-67. Red blood cells and artifacts that were present in the lumen of the vessels due to incomplete processing of the tissue were omitted from the analysis. FIG.38b shows comparison of the percentages of cells positive for HIF-1α, Iba-1, and Ki-67 within the whole AVF at 8 weeks.

[0257] The numbers of cells positive for HIF-1α, Iba-1, and Ki-67 within the AVFs were determined to evaluate overall hypoxia, inflammation, and cellular proliferation, respectively (FIG.38). The average percentage of cells positive for HIF-1α in the control, PCL, PCL+MSC, PCL-Bi, and PCL-Bi+MSC groups were 21.78 ± 4.17, 33.31 ± 9.31, 7.75 ± 5.98, 20.36 ± 10.74, and 13.02 ± 5.26, respectively. The average percentage of cells positive for Iba-1 in the groups were 22.2 ± 10.78, 90.8 ± 5.52, 61.99 ± 2.95, 74.47 ± 8.2, and 64.6 ± 4.76, respectively. The average percentage of cells positive for Ki-67 in the groups were 4.41 ± 1.66, 5.89 ± 1.24, 4.11 ± 0.98, 5.5 ± 1.32, and 3.94 ± 1.56, respectively.

[0258] For HIF-1α, the MSC-loaded groups were found to have significantly lower percentages of positive cells compared to the PCL group (p < 0.05). Although not statistically significant, the MSC-loaded groups also had a lower percentage of positive cells compared to the control group. For Iba-1, all the treatment groups were found to have a significantly higher percentage of positive cells compared to the control group (p < 0.001), but the MSC groups were found to have a significantly lower percentage of positive cells compared to the PCL group (p < 0.01). For Ki-67, there was no significant difference between the treatment groups, but the MSC groups demonstrated a non-significant trend toward reduction compared to the other treatment groups. These results suggest that the 59 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) addition of MSCs to PCL wrap can significantly reduce hypoxia and inflammation within the whole AVF and potentially reduce pathologic cellular proliferation.

[0259] A BiNPs-infused bioresorbable polymer wrap (e.g., as a perivascular wrap) that can deliver MSCs onto AVFs and can be monitored over time via conventional X-ray imaging techniques is provided. The infusion of BiNPs into the wraps resulted in significant changes in some physicochemical properties, such as fiber diameter and pore size, but these changes did not affect the wraps’ biocompatibility, capacity for loading and delivery of MSCs, and, ultimately, in-vivo efficacy.

[0260] It was found that all the treatment groups demonstrated improved wall-to-lumen and neointima-to-lumen ratios after 8 weeks compared to non-wrapped AVFs. The MSCs- loaded wraps demonstrated further reduction compared to their non-MSC-loaded counterparts, suggesting that the delivery of MSCs could potentially attenuate pathologic NIH. To further understand the effect of MSCs on the maturation of AVFs, IHC studies were conducted to evaluate the mechanisms that underlie NIH, such as endothelial dysfunction, hypoxia, and inflammation. It was found that all the treatment groups significantly attenuated hypoxia within the neointima. However, compared to the wraps without MSCs, the MSCs-loaded wraps promoted further inhibition of endothelial dysfunction and inflammation within the neointima. Moreover, an examination of all the layers of the AVFs showed that, compared to the PCL wraps, the MSCs-loaded wraps reduced the overall expression of markers associated with hypoxia and inflammation. These results indicate that although mechanical support alone can improve AVF maturation, which is concordant with recent studies in humans, the addition of MSCs could further enhance AVF maturation through the inhibition of multiple pathways that underlie NIH.

[0261] The improvement in AVF maturation that occurred with perivascular wraps can be ascribed to their capacity to regulate local hemodynamic stress. Immediately after AVF creation, the outflow vein is known to experience turbulent wall shear stress and high mechanical loads that may promote endothelial dysfunction and hyperproliferation. Therefore, the addition of a perivascular wrap, which could reduce flow turbulence and prevent overexpansion injury within the vascular wall, may enhance the patency of a maturing AVF. Although this protective mechanism can reduce endothelial dysfunction, which may also reduce local hypoxia within the neointima, it was found that it was not sufficient to attenuate the overall expression of NIH markers within the AVFs. In contrast, MSCs attenuated the NIH markers both within the neointima and across all layers, except 60 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) for Iba-1, compared to the control group. This protective phenomenon may be explained by the broad immunomodulatory and regenerative properties of MSCs that are mediated by MSCs-secreted cytokines and paracrine factors, which promote vascular homeostasis and recruitment of regulatory immune cells. The presence of more Iba-1-positive macrophages in the wrap groups compared to the control group was expected since these cells are responsible for the resorption of the wraps over time.

[0262] It was also found that the delivery of MSCs onto the AVFs promoted a significant reduction in 18F-FDG uptake as seen on PET images. It is well established that 18F-FDG accumulates in tissues with high glucose demand, such as proliferating cells and active inflammatory immune cells. In addition, glucose consumption also increases in the setting of hypoxia due to enhanced glycolysis to maintain energy production without requiring oxygen. Hence, the reduction in uptake seen with MSC-loaded wraps may be attributed to an overall reduction in hypoxia, inflammation, and cellular proliferation in the AVFs. These findings validate the role of 18F-FDG PET imaging in the surveillance of AVF failure and support the hypothesis that molecular imaging may provide insights into the underlying mechanisms of failure that are not apparent with conventional imaging and routine histology.

[0263] The experimental results described in the present disclosure support the use of a radiopaque perivascular wrap to enhance the effectiveness of MSCs in reducing NIH. Specifically, these results show that BiNP infusion of the perivascular wrap enables the long-term monitoring of AVFs via mCT while maintaining the advantages of the wraps for supporting AVF maturation. Furthermore, these results validate the utility of 18F-FDG PET in identifying underlying changes within the vascular wall that lead to NIH. Hence, PET has the potential to be used as a non-invasive indicator to evaluate individuals who are at risk of developing pathological inflammation or hypoxia and, therefore, are at risk of AVF failure. In conclusion, MSC-seeded radiopaque BiNPs-infused perivascular wraps have been shown to enhance AVF maturation in clinical settings, according to the present disclosure.

[0264] EXAMPLE 5

[0265] Overview

[0266] In some implementations, a perivascular wrap with enhanced radiologic visibility and drugs capable of attenuating NIH is provided. In some implementations, the perivascular wrap includes PCL loaded with BiNPs and an NIH-attenuating drug such as 61 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) ROSU and RAPA. Example implementations of the BiNPs-and-drug-infused perivascular wrap include the following groups: control (i.e., without wrap), PCL-Bi (i.e., wrap with BiNPs), PCL-Bi-ROSU, and PCL-Bi-RAPA. The example implementations were tested on the AVFs of a total of 24 Sprague-Dawley rats with induced CKD.

[0267] It was found that BiNPs significantly improved the BiNPs-and drug-infused perivascular wrap’s radiopacity without affecting biocompatibility. The drug release profiles of ROSU (hydrophilic drug) and RAPA (hydrophobic drug) differed significantly. ROSU demonstrated a burst release followed by gradual tapering over 8 weeks, while RAPA demonstrated a gradual release similar to that of the hydrophobic BiNPs. In-vivo investigations revealed that both drug-loaded wraps can reduce vascular stenosis on ultrasonography and histomorphometry as well as reduce 18F-FDG uptake on PET. Immunohistochemical studies revealed that PCL-Bi-ROSU primarily attenuated endothelial dysfunction and hypoxia in the neointimal layer, while PCL-Bi-RAPA modulated hypoxia, inflammation, and cellular proliferation across the whole outflow vein. In various implementations, the controlled delivery of drugs with different properties and mechanisms of action against NIH through implementations of the multifunctional, radiopaque BiNPs- infused perivascular wrap provided herein can improve imaging and histologic parameters of AVF maturation.

[0268] Prior studies have shown that drug delivery offers a promising solution for reducing AVF failure rates by enabling the administration of higher drug concentrations with reduced systemic side effects. A variety of targeted drug delivery mechanisms, including polymer scaffolds and nanoparticle carriers, have the potential for targeting AVFs. As described in detail above, electrospun biodegradable polymers, such as PCL, can create fibrous scaffolds that mimic the vascular extracellular matrix and seamlessly integrate with blood vessels. However, the radiolucent nature of most polymeric delivery systems hinders long-term in-vivo surveillance, which prevents a comprehensive evaluation of device safety and efficacy. To address this limitation, the present disclosure provides implementations in which radiopaque perivascular wraps infused with BiNPs and loaded with at least one of ROSU or RAPA, drugs with distinct properties and mechanisms against NIH.

[0269] Metallic nanoparticles, such as AuNPs and BiNPs, provide an opportunity to enhance the detectability of polymer-based devices using standard X-ray techniques. It has been known that BiNPs make excellent contrast agents as they can be effectively 62 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) differentiated from common vascular contrast agents based on iodine. ROSU is one of the more potent statins and is hydrophilic in nature, while RAPA, also known as sirolimus, is a hydrophobic agent that belongs to a class of drugs known as mechanistic target of RAPA (mTOR) inhibitors. It inhibits cell proliferation by halting the progression of the cell cycle from the G1 to the S phase, which makes it effective in inhibiting NIH associated with stent placement for the management of coronary artery disease.

[0270] The efficacy of a RAPA-eluting collagen membrane for inhibiting NIH has been previously demonstrated for arteriovenous grafts, but the potential of RAPA-loaded perivascular wraps has never been evaluated for AVFs. Specifically, the use of BiNP- infused perivascular wraps for optimizing the delivery of either ROSU or RAPA has yet to be explored. By combining these therapeutic agents with a radiopaque, mechanically supportive wrap, AVF maturation is enhanced and long-term in-vivo monitoring of device safety and efficacy is enabled. This study investigates the release profiles, biocompatibility, and efficacy of both ROSU-loaded (e.g., PCL-Bi-ROSU) and RAPA-loaded (e.g., PCL-Bi- RAPA), BiNPs-infused electrospun perivascular wraps against NIH. The experimental results described herein demonstrate that the utilization of a radiopaque wrap to deliver drugs against NIH could enhance AVF maturation.

[0271] The following quantitative data were presented as means ± standard deviations and analyzed using a two-tailed t test or analysis of variance with GraphPad Prism software, version 10.0.3 (GraphPad, San Diego, CA). All animals were included in the analysis. Statistical significance was defined as p < 0.05.

[0272] Fabrication and Characterization of an BiNP-Infused Polymer Scaffold

[0273] BiNPs were synthesized following a published protocol. BiNPs were mixed with PCL (Sigma-Aldrich, St. Louis, MO) at a 1:4 ratio in a chloroform / methanol (8:3) to prepare 12% w / v solutions. For drug-loaded wraps, either ROSU or RAPA was added to the mixtures at 40 mg / mL. The wraps were fabricated using a Spraybase system (Avectas, Maynooth, Ireland). The synthesis and characterization of wraps, in vitro assays, surgery, imaging, and histological analysis were performed as described in previous studies.

[0274] The fiber diameter and pore size of the perivascular wraps were determined using a Nova NanoSEM scanning electron microscope (SEM, Field Electron and Ion Company, Hillsboro, OR). For fiber diameter measurement, 5 SEM images were captured from random areas, and then 20 fibers were measured per image using ImageJ (National 63 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) Institutes of Health, Bethesda, MD). The porosity of each wrap was evaluated through the calculation method and liquid intrusion method. An STA PT 1000 thermogravimetric analyzer (Linseis, Selb, Germany) was used to determine the melting temperature (Tm) and glass transition temperature (Tg) of the wraps. An MTESTQuattro materials testing system (ADMET, Norwood, MA) was used to measure maximum stress, while a Skyscan 1276 CMOS micro-CT system (Bruker, Billerica, MA) was used to capture the wraps’ X-ray and micro-CT images.

[0275] FIG.39 shows representative SEM, photographic, X-ray, and micro-CT images of the electrospun polymeric wraps described herein. SEM images show that the electrospun wraps include interwoven nanofibers. The addition of BiNPs transformed the previously white wrap into dark gray wraps, enhancing its visibility on X-ray and micro-CT imaging. The physicochemical properties of the electrospun wraps are summarized in Table 9. All the BiNP-loaded wraps had a significantly reduced fiber diameter compared to PCL (p < 0.001). The PCL-Bi-ROSU wrap had the thinnest fiber diameter, but there was no significant difference between all the BiNP-loaded wraps. The PCL-Bi-ROSU wrap also had the smallest pore size, and it had a significantly smaller pore size compared to the PCL wrap (p < 0.01). The melting temperature (Tm) of the wraps is well above body temperature (approximately 37°C), ensuring that the wraps will maintain their shape and structural integrity after implantation around the vessel. The glass transition temperature (Tg) of the wraps is well below body temperature, ensuring that the wraps will not be in a crystalline state and instead will be flexible enough for the controlled release of drugs over an extended period. There was no significant difference between the wraps in terms of porosity and tensile strength. Table 9. Physicochemical properties of the perivascular wraps. Diameter Pore size Porosity (%) Tm Maximum 2 Tg(°C) (nm) (μm )Calculated Intrusion(°C)stress (mPa)320.60 ± 27.70 ± 76.75 ± PCL 93.55 ± 4.39 52.53 -69.61 9.17 ± 0.31 86.85 7.88 8.06 225.00 ± 25.91 ± 68.25 ± PCL-Bi 92.79 ± 0.87 54.58 -68.51 7.82 ± 1.07 92.89 4.78 7.07 PCL-Bi- 207.30 ± 23.51 ± 67.97 ± 92.49 ± 0.64 54.40 -68.92 8.91 ± 1.02 ROSU 85.04 3.70 4.85 64 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) PCL-Bi- 227.2 ± 24.90 ± 69.84 ± 93.83 ± 1.46 51.09 -69.58 9.07 ± 1.19 Rapa 111.8 7.57 9.72

[0276] In-Vitro Cytotoxicity, Drug Release, and Hemocompatibility Assay

[0277] The synthesized wraps were incubated in phosphate-buffered saline at 37°C with vigorous shaking for up to 8 weeks to simulate the degradation of polymers via hydrolysis. The soaking solution was removed and tested for bismuth and drug content. Then, the soaking solution was replaced with the same volume and repeated at every time point. Bismuth content was quantified using inductively coupled plasma optical emission spectrometry (Varian 720-ES, Agilent, Santa Clara, CA). On the other hand, the amount of ROSU or RAPA released was calculated from the absorption of the supernatant at 244 nm or 280 nm, respectively, using a UV–Vis spectrophotometer (Cary 60, Agilent, Santa Clara, CA), respectively. The amount of the BiNPs and drug released were divided by the theoretical total amount loaded to determine the percentage of substance released.

[0278] The wraps’ biocompatibility was assessed through the evaluation of cytotoxicity, hemolysis, and clotting time. For cytotoxicity, the gas-sterilized wraps were incubated in Dulbecco's Modified Eagle Medium (DMEM, Corning, Corning, NY) for 24 hours. Afterward, vascular cells—immortalized human vascular endothelial cells (EC-RF24, American Type Culture Collection (ATCC), Manassas, VA) and primary mouse vascular aortic smooth muscle cells (MOVAS, ATCC)—were exposed to treated media at increasing concentrations. The acute cytotoxicity of the wraps was evaluated through the alamarBlue™ cell viability assay (Invitrogen, Waltham, MA) after 72 hours.

[0279] Hemolysis was evaluated following incubation of the wraps in an erythrocyte solution extracted from heparinized rat blood for an hour at 37°C. The supernatants‘ absorbance was read at 540 nm to evaluate the lysis of erythrocytes. On the other hand, clotting time was evaluated by incubating the wraps in platelet-poor plasma prepared from citrated rat blood for 30 minutes at 37°C, and subsequently adding brain cephalin and CaCl2solution to determine the aPTT.

[0280] FIG.40 depicts In vitro nanoparticle release, drug release, and biocompatibility. FIG.40a shows cumulative BiNPs and drug release profiles of the wraps over 8 weeks. FIG.40b shows results of the evaluation of the wraps’ effect on the viability of immortalized human vascular endothelial cells (EC-RF24) and primary mouse vascular aortic smooth muscle cells (MOVAS). FIG.40c shows results of the evaluation of the 65 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) wraps’ effect on hemolysis and aPTT.

[0281] In particular, FIG.40a demonstrates the in vitro release of BiNPs and drugs from the wraps. The BiNP release from the BiNP-loaded wraps exhibited a similar gradual release pattern. At 8 weeks, PCL-Bi, PCL-Bi-ROSU, and PCL-Bi-RAPA have released 6.51 ± 2.73 %, 10.80 ± 2.19 %, and 7.72 ± 4.43 % of the loaded BiNPs, respectively. The high variability in BiNP release at week 8 may be explained by variability in fiber degradation over time. The degradation rate of thinner fibers in electrospun scaffolds is often higher compared to thicker fibers because of their greater surface area-to-volume ratio, which translates to increased contact with the surrounding environment. Since electrospinning often produces scaffolds with a mix of relatively thinner and thicker fibers, the variability in their proportions can result in variable degradation rates across different scaffolds. This, in turn, can lead to variable release profiles of embedded nanoparticles over time, as the degradation of the scaffold directly influences the release kinetics.

[0282] PCL-Bi-ROSU and PCL-Bi-RAPA demonstrated controlled drug release patterns. PCL-Bi-ROSU exhibited an initial burst release of 78.50 ± 18.10 %, and the release pattern tapered over the course of 8 weeks. At week 8, the cumulative release of ROSU was 92.90 ± 5.69 %. In contrast, PCL-Bi-RAPA demonstrated a gradual release of RAPA similar to the pattern of BiNP release. At 8 weeks, 80.60 ± 4.98 % of the loaded RAPA has been released from the wrap. FIG.40 demonstrates the wraps’ effect on the viability of EC-RF24 and MOVAS cells, as well as the wraps’ impact on hemolysis and clotting time. The wraps did not exhibit acute toxicity against EC-RF24 and MOVAS cells. Moreover, the wraps did not promote any significant changes in hemolysis and clotting time.

[0283] Formation of an AVF and Application of the Scaffold as a Perivascular Wrap

[0284] All animal experiments have been approved by the MD Anderson Cancer Center’s Institutional Animal Care and Use Committee (Protocol No.00001882-RN01). Sprague-Dawley rats (400 SAS SD, Charles River Laboratories, Wilmington, MA) underwent a two-step 5 / 6th nephrectomy procedure for the induction of CKD. After 4 weeks, AVFs were created through an end-to-side anastomosis of the external jugular vein with the common carotid artery.18 After which, 24 rats were distributed to the treatment groups—control (e.g., AVF without wrap), PCL-Bi, PCL-Bi-ROSU, and PCL-Bi-RAPA. The gas-sterilized wraps (1 mm x 5 mm) were implanted around the outflow vein 66 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) immediately after AVF creation. The rats were euthanized via thoracotomy after imaging.

[0285] In-Vivo Imaging of Scaffold as a Perivascular Wrap around the AVF

[0286] A Skyscan 1276 CMOS micro-CT system (Bruker, Billerica, MA) was utilized to determine the radiopacity of the implanted wraps in Hounsfield units (HU). A Vevo 2100-LAZR system (VisualSonics, Toronto, ON, Canada) with a 15-MHz probe was utilized to capture B-mode ultrasound (US) images to assess each AVF’s wall thickness and luminal diameter. The AVF’s wall-to-lumen ratio, which is a US marker of vascular stenosis, was derived from these measurements. An Albira Preclinical PET / SPECT / CT system (Bruker) was utilized to obtain 18F-FDG PET / CT images, and the maximum standard uptake values (SUVmax) for each AVF were determined.

[0287] FIG.41 depicts results of the monitoring of wrap placement and integrity with micro-CT. FIG.41a shows in-vivo micro-CT images of the AVFs at weeks 2 and 8. In FIG. 41b, evaluation of the wraps’ radiopacity via micro-CT showed that the incorporation of bismuth nanoparticles increased the Hounsfield unit (HU) values of the wraps, which can facilitate monitoring of the positioning and structural integrity of the wraps in vivo.

[0288] FIG.41 shows representative in vivo micro-CT images of the AVFs at weeks 2 and 8. All the BiNP-loaded wraps were significantly more radiopaque than control at weeks 2 (p < 0.001) and 8 (p < 0.01). There was no significant difference between the BiNP- loaded wraps at both time points. Among the BiNP-loaded wraps, PCL-Bi-ROSU demonstrated the highest radiopacity at week 2. However, it had the lowest radiopacity at week 8, which correlates with the in vitro BiNP release profile of the wraps. A Skyscan 1276 CMOS micro-CT system (Bruker, Billerica, MA) was utilized to determine the radiopacity of the implanted wraps in Hounsfield units (HU). A Vevo 2100-LAZR system (VisualSonics, Toronto, ON, Canada) with a 15-MHz probe was utilized to capture B-mode ultrasound (US) images to assess each AVF’s wall thickness and luminal diameter. The AVF’s wall-to-lumen ratio, which is a US marker of vascular stenosis, was derived from these measurements. An Albira Preclinical PET / SPECT / CT system (Bruker) was utilized to obtain 18F-FDG PET / CT images, and the maximum standard uptake values (SUVmax) for each AVF were determined.

[0289] FIG.42 depicts results of the evaluation of AVFs’ wall-to-lumen ratio via ultrasonography. FIG.42a shows B-mode ultrasonographic images of the AVFs with tracings representing the lumen of the external jugular vein (yellow), the wall of the external 67 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) jugular vein (cyan), and the common carotid artery (red). In FIG.42b, the evaluation of wall-to-lumen ratios showed that the drug-loaded wraps can further attenuate outflow vein stenosis when compared to PCL-Bi wraps after 8 weeks.

[0290] In particular, FIG.42 demonstrates B-mode US images of the AVFs at weeks 2 and 8. All the wraps caused a significant reduction in the wall-to-lumen ratio of the AVFs compared to control as early as 2 weeks (p < 0.001). This protective effect was maintained even after 8 weeks (p < 0.001). In addition, when compared to PCL-Bi, PCL-Bi-ROSU and PCL-Bi-RAPA caused a further reduction in outflow vein stenosis. There was no significant difference between the drug-loaded wraps (PCL-Bi-ROSU and PCL-Bi-RAPA) at both time points.

[0291] FIG.43 depicts results of the evaluation of the AVFs’ 18F-FDG uptake via positron emission tomography. FIG.43a shows PET / CT images of the AVFs (white box). In FIG.43b, evaluation of 18F-FDG uptake showed that the drug-loaded wraps can decrease the AVFs’ maximum standard uptake value (SUV Max) as early as 2 weeks..

[0292] In particular, FIG.43 illustrates the PET / CT findings for the AVFs at weeks 2 and 8. At 2 weeks, PCL-Bi-ROSU and PCL-Bi-RAPA caused a significant reduction in the AVFs’ 18F-FDG uptake than control (p < 0.05). After 8 weeks, both the drug-loaded wraps had a significantly lower uptake compared to PCL-Bi (p < 0.01). The control AVFs also had a significantly lower uptake than PCL-Bi (p < 0.05). As shown, the drug-loaded wraps had a reduced uptake than the control. There was no significant difference between the drug- loaded wraps at both time points.

[0293] FIG.44 depicts results of the evaluation of the AVFs’ neointima-to-lumen ratio via histomorphometry. FIG.44a shows hematoxylin and eosin-stained slides of the AVFs at 2 and 8 weeks. The dashed line separates the neointima from the medial layer. Red blood cells and artifacts in the lumen were excluded from the analysis. FIG.44b shows results of the evaluation of the neointima-to-lumen ratios of the AVFs after 2 and 8 weeks.

[0294] In particular, FIG.44 illustrates the histological findings for the AVFs at weeks 2 and 8. All the treatment groups had a significantly lower neointima-to-lumen ratio than control at weeks 2 (p < 0.01) and 8 (p < 0.001). For both weeks, the drug-loaded wraps also showed a non-significant trend toward further reduction in the neointima-to-lumen ratio compared to PCL-Bi.

[0295] Histomorphometry and Immunohistochemistry of a Perivascular Wrap around 68 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) the AVF

[0296] The AVFs were harvested, fixed, processed, and embedded for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining for: (1) von Willebrand Factor (vWF), highly expressed in endothelial cells activated by hemodynamic stress or injury; (2) hypoxia-inducible factor 1 alpha (HIF-1α), highly expressed in cells experiencing hypoxia; (3) ionized calcium-binding adaptor molecule 1 (Iba-1), highly expressed in macrophages, which mediate inflammation; and (4) Ki-67, a nuclear marker of cellular proliferation. An Aperio LV1 real-time digital pathology system (Leica Biosystems, Buffalo Grove, IL) was utilized to capture and analyze slide images. The neointima-to-lumen ratio, which is a histomorphometric marker of vascular stenosis, was calculated from neointimal and luminal areas. The Halo image analysis platform (Indica Labs, Albuquerque, NM) was utilized to determine the percentage of IHC-stained cells.

[0297] FIG.45 depicts results of evaluation of the AVFs’ neointimal expression of vWF, HIF-1α, Iba-1, and Ki-67. FIG.45a shows immunohistochemically stained slides of the AVFs’ neointimal layer at week 8. The dashed line separates the neointima from the medial layer. Red blood cells and artifacts in the lumen were excluded from the analysis. FIG.45b shows results of the evaluation of the percentage of cells that are positive for von Willebrand Factor (vWF), hypoxia-inducible factor 1 alpha (HIF-1α), ionized calcium- binding adaptor molecule 1 (Iba-1), and Ki-67 within the neointima after 8 weeks.

[0298] The percentage of cells positive for vWF, HIF-1α, Iba-1, and Ki-67 was identified to evaluate endothelial dysfunction, hypoxia, inflammation, and cellular proliferation within the neointimal layer, respectively. FIG.45 illustrates the IHC findings for the neointimal layer of the AVFs after 8 weeks. All the treatment groups demonstrated a reduction in the percentage of cells that are positive for vWF compared to the control. However, only PCL-Bi-ROSU demonstrated a statistically significant reduction (p < 0.05). All the treatment groups had a significantly lower percentage of cells that were positive for HIF-1α than control (p < 0.05), with PCL-Bi-ROSU showing the lowest percentage among all the groups. The drug-loaded wraps caused a non-significant trend toward reduction in the percentage of cells that are positive for Iba-1 and Ki-67 compared to PCL-Bi. These suggest that the incorporation of ROSU can significantly mitigate endothelial dysfunction and hypoxia in the neointimal layer. Moreover, both ROSU and RAPA potentially improve 69 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) the attenuation of inflammation and cellular proliferation within perivascular wraps.

[0299] FIG.46 depicts results of the evaluation of the AVFs’ overall expression of HIF- 1 α, Iba-1, and Ki-67. FIG.46a shows immunohistochemically stained slides of the AVFs after 8 weeks. The dashed line separates the neointima from the medial layer. Red blood cells and artifacts in the lumen were excluded from the analysis. FIG.46b shows results of the evaluation of the percentage of cells that are positive for hypoxia-inducible factor 1 alpha (HIF-1α), ionized calcium-binding adaptor molecule 1 (Iba-1), and Ki-67 within the whole AVF after 8 weeks.

[0300] The percentage of cells positive for HIF-1α, Iba-1, and Ki-67 within the AVFs was identified to evaluate overall hypoxia, inflammation, and cellular proliferation, respectively. FIG.46 illustrates the IHC findings for the whole AVFs after 8 weeks. For HIF-1α, PCL-Bi-Rapa demonstrated the lowest percentage, which is significantly lower compared to PCL-Bi and PCL-Bi-ROSU (p < 0.05). For Iba-1, all the treatment groups had a significantly higher percentage of positive cells than the control group (p < 0.01), but PCL-Bi-RAPA had a lower percentage of positive cells than the PCL-Bi and PCL-Bi- ROSU. For Ki-67, PCL-Bi-RAPA demonstrated the lowest percentage, which is significantly lower compared to PCL-Bi-ROSU (p < 0.05). These suggest that the incorporation of RAPA can significantly attenuate hypoxia and cellular proliferation within the whole AVF and potentially reduce pathologic inflammation within perivascular wraps.

[0301] FIG.47 depicts a cross-sectional image of a sample 800 including a perivascular wrap 820 wrapped around a vascular structure 810 (e.g., vessel), where the perivascular wrap 820 is infused with BiNPs (not depicted) and radiopaque drug particles 830 including ROSU or RAPA. The vascular structure 810 includes a vascular wall 812 surrounding a lumen 814. The perivascular wrap 820 is implemented around an outer surface of the vascular wall 812. In some implementations, the radiopaque drug particles 830 can be released from the perivascular wrap 820 through the vascular wall 812 and into the lumen 814 in a controlled manner as described herein.

[0302] Implementations of the present disclosure have utilized electrospinning to integrate drug particles that can inhibit AVF failure (e.g., ROSU and RAPA) into a BiNPs- infused wrap which can confer mechanical support and radiologic visibility. In some implementations, the BiNPs-and-drug-infused wrap is used as a multidimensional intervention for targeting AVF failure that can be monitored over time through conventional 70 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) imaging modalities.

[0303] The experimental results revealed that the addition of BiNPs significantly improved the radiopacity of the wraps. This is expected since BiNPs have demonstrated sufficient radiopacity when used as a contrast agent for polymeric medical devices in previous studies. This feature may be readily taken advantage of for AVF surveillance and device monitoring since Bi is one of the least toxic heavy metals, and its typical pharmaceutical formulations (e.g., bismuth subcitrate, bismuth subcitrate potassium, bismuth subsalicylate, and ranitidine bismuth citrate) have been safely used for the treatment of various disorders for many years. BiNPs incorporated into the wraps have an average diameter of 3.44 nm and thus are expected to be cleared by the kidneys since the capillary walls of the glomerulus usually have a filtration-size threshold of 6 nm.

[0304] It was found that the incorporation of BiNPs reduced the average fiber diameter of the wraps. This phenomenon may be attributed to the increased conductivity of the solution after the addition of metallic nanoparticles. Similarly, the addition of ROSU calcium salt led to a reduction in average fiber diameter and average pore size due to the increased conductivity conferred by calcium. Nonetheless, these differences did not affect the BiNPs-and-drug-infused wrap’s porosity, mechanical strength, and biocompatibility. However, the drug release profiles of ROSU and RAPA differed significantly. ROSU demonstrated a burst release followed by gradual tapering over the course of 8 weeks, while RAPA demonstrated a gradual release similar to that of the BiNPs. The hydrophilic nature of ROSU facilitated its more immediate release from the hydrophobic PCL wrap, while the hydrophobic character of RAPA and BiNPs promoted slower dissociation from the hydrophobic wrap over time. Nonetheless, a stable release of ROSU was still achieved after the initial burst release.

[0305] In-vivo imaging demonstrated that the multifunctional drug-loaded wraps can be monitored longitudinally through micro-CT, reduce vascular stenosis on ultrasonography, and reduce 18F-FDG uptake on PET / CT. The high radiopacity conferred by the infusion of BiNPs was maintained by the BiNP-loaded wraps over the course of 8 weeks. Despite having different drug release profiles, both PCL-Bi-ROSU and PCL-Bi-RAPA had comparable visibility on CT imaging. The PCL-Bi-ROSU scaffold had the highest radiopacity at week 2 most likely due to an increase in attenuation conferred by calcium 71 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) from the ROSU calcium salt.

[0306] All the wraps demonstrated a significant reduction in the AVFs’ wall-to-lumen ratio versus control, but both drug-loaded wraps promoted a further reduction in outflow vein stenosis when compared to PCL-Bi alone on ultrasonography. Both PCL-Bi-ROSU and PCL-Bi-RAPA also had a significantly lower 18F-FDG uptake on PET / CT compared to PCL-Bi. These results correlate with histomorphometry wherein both drug-loaded wraps showed a non-significant trend towards further reduction in neointima-to-lumen ratio compared to PCL-Bi.

[0307] While PCL-Bi-RAPA and PCL-Bi-ROSU appear to exert comparable effects on AVFs, it is possible that their respective mechanisms of action are distinct. Hence, IHC studies were conducted to examine the mechanisms that underlie NIH, such as endothelial dysfunction, hypoxia, inflammation, and cellular proliferation. All the treatment groups demonstrated a reduction in endothelial dysfunction and neointimal hypoxia. Similar to histomorphometry, both PCL-Bi-RAPA and PCL-Bi-ROSU have demonstrated a further reduction in endothelial dysfunction and neointimal hypoxia compared to PCL-Bi. Both drug-loaded wraps also had lower neointimal inflammation and neointimal cellular proliferation. In some implementations, the reduction in endothelial dysfunction, neointimal hypoxia, and neointimal cellular proliferation was most pronounced with PCL-Bi-ROSU. In some implementations, PCL-Bi-RAPA demonstrated the lowest overall hypoxia and overall cellular proliferation. It also had a significantly lower overall inflammation compared to PCL-Bi and PCL-Bi-ROSU. These results suggest that the action of PCL-Bi-ROSU mainly involves the attenuation of pathologic processes in the neointimal layer, while PCL-Bi- RAPA exerts its action via the modulation of NIH pathways across all the layers of the outflow vein.

[0308] The experiments results described herein showed that the application of the perivascular wraps provided herein could reduce AVF stenosis. This is concordant with previous studies, and this effect arises from the ability of perivascular scaffolds to reduce excessive hemodynamic stress and endothelial injury. Advantageously, incorporating therapeutic agents (e.g., MSCs, drugs, and / or metallic nanoparticles) in the perivascular wraps provided herein helps reduce the effects of one or more pathways that lead to NIH, such as hypoxia and inflammation. Indeed, it was demonstrated that the controlled delivery of drugs, such as ROSU and RAPA, have shown benefits in reducing AVF failure and 72 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) further improved the imaging and histologic parameters of AVF maturation.

[0309] In some instances, the effects of ROSU were more pronounced in the neointimal layer, and this is anticipated since it protects the endothelium from immune cell infiltration, thrombosis, and atherogenesis. In some instances, the effects of RAPA were observed across all the layers of the outflow vein because the anti-proliferative characteristics of this substance may impact a diverse range of cell types. The experimental results described herein demonstrate that the BiNPs-loaded wrap can support the loading and controlled release of drugs with different properties and mechanisms of action against NIH.

[0310] While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any inventions or of what can be claimed, but rather as descriptions of features specific to particular implementations of the systems and methods described herein. Certain features that are described in this specification in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features can be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination can be directed to a subcombination or variation of a subcombination.

[0311] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results.

[0312] Having now described some illustrative implementations and implementations, it is apparent that the foregoing is illustrative and not limiting, having been presented by way of example. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, those acts and those elements can be combined in other ways to accomplish the same objectives. Acts, elements and features 73 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) discussed only in connection with one implementation are not intended to be excluded from a similar role in other implementations or implementations.

[0313] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including” “comprising” “having” “containing” “involving” “characterized by” “characterized in that” and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additional items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components.

[0314] Any references to implementations or elements or acts of the systems and methods herein referred to in the singular can also embrace implementations including a plurality of these elements, and any references in plural to any implementation or element or act herein can also embrace implementations including only a single element. References in the singular or plural form are not intended to limit the presently disclosed systems or methods, their components, acts, or elements to single or plural configurations. References to any act or element being based on any information, act or element can include implementations where the act or element is based at least in part on any information, act, or element.

[0315] Any implementation disclosed herein can be combined with any other implementation, and references to “an implementation,” “some implementations,” “an alternate implementation,” “various implementation,” “one implementation” or the like are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic described in connection with the implementation can be included in at least one implementation. Such terms as used herein are not necessarily all referring to the same implementation. Any implementation can be combined with any other implementation, inclusively or exclusively, in any manner consistent with the aspects and implementations disclosed herein.

[0316] References to “or” can be construed as inclusive so that any terms described using “or” can indicate any of a single, more than one, and all of the described terms.

[0317] Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included for the sole purpose of 74 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) increasing the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence have any limiting effect on the scope of any claim elements.

[0318] The systems and methods described herein can be embodied in other specific forms without departing from the characteristics thereof. Although the examples provided can be useful transforming a three-dimensional point cloud to a different reference frame, the systems and methods described herein can be applied to other environments. The foregoing implementations are illustrative rather than limiting of the described systems and methods. The scope of the systems and methods described herein can thus be indicated by the appended claims, rather than the foregoing description, and changes that come within the meaning and range of equivalency of the claims are embraced therein. 75 4884-2520-8786.2

Claims

Atty. Dkt. No.642631-0045 (pMDA23-083) WHAT IS CLAIMED IS:

1. A polymer scaffold, comprising: a porous component including a network of resorbable polymer fibers; and a therapeutic component dispersed in the porous component, the therapeutic component including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

2. The polymer scaffold of claim 1, wherein the resorbable polymer fibers include at least one of polycaprolactone (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), or polydioxanone (PDO).

3. The polymer scaffold of claim 2, wherein the resorbable polymer fibers consist essentially of PCL.

4. The polymer scaffold of claim 2, wherein the resorbable polymer fibers include a blended polymer of two or more of PCL, PLA, PLGA, PEG, and PDO.

5. The polymer scaffold of claim 1, wherein the cells include mesenchymal stem cells.

6. The polymer scaffold of claim 1, wherein the drug particles include an anti- inflammatory drug, an anti-thrombotic drug, or a combination thereof.

7. The polymer scaffold of claim 6, wherein the drug particles include at least one of rosuvastatin, rapamycin, diclofenac, clopidogrel, dipyridamole, or exosomes.

8. The polymer scaffold of claim 1, wherein the metallic nanoparticles include at least one radiopaque element.

9. The polymer scaffold of claim 8, wherein the metallic nanoparticles include at least one of gold, bismuth, gadolinium, tungsten, ytterbium, barium, tantalum, or zirconium.

10. The polymer scaffold of claim 1, wherein the porous component includes a tubular structure having an inner diameter and an outer diameter greater than the inner diameter, 76 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) and wherein the inner diameter is sized to allow the polymer scaffold to wrap around a vascular structure in a subject.

11. The polymer scaffold of claim 1, wherein the porous component includes a tubular structure having an inner diameter and an outer diameter greater than the inner diameter, and wherein the outer diameter is sized to allow the polymer scaffold to extend between two ends of a vascular structure in a subject.

12. A method of fabricating a polymer scaffold, comprising: dissolving a polymer in a solvent to form a polymer solution; blending a therapeutic component into the polymer solution to form a blended polymer solution, the therapeutic component including at least one of a plurality of drug particles, or a plurality of metallic nanoparticles; electrospinning the blended polymer solution to form the polymer scaffold including the therapeutic component dispersed in a porous network of fibers containing the polymer; and incorporating a plurality of cells in the polymer scaffold.

13. The method of claim 12, wherein the polymer includes at least one of polycaprolactone (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), or polydioxanone (PDO).

14. The method of claim 12, wherein the cells include mesenchymal stem cells.

15. The method of claim 12, wherein electrospinning the blended polymer solution is configured to form the porous network of fibers being aligned in a certain direction.

16. The method of claim 12, wherein the drug particles include at least one of an anti- inflammatory drug or an anti-thrombotic drug.

17. The method of claim 12, wherein the metallic nanoparticles include at least one radiopaque element. 77 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) 18. The method of claim 17, wherein the metallic nanoparticles include at least one of gold, bismuth, gadolinium, tungsten, ytterbium, barium, tantalum, or zirconium.

19. A method of treating a subject with a polymer scaffold, comprising: providing the polymer scaffold, the polymer scaffold including: a porous component having a network of polymer fibers resorbable by the subject, and a therapeutic component dispersed in the porous component, the therapeutic component including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles; and deploying the polymer scaffold at a vascular structure of the subject.

20. The method of claim 19, wherein the polymer scaffold is configured as a perivascular wrap.

21. The method of claim 20, wherein deploying the polymer scaffold includes: surgically forming an arteriovenous fistula (AVF) coupling an artery to a vein of the subject, wherein the vascular structure is the vein of the AVF; and wrapping the polymer scaffold around the vein as the perivascular wrap.

22. The method of claim 19, wherein the polymer scaffold is configured as a vascular graft, and wherein deploying the polymer scaffold includes coupling the polymer scaffold between two open ends of the vascular structure.

23. The method of claim 19, wherein the polymer scaffold is configured as an intravascular stent, and wherein coupling the polymer scaffold includes inserting the polymer scaffold into a lumen of the vascular structure.

24. A method of fabricating a polymer scaffold, comprising: dissolving a polymer in a solvent to form a polymer solution; electrospinning the polymer solution to form the polymer scaffold a porous network of fibers containing the polymer; and 78 4884-2520-8786.2Atty. Dkt. No.642631-0045 (pMDA23-083) subsequently incorporating a therapeutic component in the polymer scaffold, the therapeutic component including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles.

25. The method of claim 24, wherein the polymer includes at least one of polycaprolactone (PCL), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), or polydioxanone (PDO).

26. The method of claim 24, wherein the cells include mesenchymal stem cells.

27. The method of claim 24, wherein electrospinning the blended polymer solution is configured to form the porous network of fibers being aligned in a certain direction.

28. The method of claim 24, wherein the drug particles include at least one of an anti- inflammatory drug or an anti-thrombotic drug.

29. The method of claim 28, wherein the drug particles include at least one of rosuvastatin, rapamycin, diclofenac, clopidogrel, dipyridamole, or exosomes.

30. The method of claim 24, wherein the metallic nanoparticles include at least one radiopaque element.

31. The method of claim 30, wherein the metallic nanoparticles include at least one of gold, bismuth, gadolinium, tungsten, ytterbium, barium, tantalum, or zirconium.

32. A polymer scaffold, comprising: a porous member including a network of resorbable polymer fibers; and a therapeutic material dispersed in the porous member, the therapeutic material including at least one of a plurality of cells, a plurality of drug particles, or a plurality of metallic nanoparticles. 79 4884-2520-8786.2

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