Thin films for promotion of vascularization and wound healing

Abstract

A film for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization includes a plurality of fibers arranged in an overlapping configuration to form a porous, thin film with an average pore size of at least 0.01 μm2 and less than 60 μm2. The plurality of fibers has an average diameter of at least 10 nm and less than 1,000 nm. Each of the plurality of fibers have an aspect ratio of length to width of at least 10, and each of the plurality of fibers is a biocompatible material.

Description

Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 THIN FILMS FOR PROMOTION OF VASCULARIZATION AND WOUND HEALING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims priority benefit to U.S. Provisional Patent Application No. 63 / 693,807, filed on September 12, 2024, the entire content of which is incorporated herein by reference. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant 141525 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND 1. Technical Field

[0003] The currently claimed embodiments of the present invention relate to films for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, vascularization, and wound healing, and devices incorporating the films and methods of using the films. 2. Discussion of Related Art

[0004] Lung transplantation is the gold standard therapeutic option for end-stage respiratory failure, performed over 4,600 times annually worldwide with over 4,000 patients on the waitlist, and over 3,000 new patients added to the waitlist every year.1-3Despite being a potentially life-Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 saving option for many patients, lung allografts face the highest failure rates compared to all other major solid organ transplants.3,4Median survival after lung transplant remains 5.8 years, compared to heart, liver, and kidney transplantation with median survival of 12, 16 years, and 19.2 years, respectively.5-8High rates of failure after lung transplantation are driven by chronic lung allograft dysfunction (CLAD), which comprises the immune and non-immune complications related to lung transplantation, notably including airway complications.9-13Airway complications, namely airway anastomotic stenosis and dehiscence, and bronchial infection occur in as many as 33% of lung transplant recipients, and contribute significantly to morbidity and mortality post-lung transplantation, often necessitating additional surgical or bronchoscopic interventions, and requiring aggressive antibiosis.14-19These complications are primarily driven by ischemia and resultant hypoxia to the transplanted airway due to sacrifice of the bronchial vasculature and immune-mediated microvasculature destruction.10,12,20Lungs are typically perfused by both the pulmonary artery and bronchial circulation, but in conventional lung transplantation, the bronchial circulation is sacrificed and not re-anastomosed. The resultant aberrant circulation to the lungs post-transplant results in regional ischemia and irreversible fibrotic remodeling, which can disrupt the anastomosis, and make the allograft and recipient susceptible to devastating infections.10,15While immunosuppression can manage or at least mitigate the late-stage immune-driven microvascular destruction, there are no adequate, widely available, or durable solutions to address the aberrant circulation post-lung transplant. An urgent unmet need exists in addressing persistent ischemia of the airways after lung transplantation, which drives morbidity, lethal airway complications, and prompts downstream CLAD.

[0005] Several strategies have been tested to address airway devascularization in lung transplantation to prevent downstream airway complications and CLAD. Surgical revascularization of the bronchial arteries is employed by a limited number of transplant centers and utilizes a left internal mammary artery to donor aortic patch anastomosis.21Results from select centers performing this adjunct to lung transplantation show that fewer airway complications, and improved 5-year survival of 69% compared to 57% in traditional lung transplantation.12,21,22However, this comprises an incredibly narrow proportion of lung transplant recipients with most studies reporting 20-30 patients total. As the technique to perform surgical bronchial artery revascularization significantly deviates from conventional lung transplantation, and introduces additional bleeding risk, and a significant chance for technical failure in anastomotic failureAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 between the left internal mammary and the aortic patch, there has been very limited adoption.21There is a need for therapeutic options that improve upon clinical data showing the benefit of restoring airway circulation and can do so while maintaining the existing surgical workflow without complications. Thus, there is interest in therapeutics that upregulate local angiogenic transcription factors such as hypoxia inducible factor (HIF).10,23-25This includes iron chelating agent deferoxamine (DFO), which ultimately upregulates HIF and promotes microvascular regeneration at the anastomosis in a rodent airway transplant model.11DFO also reduced aspergillus infections, and is hypothesized to reduce downstream CLAD, as better perfusion allows for penetration of immunosuppressive medication to the anastomosis.24However, there are no platforms for sustained delivery of proangiogenic agents to this area and very few agents that have been explored in upregulating vascular transcription factors.

[0006] Consequently, there remains a need for improved films for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, vascularization, and wound healing, and devices incorporating the films and methods of using the films. SUMMARY

[0007] A film for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization according to an embodiment of the current invention includes a plurality of fibers arranged in an overlapping configuration to form a porous, thin film with an average pore size of at least 0.01 µm2and less than 60 µm2. The plurality of fibers have an average diameter of at least 10 nm and less than 1,000 nm. Each of the plurality of fibers have an aspect ratio of length to width of at least 10, and each of the plurality of fibers is a biocompatible material.

[0008] A device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization according to an embodiment of the current invention includes a film according to an embodiment of the current invention, and an oxygen sensor incorporated into the film. TheAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 oxygen sensor is configured to monitor status of vascularization and perfusion at the treatment site and transmit data for remote monitoring.

[0009] A device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization according to an embodiment of the current invention includes a first film according to an embodiment of the current invention and a second film according to an embodiment of the current invention. The second film has at least one physical parameter that is different from said first film. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Embodiments of the present invention, as well as the methods of operation and functions of the related elements of structure and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the invention.

[0011] FIGS.1A-1F show examples of degradable thin films for airway transplantation: (A) smooth polyglycolide (PGS), (B) random nanofiber PGS, (C) aligned nanofiber PGS, (D) random nanofiber PGS / 20% DFO, (E) composite PGS / 40% DFO, (F) microfiber poly-l-lactic acid (PLLA) / 40% tacrolimus. Scale bar = 10 µm.

[0012] FIG. 2 shows cumulative in vitro DFO release per mg of randomly aligned nano- structured PGS thin films with 10% and 20% DFO (w / w) loading.

[0013] FIGS.3A-3C show (A) Tracheal devascularization and auto-transplantation model in rats. (Row B) Left: Gross image of native trachea. Right: LASCA color image of native trachea, with heatmap correlating greater perfusion to red, and lower perfusion to blue. (Row C) Left:Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Trachea immediately post auto-transplantation. Right: LASCA color image showing a steep drop in perfusion in the auto-transplanted segment of trachea.

[0014] FIGS.4A-4D show (Row A) Operated control at day 10 showing persistent perfusion loss. (Row B) Autotransplanted trachea at day 10 with random nano-PGS applied during index procedure with improvement in perfusion. (C) Smooth PGS at the time of surgery does not improve perfusion at day 10. (D) nano-PGS-DFO thin film applied to the post-transplant trachea, demonstrating robust perfusion throughout the graft, with speckle angiography demonstrating native appearing tracheal vasculature 10-days post-operatively.

[0015] FIGS. 5A-5D show (A) H&E stain of a control auto-transplanted trachea on day 10 (4x), with 20x zoom in right panel, focusing on sloughed ciliated epithelium with loss of architecture, and scant goblet cells. (B) MTC staining of control auto-transplanted trachea at day 10 (4x) with 20x zoom panel on the right demonstrating loss of epithelial structure and thinned out cartilaginous tissue. (C) H&E stain of random nano-PGS thin film treated auto-transplanted trachea (4x), with 20x zoom in right panel demonstrating preserved ciliated epithelium with intact goblet cells. (D) MTC of random nano-PGS treated auto-transplanted trachea at day 10 (4x), with 20x zoom on the right demonstrating robust ciliated epithelium.

[0016] FIG. 6 shows perfusion unit loss between native and autografted rat trachea. Nano- PGS / 20% DFO had the lowest perfusion loss following surgery, indicative of near native perfusion in the autograft. ****p<0.005.

[0017] FIG. 7 shows average % perfusion loss between native and autografted trachea. Random Nano PGS performed significantly better than Smooth, Random Micro, and Aligned Nano films. DFO-loaded Random Nano PGS films had the lowest reduction in perfusion.

[0018] FIG. 8 shows representative images of the cross-section of the trachea at the anastomosis site: (left) lectin-positive vessels; A: native healthy trachea, B: operated control at day 10, C: Random Nano PGS-20% DFO at day 10 post-op showing new, functional blood vessels, and (right) MTC staining of 20% DFO.

[0019] FIGS. 9A-9F show key steps in auto-transplantation. A schematic overview of key procedural steps in the rat tracheal auto-transplantation model are: (A) incision, (B) exposure ofAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 the vertical strap muscles, (C) exposure of the trachea after division of the strap muscles, (D) surgical devascularization with care taken to ligate the tracheal vessels, (E) excision of the autograft, and (F) auto-transplantation.

[0020] FIGS. 10A-10E show polydioxanone (PDO) thin film morphology and orientation. SEM and AFM-based characterization of PDO film morphology and orientation. (A) Representative, high magnification SEM image of electrospun PDO thin film revealing randomly oriented, defect-free fibers with an average diameter of 915.6 ± 281 nm (n=100). (B) AFM analysis revealed uniform surface roughness and topography (n=3). Fiber orientation was assessed using (C) color-coded images and (D) Fast Fourier Transform (FFT) spectra, revealing (E) a broad distribution of intensities, indicating a lack of a predominant angle of orientation (n=3).

[0021] FIGS. 11A-11D show Pre- and Post-Operative Perfusion of the Rat Trachea. Rows A-C show representative images, from left to right, of a gross image of the trachea, LSCI perfusion mapping, and angiography. (A) Native trachea with perfusion mapping including red hues that indicate greater perfusion and angiography demonstrating high resolution visualization of blood vessels. (B) Trachea following surgical ligation of the tracheal arteries, with demonstrably lower perfusion in the trachea, with some likely collateralization from the thyroid. (C) Tracheal autograft demonstrating severely diminished immediate post-operative perfusion. (D) Graph of perfusion percent loss at each stage relative to start of the procedure, with persistently decreased perfusion after each measure of devascularization (n=8). Statistically significant lower perfusion at each point was observed with ANOVA and Tukey’s test (***p<0.001). Scalebar = 3 mm.

[0022] FIGS. 12A-12F show Tracheal Perfusion Following PDO Thin Film Application. Representative perfusion maps of the auto-transplanted trachea at (A) day 3 (n=2) and (B) at day 10 (n=8). Both autografts demonstrate poor perfusion indicated by the cool blue hue in the autograft. (C) Representative surgical image of the trachea at day 10, with notable blue hue to the autograft, which is a gross sign of ischemia. (D) Representative perfusion map of an auto- transplanted trachea receiving a random nanofiber PDO thin film at day 10 (n=8) and (E) corresponding surgical image, revealing degradation of the film at 10 days. (F) Auto-transplanted tracheas treated with a random nanofiber-based PDO thin film demonstrated significantly reduced perfusion loss in comparison to operated control tracheas (**p<0.01).Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0023] FIGS. 13A-13C show Histological Analysis and Injury Scoring of Rat Tracheas. Representative 4x and 20x magnification images of H&E-stained sections of (A) non-operated control trachea, (B) operated autograft (day 10), and (C) random nano PDO film-treated autograft (day 10). The unoperated control images (n=5) reveal organized cartilage and organized ciliated epithelium with intact goblet cells. Intact blood vessels are present throughout the tissue. Images of untreated autografts (n=5) show notable signs of ischemic injury, including dense inflammation of the subepithelial region, disruption of the ciliated epithelium, lack of nuclei in the cells of the membranous trachea suggestive of necrosis, and major disruption to the cartilaginous trachea. PDO-treated tracheas (n=4) show intact cartilaginous rings, with preserved basophilia, and live goblet cells in healthy appearing ciliated epithelium.

[0024] FIG.14 shows Immunoassay Profiling for Pro-angiogenic Proteins. Quantification of a set of inflammatory cytokines and cell adhesion molecules in operated control and treated rats demonstrates a significantly higher mean fold increase in expression of VCAM-1 in the PDO thin film implantation group (*p<0.05).

[0025] FIGS.15A-15F show representative SEM images of PGS-based films (A) smooth, (B) Random Micro, (C) Aligned Nano (D) Random Nano, (E) Random Nano-DFO 10%, and (F) Random Nano-DFO 20%. Scale bar = 10 µm.

[0026] FIGS.16A-16C show (A) Cumulative in vitro DFO release and (B) total drug loading per 1 mg of 10%, 20% and 40% DFO nano-PGS films, (C) Pharmacokinetic (PK) profile of DFO release from electrospun PGS films containing 10% DFO (w / w). DFO was detected in the tracheal tissue at all time points through day 10; however, no drug was detected in the plasma at any timepoints, demonstrating the utility and safety of local drug delivery via film. The graph shows the cumulative concentration of DFO over time. Data are presented as mean ± standard deviation from three independent experiments.

[0027] FIGS. 17A-17B show A: Illustration of rat tracheal auto-transplantation, demonstrating specific measures taken to devascularize the airway. Tracheal arteries are ligated. Then, a 3-4 ring segment of trachea is excised, and then re-anastomosed. If the rat is in a treatment group, a thin film measuring 1x1cm is wrapped circumferentially on the surface of the autograft. The thin films do not require glue or suture to stay in place and adhere when wettened. B: (TopAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Row): Imaging of the native rat trachea with laser speckle contrast angiography demonstrating bright regions where there is active blood flow. (Bottom Row): imaging of the rat trachea immediately post-auto-transplantation, demonstrating hypoperfusion within the autograft, as indicated by the diminished light pattern on LSCI. Scale bar= 10mm.

[0028] FIGS.18A-18B show A: Representative laser speckle contrast angiography of healthy unoperated rat trachea, and rat tracheas on Post-Operative Day 10 after autotransplantation, and application of a PGS-based thin film if applicable. Thin films are imperceptible at 10 days post- auto-transplant. The autograft is highlighted by the yellow box, demonstrating where thin film was wrapped around the exterior of the trachea. Bright patterns denote regions of increased blood flow. Scale bar=5mm. B: Graph of percent decrease in perfusion from the native trachea to the tracheal autograft. Lower percentages suggest lower perfusion losses between native trachea, and the autografted trachea. Rats that had a nano-DFO 10% thin film placed on the autograft at the index surgery demonstrated the lowest loss in perfusion at the terminal end point. N=6-9 rats per group. Only P values <0.0001 are shown for clarity of the graph.

[0029] FIGS.19A-19B show A: Representative lectin perfusion imaging for select treatment groups after tracheal auto-transplantation on POD 10. Lectin is only expressed where there is blood flow given that it is injected remotely in the rat and must perfuse the trachea to produce a staining pattern. Polarity of staining is demonstrated in the healthy control corresponding to the lateral tracheal arteries. Scale bar=1mm. B: Area of trachea with positive lectin staining (calculated using ImageJ). Tracheas treated with PGS Nano-DFO 10% demonstrated the highest lectin positivity at the end point compared to other treatment groups. N=3-5 per treatment group. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0030] FIG.20 shows qPCR data among healthy control, operated control, random nano PGS, and random nano PGS / 10% DFO rats of HIF-1a, IL-10, PTGS-2, and VEGF. N=3-5 per group. *: p<0.05; **: p<0.01; ****: p<0.0001.

[0031] FIGS. 21A-21B show A: Illustration of pig tracheal patch allotransplantation model, demonstrating that a 3x3 cm patch of trachea is excised from a donor pig, and anastomosed as an interposition in a 3x3 cm excised hole in the recipient trachea. In treatment groups, a thin film is then overlain on top of the patch allograft, as demonstrated in the photos.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0032] FIG.22 shows representative LSCI images from operated control, random nano-PGS, and random nano PGS DFO-10% treated swine. The yellow box encompasses the patch transplant region, and brighter areas indicate increased perfusion. The graph represents percent perfusion loss between native trachea and the allograft, with lower percentages indicating favorable perfusion. N=4 in each treatment group, *:P<0.05; **:P<0.01.

[0033] FIG.23 shows bronchoscopic images at 14 days post allotransplantation. Granulation tissue, inflammation, and necrotic changes evident in the operated control arm. Hypoperfusion notable in the no drug randon nano PGS arm. Well-healed appearance with intact blood vessels noted in the random nano PGD DFO 10% arm.

[0034] FIG.24 shows qPCR data among swine. N=2-3 per group.

[0035] FIGS. 25A-25C show representative H&E sections of (A) operated control, (B) Random Nano PGS, and (C) Randon Nano PGS DFO 10% tracheas on day 10. A shows notable pallor and fracture of the cartilaginous rings with loss of staining in chondrocyte nuclei consistent with ischemic injury, and flat, blunted ciliated epithelium with few goblet cells. B shows signs of ischemia present in the cartilaginous rings with pallor and cracking. Inflammation throughout the graft indicated by lymphocyte presence, but improved appearance of ciliated epithelium with increased density of goblet cells. C shows healthy appearing cartilage with nuclei staining throughout, as well as preservation of topography in the ciliated epithelium. Multiple endothelial channels consistent with microvessels on perimeter of graft. Lymphocytes are present in all layers. Scale bar = 200 μm. DETAILED DESCRIPTION

[0036] Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed, and other methods developed, without departing from the broad concepts of the present invention. All references cited anywhere in this specification are incorporated by reference as if each had been individually incorporated.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0037] It is known that implant composition and topography influence cell adhesion, proliferation and behavior in patients; however, implant characteristics have not been rigorously evaluated for angiogenesis induction in vivo, which is critical for tissue engineering and transplant applications.26-34Our novel nano-fabrication platform, according to some embodiments of the current invention, allows for reproducible, high resolution modification of implant composition, surface topography, architecture, porosity, and degradation in order to provide thin films for the promotion of vascularization.35,36Moreover, biocompatible thin films or other implants that can be applied directly at the surgical site without affecting the surgical workflow can be provided according to some embodiments of the current invention.

[0038] Ensuring robust local vascularization is a major, foundational challenge in tissue engineering / repair.37-39In lung transplantation specifically, bronchial artery revascularization has been attempted to improve perfusion post-operatively, but has had limited adoption due to the technical skill required, increased bleeding risk, and extended operation time.40Several engineering approaches have been developed to provide local proangiogenic cues post- operatively, primarily focused on local drug administration; however each has its own limitations for use, including off-target side effects from lack of localization, poor residence time or inability to sustain release, poor potency or stability of active agents, high cost, or incompatibility with the surgical workflow. For example, small animal model studies have directly applied DFO topically during the procedure, but this approach has limited residence time and penetration, and may not be as effective in larger animal models or humans.10Nanoparticle-based delivery systems face similar challenges.41Delivery of VEGF or other growth factors is of interest, but it is challenging to load them at high doses, maintain biological activity, and achieve sustained release, and they may lead to abnormal vascular growth, edema, and inflammation.42Cell implantation-based approaches have also been developed, but these have a significant regulatory burden, cost, and depending on the cell type(s), safety and ethical concerns. In contrast, application of nano- structured thin films at the anastomosis site could provide a scalable, low cost, and easy-to- administer approach that fits into the existing surgical workflow according to some embodiments of the current invention. Nanofiber-based thin films are particularly attractive due to their biocompatibility, biomimetic architecture, porosity, and high surface area-to-volume ratio, thereby enhancing endothelial cell adhesion and proliferation, and providing a template extracellular matrix (ECM) to support ingrowth of a structured vascular network.43Promotion ofAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 revascularization through modification of thin film architecture and material properties offers a striking advantage over drug delivery-based approaches according to some embodiments of the current invention, as the proangiogenic effect is intrinsic to the device and is active as long as it is present. To date, this approach has been limited by a lack of understanding of the specific material properties and architectural design to promote microvascular perfusion.34,39,43Our novel thin film manufacturing approach provides precise control of thin film composition and architecture using generally regarded as safe (GRAS) polymers with controlled degradation rates.33,35,36Moreover, the thin film also provides a platform for local, sustained drug delivery directly at the anastomosis site to further enhance vascularization according to some embodiments of the current invention. Our platform can allow for loading of a wide range of small molecules into nanofibers capable of sustained drug delivery while maintaining excellent mechanical properties at high drug loading.44,45Ultimately, the thin films we describe could provide surgeons with a easy-to-apply tool that integrates at the anastomosis site to improve wound healing, provide local, sustained delivery of proangiogenic agents, and promote vascularization prior to safely degrading according to some embodiments of the current invention.

[0039] We recently adapted our versatile manufacturing system to determine the potential benefit of nano-structured thin films (with or without drug) to promote vascularization at the tracheal anastomosis site following airway transplantation according to an embodiment of the current invention. We manufactured degradable thin films composed of polyglycolide (PGS), polydioxanone (PDO; not shown), and Poly(L-lactide) (PLLA) to evaluate films with degradation profiles ranging from weeks to months to years, respectively. We also manufactured smooth, nano-structured, and micro-structured films with optional loading of DFO or tacrolimus to understand the effect of implant architecture and drug functionality on perfusion. Selected films are shown in FIGS.1A-1F, including smooth PGS (produced via solvent casting), nano-structured random and aligned PGS, nano-structured random PGS / 20% DFO, composite scaffold consisting of a layer of PGS / 40% DFO in between two layers of nano-structured random PGS, and micro- structured random PLLA / 40% tacrolimus. PGS and PLLA were dissolved at 12 wt% (w / v) in HFIP. Aligned fibers were collected by rotating the collector at 1,500 rpm. Material characteristics of selected thin films are shown in Table 1, demonstrating modification of thin film porosity and fiber diameter and alignment (n=4). PGS random, PGS / 10% DFO, and PGS / 20% DFO had tensile strengths of 0.39 ± 0.0, 0.41 ± 0.2, and 0.23 ± 0.7 (n=4), respectively.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Notably, DFO loading significantly reduced fiber diameter, and doubling the drug loading enabled tuning of the release profile from sustained release over 7 days at 10% loading to burst release in the first 24 h at 20% (n=3), which also correlated with a loss in strength (FIG. 2). Drug loading for PGS / 10% DFO and PGS / 20% DFO were 17 ± 1 and 35 ± 1 µg / mg thin film, respectively (n=3). Smooth, nano-structured, and DFO-loaded PGS were evaluated in a rat auto- transplantation model (described below). Smooth and nano-structured films were tested to understand the role of nanoarchitecture on vascularization. DFO-loaded films were tested to evaluate the effect of local delivery of an iron chelator for stabilization of airway HIFs. Table 1. Material characteristics of degradable thin films for airway transplantation showing variation of fiber diameter, alignment, and porosity.

[0040] Rodent tracheal allotransplantation models have been utilized to study the immune- mediated destruction of airway microvasculature, wherein non-immunosuppressed tracheal allografts in mice showed complement-dependent destruction of tracheal micro-vessels.11,20,24These methods showed convincing microscopic destruction of micro-vessels in the allografts, that could be rescued and even regenerated when immunosuppression was introduced. This seminal work uncovered the relationship between rejection, chronic airway allograft ischemia, and CLAD, but it notably lacked gross perfusion data on the tracheal allografts, leaving unclear whether the concurrent pathology of surgical devascularization that routinely occurs in lung transplantation was modeled by the rodent allotransplant model, and whether microvascular regeneration alone provided meaningful perfusion to the allografts. The ischemic insult that comes from lack ofAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 revascularization of the airways after surgical devascularization inherent to conventional lung transplant causes an injury pattern that prompts downstream CLAD even without immune injury.12As such, we developed a rat tracheal auto-transplant model that emphasized measures for complete surgical devascularization (FIG. 3A). Male Wistar rats underwent surgical devascularization of a 4-5 ring segment of trachea that was then excised and auto-transplanted. Perfusion pre- and post-auto-transplantation was assessed with laser speckle contrast angiography (LASCA), an imaging technique that visualizes blood flow via light scattering off of moving blood cells, with red areas demonstrating higher perfusion than blue on images.46,47LASCA provides a useful method of assessing perfusion as it does not require sacrifice of the animal, and as such, multiple post-operative time points can be studied with each animal. Representative pre- transplant LASCA is shown in FIG.3B, demonstrating intact tracheal vascular architecture, with notable circumferential tracheal arteries demonstrated between cartilaginous rings. Immediately post-transplant, there is a marked region of hypoperfusion restricted to the autograft (FIG. 3C), with a significant drop in perfusion from the native trachea to the autograft. Necks were closed and then re-explored at day 10 to assess whether devascularization persisted. At day 10, the tracheal autografts remained hypoperfused (FIG.4A), with little-to-no improvement in perfusion unit drops across the native to the auto-transplanted trachea. The trachea also appeared dusky, characteristic of malperfusion. Hematoxylin and eosin (H&E) and Masson’s trichrome (MTC) tracheal staining showed loss of ciliated endothelium (FIG. 5A-B). This suggests that even without rejection, devascularization and auto-transplantation of the trachea causes acute-onset, and persistent ischemia to the airways.

[0041] We hypothesized that nano-structured thin films would integrate at the anastomosis site and prompt vascular collateralization. Thus, we applied various formulations of thin films on the auto-transplanted tracheas at the anastomosis site, assessed them for perfusion at day 10, and performed histopathological assessment and immunoassays. First, we tested thin films with varied architecture and degradation rates starting with nano-structured PGS (FIG. 1B), nano-structured PDO, and a smooth PGS thin film (FIG. 1A). We found that at day 10, smooth PGS films performed similarly to our operated control specimens that were not treated with any film, with similarly reduced perfusion in the tracheal autograft. In contrast, randomly aligned, nano- structured films universally improved perfusion in the tracheal autograft, with PGS performing best (FIGS. 4B-4C). Random Nano-PGS demonstrated the lowest drop in perfusion betweenAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 native trachea and the autograft, with some restoration of horizontal tracheal artery architecture. Histologically, there was clear preservation of the complex architecture of the ciliated tracheal epithelium, with intact goblet cells, and perfusing vessels throughout the autograft (FIG. 5B). This suggests that the increased perfusion seen with LASCA may have led to better preservation of the trachea, as epithelial injury is an indicator of ischemia in the airways.19

[0042] Given the unprecedented improvements seen with random nano-PGS, we chose to load DFO into nano-structured PGS film. Nano-PGS / 20% DFO (FIG.3D) films were applied to our tracheal auto-transplant model and assessed at day 10. Nano-PGS / 20% DFO showed a 4x improvement in perfusion compared to operated control, and most native-like return of tracheal vascular architecture (FIG.4D). When quantitative perfusion unit differences between the native trachea and the autograft were compared among the various thin film treatment groups (n=4 per group), the random nano-PGS / DFO group demonstrated the lowest perfusion loss between native trachea and transplanted autograft suggesting best improvement in perfusion (p<0.005) (FIG.6). Notably, random nano-PGS / DFO performed significantly better than random nano-PGS alone (p<0.005), suggesting that DFO and random nano-PGS have a synergistic role in revascularization.

[0043] We have further evaluated the benefit of random nano-structured, DFO-loaded films against operated control, smooth films, random micro-structured films, aligned nano-structured films, and random nano-structured films composed of PGS (FIG. 7). Film breaking strengths were 0.39 ± 0.04 N for random nano-PGS, 0.41 ± 0.17 N for random nano-PGS-10% DFO (335 ± 110 nm), 0.23 ± 0.70 N for random nano-PGS-20% DFO (131 ± 36 μm), 0.51±0.06 N for random micro-PGS (1,925 ± 220 nm), 4.91±0.29 N for aligned nano-PGS, and 3.03±0.93 N for smooth PGS.

[0044] We found that both the architecture / patterning and drug functionality of the films significantly affected post-operative perfusion (FIG.7). Smooth PGS, Random Micro PGS, and Aligned Nano PGS films had no effect on perfusion and demonstrated comparable levels of perfusion at day 10 to an operated control. However, application of Random Nano PGS films lowered perfusion loss to only almost 10%, and DFO loading into these films further reduced percent loss to below 5%, demonstrating close to healthy control levels of perfusion. FIG. 8 shows lectin staining of blood vessels at cross-sections of the trachea at the anastomosis site (left;Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 A: native healthy trachea, B: operated control at day 10 post-op, C: Random Nano PGS-20% DFO at day 10 post-op) showing development of functional vasculature and (right) MTC staining of cross-sections of the trachea at the anastomosis site 10 days after application of Random Nano PGS-20% DFO showing organized formation of vasculature surrounding the trachea.

[0045] We tested the mechanism of improved airway perfusion using Luminex Multiplex assays (Thermo-Fisher, Waltham, MA) on post-operative samples. Notable results included a significant 0.9 mean fold decrease in interleukin-1 alpha (IL-1a) among the DFO-treated tracheas (p=0.02). IL-1a is associated with the downstream development of bronchiolitis obliterans syndrome, the main airway manifestation of CLAD in patients.48Both random nano-PGS and DFO thin films were associated with a trend toward increased interleukin-10 (IL-10) expression (mean fold increase [MFI] of 2.2, p=0.2, and 1.2, p=0.2 for nano-PGS and DFO respectively). Increased expression of IL-10 is associated with decreased rates of bronchiolitis obliterans and downstream airway complications in lung transplantation.49-51In Random Nano-PGS-treated tracheas, vascular cell adhesion molecule-1 (VCAM-1) was significantly increased (MFI: 2.8, p=0.001). Increased VCAM-1 is associated with angiogenesis.52

[0046] In summary, we have advanced our fully polymeric, nanofiber-based thin film system for application to lung transplantation according to an embodiment of the current invention through modification of film architecture, surface properties, degradation rate, and loading of proangiogenic agents. We have shown that rapidly degrading nano-structured thin films met strength and handleability requirements for application to the anastomosis site. These films significantly improved perfusion in comparison to an operated control and to smooth, random microfiber, and aligned nanofiber thin films of the same polymer composition and thickness. We believe that specific, non-obvious implant architectures and surface chemistries in combination with local delivery of a proangiogenic agent will effectively promote vascularization and prevent the downstream complications related to ischemia of the airways following transplantation. To date, this approach has been limited by a lack of understanding of biomaterial properties that promote vascularization and a lack of highly biocompatible and durable local drug delivery modalities that fit within the existing surgical workflow. In fact, the prior art suggests that materials with a slow degradation rate, high stiffness, aligned fibers, and microarchitecture with high porosity will most significantly promote vascularization; however, these findings are in contrast with our non-obvious data demonstrating robust vascularization with rapidly degrading,Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 randomly aligned, nano-structured PGS- and PDO-based thin films.34,43Identifying approaches that promote local vascularization and prevent / address hypoxia / ischemia is a major challenge in organ transplantation, reconstructive surgery, wound healing, treatment of peripheral artery disease, and the success / longevity of tissue engineered constructs and cell therapies. Some embodiments of the current invention can have broad applicability across these unmet needs.

[0047] Table 2. Material and morphological properties of PGS and PDO-based thin films Film Type Fiber Directionality Porosity Strength Contact Diameter (Coherency) Pore size Area (N) angle (nm) ( m2) (%) (degree) 7 6 6 4 8 0 1 3

[0048] Accordingly, a film for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, vascularization, and wound healing, according to some embodiments of the current invention includes a plurality of fibers arranged in an overlapping configuration to form a porous, thin film with an average pore size of at least 0.01 µm2and less than 60 µm2. The plurality of fibers have an average diameter of at least 10 nm and less than 1,000 nm. Each of the plurality of fibers have an aspect ratio of length to width of at least 10. In some embodiments, the fibers can generally extend essentially the entire dimension of the film, from edge to edge, for example, depending of the particular orientation. However, the general concepts of the current invention are not limited to this example. Without limitation, some fibers can extend across only portions of the film. Each of the plurality of fibers is a biocompatible material.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0049] In some embodiments, the plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.71. In some embodiments, the plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.51. In some embodiments, the plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.36.

[0050] In some embodiments, the average pore size is at least 0.01 µm2and less than 40 µm2. In some embodiments, the average pore size is at least 0.01 µm2and less than 30 µm2. In some embodiments, the average pore size is at least 0.01 µm2and less than 10 µm2. In some embodiments, the average pore size is at least 0.02 µm2and less than 2.2 µm2.

[0051] In some embodiments, the plurality of fibers have an average diameter of at least 50 nm and less than 900 nm. In some embodiments, the film has a thickness greater than 25 µm and less than 1 mm. In some embodiments, the film has an average Young’s modulus of at least 20 kPa and less than 1 GPa. In some embodiments, the film has an average breaking strength greater than 0.2 N.

[0052] In some embodiments, the plurality of fibers are synthetic degradable fibers that are composed of one or more polymer or copolymer of at least one of Polylactic Acid (PLA), Polyglycolic Acid (PGA), Polycaprolactone (PCL), Poly(lactic-co-glycolic Acid) (PLGA), Polydioxanone (PDO), Poly(ε-caprolactone), Polyorthoesters (POEs), Polyanhydrides, Polyhydroxyalkanoates (PHAs), Poly(β-amino esters), and biodegradable Polyurethanes with molecular weights of at least 1 kDa and less than 2,000 kDa. In some embodiments, the molecular weights of the polymers or copolymers are at least 25 kDa and less than 500 kDa. In some embodiments, the film further includes at least one proangiogenic moiety loaded into at least some of said plurality of fibers. In some embodiments, the proangiogenic moiety is a small molecule drug consisting of Deferoxamine, CEP03, SC-3-149, Rocaglamide, Fasudil, Deforolimus, Statins, Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II Receptor Blockers (ARBs), Sildenafil, Tadalafil, Metformin, Nitrates, and any combination thereof. In some embodiments, the proangiogenic moiety is vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor, angiopoietin-1, heparin, oxygen, oxygen releasing moiety, or any combination thereof.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0053] An embodiment of the current invention provides a device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, vascularization, and wound healing that includes at least one film according to any one of above-noted embodiments of the current invention and an oxygen sensor incorporated into at least one of the films. The sensor is configured to monitor a status of vascularization and perfusion at the treatment site and transmit data for remote monitoring. In some embodiments, the sensor transmits the data wirelessly.

[0054] In some embodiments, the above-noted device includes a second film according to any one of above-noted embodiments of films. In some embodiments, the second film has at least one physical parameter that is different from a corresponding physical parameter of the first film.

[0055] In some embodiments, the above-noted device further includes at least one of a coating, a protective layer, an adhesive layer or any combination thereof in contact with at least one of the first and second films.

[0056] Another embodiment of the current invention is directed to a device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization that includes a first film according to any one of the above-noted embodiments of the current invention and a second film according to any one of the above-noted embodiments of the current invention. The second film has at least one physical parameter that is different from said first film. In some embodiments, the device further includes at least one of a coating, a protective layer, an adhesive layer or any combination thereof in contact with at least one of said first and second films. Pore size measurement:

[0057] High-resolution SEM images of the samples were acquired. The images were analyzed using ImageJ software. Images were converted to 8-bit grayscale and thresholded to isolate pores. The thresholded images were binarized, and pore size was measured using the Analyze Particles. Coherency measurement and definition:Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0058] 53Alignment quantification is often performed utilizing 2D image analysis obtained by scanning electron microscopy. Current open access image analysis software that has been used for quantification of alignment includes Directionality and Orientation J plugins for ImageJ. Fourier-based or Local Gradients methods can be used in Directionality to determine angular distributions. The program provides the overall direction of alignment based on the distribution peak. In addition, the plugin provides a dispersion and amount value. The dispersion is the standard deviation of the Gaussian fit that can be used to provide information on the angular distribution, and the amount value can provide information on the proportion of structures in a preferred direction. An alternative ImageJ plugin for characterizing anisotropy is Orientation J, which utilizes a structure tensor method. Similar to Directionality, Orientation J provides a histogram for visual representation of fiber distribution. The plugin also provides a measure of the predominant direction of orientation and a measure of coherency. Coherency is a measure of the degree to which structures are oriented with a coherency of 0 for a completely isotropic image and a value of 1 for highly oriented structures.

[0059] The following outlines some aspects of the current invention:

[0060] In some embodiments, the films may be acting at three different levels: cell level (adhesion, migration, proliferation), vessel level (angiogenesis, vascularization, infiltration, promoting vascular stability), and / or at the microvascular network level (microvascular growth, microvascular expansion, network maturation and remodeling). 1. Film architecture and material properties that safely integrate with tissue and promote endothelial cell adhesion, migration, proliferation, ingrowth, and vascularization locally. a. Random architecture orientation 1. Coherency of 0-0.7, more preferably 0-0.5, or more preferably 0.05-0.35 b. Porosity and pore size 1. 0.01 – 60 µm, preferably 0.01- 10 µm and 30-40 µm, or more preferably 0.01-2 µm2c. Fiber diameter 1. Diameter of 10-999 nm, more preferably 50-900 nm d. Contact angle / wettability 1. Angle of 0-145 degrees, more preferably 45-135 degrees e. Stiffness 1. Young’s Modulus of 20 kPa-1 GPa f. Strength 1. Breaking strength of greater than 0.2 N g. ThicknessAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 1. Thickness of greater than 25 µm (for lung transplant 25 µm-1 mm preferred) h. Appropriate fiber density and connectivity Biocompatible film formulation that safely integrates with tissue and promotes vascularization a. Synthetic degradable polymers or copolymers thereof 1. Polylactic Acid (PLA), Polyglycolic Acid (PGA), Polycaprolactone (PCL), Poly(lactic-co-glycolic Acid) (PLGA), Polydioxanone (PDO), Poly(ε- caprolactone), Polyorthoesters (POEs), Polyanhydrides, Polyhydroxyalkanoates (PHAs), Poly(β-amino esters), and biodegradable Polyurethanes 2. Preferably PGA, PLA, PLGA, PDO, PCL, Polyorthoesters (POEs), Polyanhydrides, and biodegradable Polyurethanes with molecular weights ranging from 1-2000 kDa (preferably 25-500 kDa) b. Synthetic non-degradable polymers 1. Polyethylene (PE), Polypropylene (PP), Polytetrafluoroethylene (PTFE), Polyurethanes, and Silicone c. Biological materials or combinations thereof 1. Collagen, Hyaluronic Acid, Chitosan, Alginate, Fibrin, Silk Fibroin, and Keratin d. Small molecule proangiogenic drugs 1. Deferoxamine, CEP03, SC-3-149, Rocaglamide, Fasudil, Deforolimus, Statins, Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II Receptor Blockers (ARBs), Sildenafil, Tadalafil, Metformin, Nitrates e. Other proangiogenic moieties 1. vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), epidermal growth factor, angiopoietin-1, heparin, oxygen or oxygen releasing moieties Degradation timeline a. Preferably 1 day to 1 year, more preferably 5 days to 6 months (for lung transplant, preferably 1-6 weeks) Different ways of incorporating proangiogenic moieties a. Direct drug loading within fibers (1-80% w / w, more preferably 5-40% w / w) b. Coaxial drug loading within fibers c. Drug loading within specific layers of the thin film 1. Multi-layered thin film, with film layer in contact with the tissue releasing drug, and exterior surface providing a barrier for release 2. Multi-layered film with different degrees of porosity in each layer, with certain layers having a porosity that supports tissue integration and angiogenesis, with other layers designed to prevent immune cell invasion d. Drug patterned in specific geometries or gradients on the thin film to directionally stimulate vascularization 1. For example, drug patterned perpendicular to the trachea or patterned circumferentially around the trachea e. Film filled with drug and then coated 1. Film composed of nanofibers soaked in drug solution and then coated with a polymer or copolymer from the below list:Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 1. Silicone, Polybutilate, Polyglycolic Acid, Polylactic Acid, Polyethylene Glycol (PEG), Polyglactin, Polytetrafluoroethylene (PTFE), Calcium Stearate, Poly(lactide-co-glycolide) (PLGA), Poly(caprolactone), Polydioxanone f. Drug included within coating on the film g. Uniaxial delivery of proangiogenic moieties 1. Significant drug only released in the direction of the damaged or injured tissue 5. Timeline for sustained delivery of proangiogenic moieties a. Preferably 1 day to 1 year, more preferably 3 days to 6 months (for lung transplant, preferably 3 days to 6 weeks) b. Stimuli-responsive delivery based on level of hypoxia / ischemia 6. Architecture that allows for nutrient diffusion and endothelial cell adhesion / proliferation, but prevents immune cell activation or infiltration 7. Incorporation of hydrogels or adhesives to ensure adhesion at the intervention site and / or sustained release of angiogenic moieties 8. Incorporation of sensors capable of monitoring the status of vascularization and perfusion at the treatment site, and wherein the data collected by the sensors can be transmitted wirelessly for remote monitoring 9. Methods of manufacture a. Processes for manufacturing thin films, including solvent casting, electrospinning, and 3D printing b. Specific electrospinning parameters 10. Methods of use a. A method for promoting vascularization and perfusion in a surgical site, involving applying the thin film to the site, wherein the film gradually releases chemical cues over a predetermined time period to stimulate the growth of new blood vessels and enhance blood flow to the site. b. Use of the thin film in clinical indications selected from the group consisting of: organ transplantation, reconstructive surgery, wound healing, diabetic wound healing, treatment of peripheral artery disease, and implantation of tissue engineered constructs. c. A kit for promoting vascularization and perfusion post-operatively, comprising: the thin film, instructions for applying the thin film to a surgical site; and optionally, tools for the application of the thin film.

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[0062] MATERIALS AND METHODS Materials

[0063] For the preparation of PGS and PGS / DFO meshes, polyglycolide (PGS), with an inherent viscosity of 1.05-1.25 dL / g, hexafluoroisopropanol (HFIP), deferoxamine (DFO) were purchased from Sigma Aldrich, and Cayman Chemical, respectively, and used as received.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Preparation of the Scaffolds Using Electrospinning

[0064] Electrospinning solutions were prepared by dissolving 12% and 18% w / v of PGS in HFIP under heated stirring at 70 °C overnight. Regarding the preparation of fibers containing varied drug concentrations (10, 2040%w / w), DFO and PGS were dissolved in HFIP separately and added and vortexed together at room temperature to form a homogeneous solution. Electrospinning was performed using a needle-based device equipped with controllers for humidity and temperature. The solution was loaded in a syringe (diameter d= 8.7 mm, BD) positioned horizontally. A Gamma high-voltage research power supply was employed to charge the solution positively, with the positive electrode connected to a 20 G blunt-tip needle (0.9 mm diameter) and the ground electrode attached to the collector. A programmable syringe pump controlled the flow rate. A circular flat collector and rotating drum (2000 rpm) covered by non- stick aluminum foil were utilized to fabricate 50 μm (thickness) random and aligned meshes, respectively. PGS RRR was fabricated by a layer-by-layer deposition process, consisting of PGS 40% DFO sandwiched between two layers of PGS. The applied parameters for nano-PGS random, PGS aligned, PGS / DFO (10, 20, 40%w / w), and PGS RRR were voltage tension = 12 kV, tip- collector distance = 15 cm, flow rate = 850 μl / h, deposition time = 60-120 min, humidity = 30%, temperature = 22 °C, while for micro-PGS were voltage tension = 10 kV, tip-collector distance = 10 cm, flow rate = 2000 μl / h, deposition time = 45 min, humidity = 30%, temperature = 22 °C. Scanning Electron Microscopy (SEM)

[0065] SEM imaging was conducted with the JSM-IT700HR InTouchScope™ Field Emission SEM to investigate the samples’ morphology. All samples were sputter-coated with a 10 nm thin film of Au / Pd (Desk II, Denton Vacuum, Moorestown, New Jersey). Fiber diameter, porosity, and directionality were measured using ImageJ 1.52a.

[0066] Fiber Diameter Measurement Using ImageJ: SEM images were imported into ImageJ software. Before beginning measurements, the scale was set using with the known distance from the image scale bar to ensure accurate measurements. After setting the scale, the images were converted to 8-bit grayscale to facilitate clearer visualization of the fibers. Fiber diameters were measured manually using the Straight Line tool. Multiple measurements were taken along different regions of each fiber to ensure accuracy.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0067] Pore Size Measurement Using ImageJ: 8-bit grayscale format SEM images were used for pore size analysis. To enhance pore visibility, the brightness and contrast of the images were adjusted uniformly. Thresholding was applied to the grayscale images to differentiate the pores from the fibers using the Threshold function. The threshold settings were manually adjusted to ensure accurate identification of the pores as red regions against a white background. Pores were then identified and analyzed using the Analyze Particles tool. The size parameter was set to 0– Infinity pixels and circularity was adjusted from 0.00 to 1.00 to account for irregularly shaped pores.

[0068] Fiber Directionality and Coherency Measurement Using ImageJ: To assess the orientation of the fibers, the OrientationJ plugin was applied to the SEM images. Coherency, a metric that quantifies the degree of alignment within the fiber network, was calculated as part of the orientation analysis. Coherency values range from 0, indicating random orientation, to 1, signifying perfect alignment. The resulting coherency values provide an assessment of the overall alignment and organization of the fibers within the sample. Mechanical properties

[0069] PGS meshes were cut to 3 cm long and 0.5 cm wide, clamped vertically, and pulled until yielding at a rate of 16 mm / min using a 5966 Dual Column Tabletop Testing System (Instron, Norwood, MA). Water contact angle

[0070] The contact angle analysis was performed using the Ossila contact angle goniometer setup, consisting of the camera as an optical lens and backlight illumination. Contact angles were measured by dispensing 3 μl deionized water with a syringe pump at a rate of 10 μl / min. Obtained images at 5 seconds were analyzed with ImageJ 1.52a. Statistical Analysis

[0071] Data was processed using Excel and OriginPro 1.52a software. Reported values are as means ± standard deviation (SD). All experiments were repeated three times. To calculate the average diameter of the fibers, a sample size of N=100 fibers was measured.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0072] EXAMPLE 1

[0073] The following provides further examples according to some embodiments of the current invention. The general concepts of the current invention are not limited to these examples.

[0074] Objective: Lung transplant recipients experience the lowest long-term survival among all solid organ transplant recipients. Airway complications contribute significantly to morbidity and mortality post-lung transplant and may be driven by airway devascularization inherent to procurement and implantation of the lungs. We studied application of biodegradable, nanofiber- based thin films according to an embodiment of the current invention to devascularized auto- transplanted airways to mitigate airway ischemia.

[0075] Methods: We utilized a rat tracheal auto-transplantation model that replicates airway ischemia. Rats were divided into an operated control group (n=18) and a treatment group (n=12) receiving an electrospun film composed of randomly aligned polydioxanone (PDO) nanofibers applied to the circumferential surface of the transplanted trachea. Airway perfusion was assessed via laser speckle contrast analysis at 0, 3, and 10 days. Differences in perfusion units were calculated between the non-transplanted and transplanted segments of the trachea. Multimodal analysis of angiogenesis in tracheal autografts included immunoassay profiling for pro-angiogenic cytokines, histological injury grading, and speckle angiography.

[0076] Results: Qualitative and quantitative perfusion differences were demonstrated at days 0, 3, and 10. Nanofiber-based, PDO thin films significantly improved perfusion in the transplanted segment of trachea (p<0.05). Histological injury scoring was significantly worse in the operated controls compared to the treatment group (p<0.01). Immunoassays demonstrated increased expression of vascular cell adhesion molecule 1 in the treatment group (p<0.05).

[0077] Conclusions: Application of a nanofiber-based PDO thin film induced a local tissue response that improved perfusion and histological injury scoring of the transplanted airway in an auto-transplant model of airway devascularization. Immune multiplexing suggests local inflammatory responses may drive angiogenesis. Introduction:Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0078] Despite major advancements in immunosuppression that have improved outcomes after solid organ transplantation, lung transplantation has lagged far behind in long-term survival.1,2The primary cause of mortality after lung transplant is chronic rejection due to bronchiolitis obliterans syndrome (BOS) which manifests in the airways post-operatively.3,4A significant driver of BOS is post-transplant airway hypoxia, the result of surgical devascularization that occurs during conventional lung transplantation, as well as immune- mediated destruction of microvasculature post-transplantation.3,5-8Unlike other solid organs, lungs are not completely revascularized during conventional transplantation.8,9The bronchial arteries are routinely sacrificed during procurement of donor lungs, leaving the transplanted airways, which rely on this circulation, at risk for ischemia.8Thereafter, immune mediated rejection perpetuates the destruction of blood vessels in the allograft, ultimately resulting in BOS and allograft dysfunction.5

[0079] There are limited therapeutic options to address the surgical devascularization that occurs in lung transplantation. While bronchial artery revascularization has been described, it is not widely adopted and there is a clear unmet need for a universally applicable and feasible treatment modality.9Nanofiber-based materials have gained significant traction as a tool to improve wound healing due to their ability to effectively mimic the extracellular matrix (ECM).10-12In particular, electrospun thin films consisting of ultra-fine fibers with a high surface area-to- volume ratio and interconnected porosity are ideal for facilitating essential cellular activities such as adhesion, proliferation, and migration.13-15Electrospun films have been reported previously to promote angiogenesis and assist in wound healing.10-12,16We hypothesized that application of nanofiber-based thin films will promote vascularization and improve the perfusion of transplanted airways. Here, we validated a rat auto-transplant model of airway hypoxia seen in lung transplantation and studied the application of nanofiber-based, polydioxanone (PDO) thin films in promotion of perfusion to devascularized airways. Methods: Preparation of Thin Films Using Electrospinning

[0080] Electrospinning solution was prepared by dissolving 12% w / v of PDO (Sigma-Aldrich, St. Louis, MO) with an inherent viscosity of 1.5-2.2 dL / g in hexafluoroisopropanol (Sigma-Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Aldrich) by shaking overnight at 37 °C. Electrospinning was performed using a needle-based device equipped with controllers for humidity and temperature. The solution was loaded in a syringe (diameter = 8.7 mm; BD, Franklin Lakes, NJ) positioned horizontally. A high-voltage power supply was used with the positive electrode connected to a 20 G blunt-tip needle (0.9 mm diameter) and the ground electrode attached to the collector. Fibers were collected on a flat target covered by non-stick aluminum foil. The applied parameters were voltage tension = 12 kV, tip- collector distance = 15 cm, flow rate = 850 μL / h, deposition time = 45 min, humidity = 30%, and temperature = 22 °C. Films were UV-sterilized on each side for 15 min prior to evaluation. All films were evaluated at n=3 for material and mechanical characterization. Evaluation of Thin Film Morphology and Architecture

[0081] Scanning Electron Microscopy (SEM) imaging was conducted with the JSM-IT700HR InTouchScope™ Field Emission SEM to investigate sample morphology. All samples were sputter-coated with a 10 nm thin film of Au / Pd (Desk II, Denton Vacuum, Moorestown, New Jersey). Fiber diameter, pore size, and directionality were measured using ImageJ 1.52a (n=3 thin films; n=100 fibers). Surface roughness of the electrospun PDO thin films was evaluated using an Asylum MFP-3D-BIO™ Atomic Force Microscopy (AFM) instrument operating under Tapping Mode using silicon cantilevers (RTESPA-300; Bruker Nano, Germany) with a spring constant of ~40 N / m, resonance frequency of 200-400 kHz, and an apex radius of curvature ~8 nm. Evaluation of Thin Film Mechanical Properties

[0082] PDO films were cut to 3 cm long and 0.5 cm wide, clamped vertically, and pulled until yield at a rate of 16 mm / min using a 5966 Dual Column Tabletop Testing System (Instron, Norwood, MA) to determine elongation, breaking strength, and modulus (n=3). Animal Ethics Statement

[0083] All animal procedures were approved by Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC), and Institutional Animal Care and Use Committee (IACUC) (Protocol #34143). Humane care was provided to all animals in accordance with institutional Laboratory Animal Care guidelines, and the “guide for the Care of Laboratory Animals” from the National Institutes of Health. Male Wistar rats (300-500g) (Charles RiverAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Laboratories, Wilmington, MA) were utilized for these studies. Male rats were chosen in accordance with observed sex-based differences in immune response favoring greater innate immune responses in males which may contribute to microvascular destruction in the airways.6,17Anesthesia and Surgical Model

[0084] Rats (n=30) weighing on average 300-400 g were anesthetized via intraperitoneal injection of xylazine (10 mg / kg) (NexGen Pharmaceuticals, Weatherford, TX), ketamine (70 mg / kg) (NextGen Pharmaceuticals), and atropine (0.5 mg / kg) (NextGen Pharmaceuticals) (to limit secretions).18The general operative steps are shown in FIGS.9A-9F. After sufficiently deep anesthesia was confirmed, a midline neck incision was performed, and dissection carried down to the strap muscles. The strap muscles were divided vertically, and the trachea exposed below. Next, a cotton-tipped applicator was used to sweep off adventitial tissue. A 4-5 ring segment was then selected, and the trachea was excised. A cotton tip applicator was then applied to the adventitia of the trachea to sweep away small blood vessels. A dissector was then used to dissect the lateral tracheal arteries off the trachea, which were subsequently ligated. A 4-5 ring region of trachea was then excised and washed with phosphate-buffered saline (PBS) (Sigma Aldrich). This section of trachea was then re-anastomosed in place with 8-0 PROLENETM(Ethicon, Raritan, NJ) suture. In the treatment group (n=12), a 2 cm x 2 cm PDO thin film was applied externally and circumferentially to the auto-transplanted tracheal graft. The strap muscles were then re- approximated and closed with a single interrupted 5-0 PDS®II suture (Ethicon) and the skin closed with 4-0 chromic suture (Ethicon). Measurement of Perfusion

[0085] Laser speckle contrast imaging (LSCI) was obtained using the RFLSI-ZW Laser Speckle Contrast Imaging System (RWD Systems, Sugar Land, TX). Speckle imaging measures blood flow and perfusion by analyzing scattered light refracted from moving blood. This causes a degree of blurring which can be resolved as blood flow. It does not rely on external contrast agents and can resolve blood flow down to 10 µm.19,20Perfusion was then quantified as dimensionless perfusion units.

[0086] LSCI measurements were obtained on unoperated control rat tracheas, and at three specified endpoints: immediately post-operative day 0 rat tracheas, post-operative day 3 ratAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 tracheas in operated control rats, and post-operative day 10 rat tracheas in both operated control and PDO treatment tracheas. The day 3 endpoint was used to validate devascularization in the auto-transplant model, and thus was avoided in the treatment group. Two-second clips measuring 81-101 frames were obtained with each capture, from which average perfusion units were measured in the native, non-auto-transplanted trachea, and the auto-transplanted trachea in operated rats. Percent perfusion loss was calculated using the following formula: ^^^^^^^^^^^^ ^^^^^^^^ℎ^^^^^^ ^^^^^^^^^^^^^^^^^^ െ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^ℎ^^^^^^ ^^^^^^^^^^^^^^^^^^^^100 ^^^^^^^^^^^^ ^^^^^^^^ℎ^^^^^^ ^^^^^^^^^^^^^^^^^^Histological Analysis

[0087] Unoperated tracheal control samples and day 10 tracheal samples were preserved in optimal cutting temperature (OCT) solution, snap frozen, and stored at -80 ºC. Subsequently, tissues were sectioned in 3-4 µm thick sections and stained with hematoxylin-eosin (H&E; Epredia, Kalamazoo, MI). Histological sections were qualitatively analyzed for evidence of loss on previously validated markers of airway injury including derangements to tracheal epithelium, subepithelium and cartilage, and peritracheal region.21Damage to the epithelium included loss, derangement, or atrophy of ciliated epithelium and goblet cells. Subepithelial changes of interest included inflammation, edema, and loss of vasculature. Peritracheal and cartilaginous changes included evidence of fibrosis, edema, and inflammation.21Scores of 0-3 were assigned to each sample based on derangements in each of the three locations and compared. Scores of 0 suggest healthy tissue with no apparent damage. Scores of 1 and 2 suggest concentrated regions of injury in the airway, and a score of 3 corresponds to global damage consistent with ischemic injury. Two cardiothoracic surgery residents independently assigned scores to each sample with a senior cardiothoracic surgeon designated to adjudicate discrepancies in assigned scores. Immunoassay protein quantification

[0088] Luminex®multiplex immunoassays were used to assess mean fold increase in cytokines and inflammatory markers from tissue homogenates between operated control and PDO thin film-treated tracheas at day 10. Protein was extracted from tissue samples after mechanical digestion on ice in 300 µL of extraction buffer containing 20 mM Tris HCl (Invitrogen, GrandAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Island, NY), 0.5% Tween-20 (Sigma-Aldrich), 150 mM NaCl (Invitrogen), and a cOmplete Mini Protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). A Bio-Gen PRO200 Homogenizer (PRO Scientific, Oxford, CT) was used for homogenization. The tissue lysate was then centrifuged at 14,000 xg for 10 min at 4 °C to isolate the protein in the supernatant. Protein concentrations were determined using the PierceTMBCA Protein Assay (ThermoFisher Scientific, Waltham, MA), and all tissue samples were normalized to 1 µg / µL before submission.

[0089] The Human Immune Monitoring Center at Stanford University conducted the assays. Kits were sourced from EMD Millipore Corporation (Burlington, MA) and executed according to the manufacturer’s protocol. Briefly, samples were diluted 1:1 with assay buffer, and 25 µL of the diluted sample was mixed with antibody-linked magnetic beads in a 96-well plate. This mixture was incubated overnight at 4 °C with shaking. Cold and room temperature incubations were performed on an orbital shaker at 500-600 rpm. Plates were washed twice with wash buffer using a BioTek ELx405 washer (BioTek Instruments, Winooski, VT). Following a 1 h incubation at room temperature with biotinylated detection antibody, streptavidin-PE was added for 30 min with shaking. Plates were washed again, and PBS was added to the wells for reading in the Luminex®FlexMap3D Instrument, ensuring a lower bound of 50 beads per sample per cytokine. Each sample was measured in duplicate. Custom Assay Chex control beads (Radix BioSolutions, Georgetown, TX) were added to all wells. Statistical Analysis

[0090] Statistical tests are performed with Student’s T-test, with a pre-specified alpha level of 0.05, when comparing two characteristics, and unpaired one-way ANOVA with Tukeys when comparing more than two unpaired characteristics. When comparing more than two characteristics in paired samples, a paired one-way ANOVA was used. All statistical analyses were performed in Prism Version 10.2.3 (GraphPad, Boston, MA), Excel, and OriginPro Version 1.52a (OriginLab Corporation, Northampton, MA). Film data are represented as mean ± standard deviation. Results: Characterization of PDO Thin Film Morphology and Mechanical PropertiesAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0091] Chemistry, microscopic characteristics (e.g., fiber diameter and porosity), and macroscopic features (e.g., fiber orientation), can be rationally designed to modulate the physicochemical properties of fiber-based devices and the resulting biological response to implantation.22-24This includes fiber composition, dimensions, orientation and overall thin film architecture. We hypothesized that degradable, nanofiber-based thin films may integrate with tracheal tissue and provide a temporary support structure to facilitate angiogenesis in the critical post-operative period. We fabricated nanofiber-based thin films composed of PDO, a highly biocompatible, biodegradable polymer used in fiber production for sutures and other commercial biomaterials.25FIG. 10A provides a representative, high magnification SEM image of the electrospun thin film, revealing fabrication of a porous scaffold composed of uniform, defect-free, randomly aligned fibers with a mean diameter of 915.6 ± 281 nm (Table 1.1). Fiber size and arrangement within the construct are crucial for optimizing function as an implantable film. Smaller fiber diameters enhance surface area, which may improve integration with surrounding tissues. Nanofiber-based PDO films had an average pore size of 0.95 ± 0.1 µm², which is critical for oxygen permeability and promotion of vascularization at the implantation site.26The controlled porosity and fiber dimensions of the PDO film support adequate nutrient diffusion and facilitate interactions between the film and surrounding tissues, enhancing local perfusion and healthy integration with host tissue.27AFM analysis (FIG. 10B) revealed uniform surface root- mean square roughness of 1.30. Color-coded SEM images and derivatives of Fast Fourier Transformations (FFT) were utilized to evaluate fiber alignment, resulting in an average coherency of 0.14 ± 0.09, indicating random orientation (FIGS.10C-10E). PDO films exhibited an average breaking strength of 1.41 ± 0.26 N, tensile modulus of 0.06 ± 0.01 N / tex, and elongation at break of 242 ± 62.4% (Table 1.1). The balanced mechanical properties of the nanofiber-based PDO thin films enable sufficient elasticity and flexibility to confirm to the shape and environment of the trachea while simultaneously providing sufficient strength for handleability and placement. Surgical Model

[0092] After a training phase, 30 rats underwent successful tracheal auto-transplantation, including 18 (60.0%) operated controls and 12 (40%) receiving PDO thin films. Eight of the operated controls were sacrificed at day 0 post-procedure. Two additional rats were sacrificed at day 3 to validate short term devascularization, and the remaining eight were survived to 10 days.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Laser Speckle Contrast Imaging

[0093] Laser speckle contrast imaging at day 0 in 8 rats pre-operatively (FIG. 11A), after sweeping adventitia off and ligating the tracheal arteries (FIG.11B), and immediately post-auto- transplant (FIG. 11C) demonstrated significant, progressive decrease in perfusion with each measure of devascularization (FIG. 11D) (p<0.001). The mean percent perfusion loss pre- to post-operatively was 28.9 ± 16%. At post-operative day 3, four control rats were re-opened and LSCI obtained (FIG.12A), demonstrating persisting perfusion loss with a mean percent perfusion loss of 56.7 ± 28%. At post-operative day 10, operated control rats (n=8) underwent LSCI revealing a significant perfusion defect in the auto-transplanted segment of trachea (FIGS. 12B- 12C). The mean percent perfusion loss was 28.4 ± 12% for an average 257 ± 19.3 perfusion unit loss between native and transplanted trachea (p<0.01). Auto-transplanted rats in the PDO film treatment group (n=10) at day 10 demonstrated significantly less perfusion loss between the native trachea and the transplanted trachea 72.8 ± 15 perfusion units (p<0.05). The mean percent loss was also significantly lower than the operated control group at 11.1 ± 9.7% (p<0.01), representing a 2.5x increase in perfusion in the PDO thin film treatment group (FIGS.12D-12F).

[0094] Histological Analysis

[0095] H&E staining of unoperated control tracheas (n=5, FIG. 13A) demonstrated healthy appearing ciliated epithelium, with intact goblet cells. H&E analysis of post-operative day 10 auto-transplanted tracheas (n=5, FIG.13B) revealed dense proliferation in the subepithelial space of the tracheas, lack of basophilia in the cartilaginous rings and disruption of ring integrity, narrowing of the airway, disruption of ciliated epithelium, and apparent loss of goblet cells. The mean histological injury score was 2.6 ± 0.5. In the PDO thin film treatment group, H&E-stained sections of tracheas at post-operative day 10 revealed intact cartilaginous rings with normal basophilia, preserved ciliated epithelium, albeit with present epithelial inflammation (n=4, FIG. 13C). The mean histological injury score (1.2 ± 0.5) was significantly lower than operated controls (p<0.001). These histological results correlate with the noticeable difference in perfusion between operated control and treatment groups. Immunoassay ProfilingAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0096] Results of the Luminex®multiplex immunoassay are shown in FIG.14, demonstrating mean fold increase relative to reference beads. Immunoassays were performed to assess interleukin (IL) 1 alpha and beta, chemokine (C-C) ligand 5 (CCL5), monocyte chemoattractive protein 3 (MCP-3), IL-17, intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor (VEGF) expression. When compared to the operated control group (n=3 per assay, 2 replicates each), the PDO thin film implantation group (n=7 per assay, 2 replicates each) demonstrated significantly higher expression of VCAM-1 (p<0.05). There was no significant difference in expression of the other proteins assayed. Discussion:

[0097] In this study, we demonstrated with speckle perfusion imaging that surgical devascularization produces a persistent perfusion defect in transplanted rat airways even in the setting of syngeneic immunology. We then fabricated and tested the application of a biodegradable, nanofiber-based thin-film, which demonstrated significantly improved graft perfusion compared to operated controls at 10 days post-operatively. Hypoxia resulting from compromised circulation contributes to poor long-term survival after lung transplantation, particularly in its contribution to airway complications, and ultimately the development of BOS.5These complications contribute to the 5.8 year median survival after lung transplantation, the lowest among all conventional solid organ transplants.28Airway complications can affect up to 1 in 3 lung transplant recipients and BOS is the primary cause of long-term mortality after lung transplantation.8,29Surgical models of hypoxia after lung transplantation have characterized the immune-mediated vascular destruction contributing to persisting hypoxia after lung transplantation. They typically do not address the surgical devascularization that is inherent to procurement and transplantation of lungs, given the routine sacrifice of the bronchial arteries.6,8,9Tracheal Auto-Transplantation Produces a Persistent Perfusion Defect

[0098] Though auto-transplantation of the trachea in rodents has been described in the literature, questions remain about how persistently devascularized the grafts remain and whether functional perfusion can be measured.21In this study, we utilized an autotransplant model, but incorporated specific measures to ensure successful devascularization. Namely, we ligated theAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 lateral tracheal arteries, and intentionally swept off any adventitia and associated microperfusion vessels surrounding the trachea, prior to completing circumferential dissection, and excision of an autograft. The autograft was washed in PBS and re-anastomosed to ensure that any connection in vasculature is entirely severed. We confirmed immediate drops in perfusion at each critical step from native trachea, to ligation of the arteries, and immediately post-operatively using LSCI, a method of dynamic perfusion measurement. Perfusion in the autograft was signfiicantly lower than in native trachea pre-operatively. Devascularization was evident at day 3 and at day 10 post- operatively, with significant reductions in perfusion units between the untransplanted native trachea and the autograft within the same animal.

[0099] The finding of persistent perfusion defects 10 days post-operatively is novel in the setting of syngeneic immunology.6In a study by Jiang, et al, on airway anastomotic revascularization in an allotransplant model of rodent tracheal transplantation, use of immunosuppression resulted in regeneration of microvessls demonstrated by histological analysis.6Microvascular depletion was deemed to be partly related to complement mediated destruction, and immunosuppression rescued those vessels that otherwise would be destroyed. However, it was unclear how significantly these microvessels contributed to perfusion of the graft. Using LSCI, we show that even in the setting of microvessel regeneration, as there would be in syngeneic physiology without an allo-immune response, there is no significant improvement in perfusion compared to baseline measurements. Application of a Nanofiber-based PDO Thin Film Improves Airway Perfusion

[0100] It is known that implant composition and topography influence cell adhesion, proliferation and behavior in patients; however, implant characteristics have not been rigorously evaluated for angiogenesis induction in vivo, which is critical for tissue engineering and transplant applications.24,30-37Nanofiber-based thin films are particularly attractive due to their biocompatibility, biomimetic architecture, porosity, and high surface area-to-volume ratio, thereby enhancing endothelial cell adhesion and proliferation, and providing a template extracellular matrix (ECM) to support ingrowth of a structured vascular network.38We fabricated biodegradable nanofiber-based thin films with uniform surface roughness and random orientation. Films were successfully applied to the anastomosis site and integrated with the trachea, as shown in gross images and histology. When applied circumferentially to the surface of the trachea duringAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 the autotransplant procedure, randomly aligned, nanofiber-based PDO thin films attenuated the hypoperfusion seen in operated controls at post-operative day 10. This was demonstrated with speckle angiography which showed significant improvements in the perfusion loss between the native and the auto-transplanted trachea. Additionally, there was reconstitution of the horizontal tracheal arteries suggesting improved microvascular and macrovascular circulation.

[0101] The improvements in perfusion seen on speckle angiography appear to be validated in the preservation of the structural integrity of the tracheal cartilaginous rings, which appeared necrotic, cracked, and weak in the operated control group. Furthermore, whereas the operated control group showed loss of goblet cells and blunting of ciliated epithelium, treatment group tracheas at post-operative day 10 had intact goblet cells and preserved ciliated epithelial architecture. These differences between operated control and treatment groups were reflected in histological injury scores. We observed similar changes described by Hyytinen, et al, and used their scoring method for grading injury in the transplanted tracheas.21Ischemic changes in their study included loss of structural integrity of the cartilaginous rings, disruption of the ciliated epithelium, and dense subepithelial inflammation. In the treatment group, inflammation likely resulted from similar ischemic injury. Immune Pathways May Modulate Airway Perfusion

[0102] Multiplex immunoassays revealed that VCAM-1 expression was higher in the treatment group compared to operated controls. This may be related to an increased number of viable endothelial cells in the treatment group compared to operated control. VCAM-1 is an inducible protein found in endothelial cells and a known inflammatory cytokine implicated in tumor angiogenesis and retinal hypoxia-induced neovascularization.39-41Kaur, et al, demonstrated that VCAM-1 regulates IL-8 promoter activity and further showed that intravitreal injection of VCAM-1 led to increased IL-8 and vascular sprouting and neovascularization in a murine model of hypoxic retinal disease.39Despite the increased expression of VCAM-1, VEGF levels were not significantly different between the operated control and treatment groups. Given that the angiogenic properties of VCAM-1 are potentially attributed to IL-8 upregulation, which itself promotes VEGF expression, there may be separate pathways that participate in the potential angiogenesis observed in this experiment.42Inflammation appears to be a key mediator of this effect. Despite the role of VCAM-1 in promotion of inflammatory cell migration, increasedAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 expresssion of IL-1 was not seen in our treatment group.43IL-1 upregulation is associated with chronic lung allograft disease, lung fibrosis, and rejection in lung transplantation.44-46The random nanofiber PDO thin film may provide ECM-like structural support for repair of existing microvasculature in the autograft. This may rely on IL-1 naïve inflammatory pathways that lead to increased endothelial cell viability. Favoring pathways that promote angiogenesis without activating pathways that prompt allograft dysfunction is an important characteristic of a potential therapeutic intervention for lung transplant airway devascularization. Conclusion

[0103] This study found that rat tracheal auto-transplantation adequately models persistent surgical devascularization of airways, a phenomena that occurs in clinical lung transplantation. Application of a degradable, randomly-aligned, nanofiber-based thin film significantly improved perfusion to transplanted airways, which was validated by speckle angiography and histological injury scoring. Multiplex immunoassay results demonstrated that inflammatory cytokines may play a role in promoting angiogenesis in the tracheal auto-transplants, without upregulating traditionally deleterious chemokines. There are limited therapeutic approaches to addressing post- transplant airway hypoxia; however, degradable, nanofiber-based thin films may provide a convenient new modality to attenuate hypoxia after lung transplantation.

[0104] Table 1.1. Characterization of PDO film morphology and mechanical properties (n=3, each). Sample Fiber Diameter Pore Size Directionality Breaking Tensile Elongation (nm) (µm2) (Coherency) Strength Modulus (%)e e e ces o a p e 1. Holt CD. Overview of Immunosuppressive Therapy in Solid Organ Transplantation. W.B. Saunders; 2017. p.365-380. 2. Rana A, Gruessner A, Agopian VG, et al. Survival Benefit of Solid-Organ Transplant. JAMA Surgery.2015 / 03 / 01;150(3)doi:10.1001 / jamasurg.2014.2038 3. Khan MA, Nicolls MR. Complement-mediated microvascular injury leads to chronic rejection. Adv Exp Med Biol.2013;735:233-46. doi:10.1007 / 978-1-4614-4118-2_16Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 4. Lee JC, Christie JD. Primary graft dysfunction. Proceedings of the American Thoracic Society.2009;6(1):39-46. doi:10.1513 / PATS.200808-082GO 5. Pasnupneti S, Nicolls MR. Airway hypoxia in lung transplantation. Curr Opin Physiol. Feb 2019;7:21-26. doi:10.1016 / j.cophys.2018.12.002 6. Jiang X, Malkovskiy AV, Tian W, et al. Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles. Biomaterials. 2014;35(2):803-813. doi:10.1016 / j.biomaterials.2013.09.092 7. Jiang X, Hsu JL, Tian W, et al. Tie2-dependent VHL knockdown promotes airway microvascular regeneration and attenuates invasive growth of Aspergillus fumigatus. J Mol Med (Berl). Sep 2013;91(9):1081-93. doi:10.1007 / s00109-013-1063-8 8. Nicolls MR, Zamora MR. Bronchial blood supply after lung transplantation without bronchial artery revascularization. Current Opinion in Organ Transplantation. 2010;15(5):563- 567. doi:10.1097 / MOT.0b013e32833deca9 9. Unai S, Yun J, Pettersson GB. Bronchial Artery Revascularization: Surgical Technique. Operative Techniques in Thoracic and Cardiovascular Surgery. 2023;28(2):139-153. doi:10.1053 / j.optechstcvs.2022.11.004 10. Zhang XT, Wang YX, Gao ZY, et al. Advances in wound dressing based on electrospinning nanofibers. J Appl Polym Sci. Jan 52024;141(1)doi:10.1002 / app.54746 11. Bhardwaj N, Kundu SC. Electrospinning: A fascinating fiber fabrication technique. Biotechnol Adv. May-Jun 2010;28(3):325-347. doi:10.1016 / j.biotechadv.2010.01.004 12. He C, Nie W, Feng W. Engineering of biomimetic nanofibrous matrices for drug delivery and tissue engineering. 10.1039 / C4TB01464B. Journal of Materials Chemistry B. 2014;2(45):7828-7848. doi:10.1039 / C4TB01464B 13. Reddy VS, Tian Y, Zhang C, et al. A Review on Electrospun Nanofibers Based Advanced Applications: From Health Care to Energy Devices. Polymers (Basel). Oct 29 2021;13(21)doi:10.3390 / polym13213746 14. Ganesh SS, Anushikaa R, Swetha Victoria VS, Lavanya K, Shanmugavadivu A, Selvamurugan N. Recent Advancements in Electrospun Chitin and Chitosan Nanofibers for Bone Tissue Engineering Applications. J Funct Biomater.2023;14(5):288. 15. Zhang X, Wang Y, Gao Z, et al. Advances in wound dressing based on electrospinning nanofibers. J Appl Polym Sci.2024;141(1):e54746. doi:https: / / doi.org / 10.1002 / app.54746 16. Pal D, Das P, Mukherjee P, et al. Biomaterials-Based Strategies to Enhance Angiogenesis in Diabetic Wound Healing. Acs Biomater Sci Eng. Apr 17 2024;10(5):2725-2741. doi:10.1021 / acsbiomaterials.4c00216 17. Klein SL, Flanagan KL, Klein SL, Flanagan KL. Sex differences in immune responses. Nature Reviews Immunology 201616:10.2016-08-22;16(10)doi:10.1038 / nri.2016.90 18. Oh SS, Narver HL. Mouse and Rat Anesthesia and Analgesia. Current Protocols. 2024 / 02 / 01;4(2)doi:10.1002 / cpz1.995 19. Li D-Y, Xia Q, Yu T-T, et al. Transmissive-detected laser speckle contrast imaging for blood flow monitoring in thick tissue: from Monte Carlo simulation to experimental demonstration. Light: Science & Applications 202110:1. 2021-12-03;10(1)doi:10.1038 / s41377- 021-00682-8 20. Laser Speckle Contrast Imaging: theory, instrumentation and applications - PubMed. IEEE reviews in biomedical engineering.2013;6doi:10.1109 / RBME.2013.2243140 21. Hyytinen T, Paavonen T, Inkinen K, Ahonen J, Mattila S. Airway anastomotic healing in the rat tracheal autograft. Eur Surg Res.1999;31(2):155-61. doi:10.1159 / 000008634Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 22. Josyula A, Mozzer A, Szeto J, et al. Nanofiber-based glaucoma drainage implant improves surgical outcomes by modulating fibroblast behavior. Bioengineering & translational medicine. 2023;8(3):e10487. doi:https: / / doi.org / 10.1002 / btm2.10487 23. Parikh KS, Josyula A, Omiadze R, et al. Nano-structured glaucoma drainage implant safely and significantly reduces intraocular pressure in rabbits via post-operative outflow modulation. Scientific Reports.2020 / 07 / 312020;10(1):12911. doi:10.1038 / s41598-020-69687-4 24. Josyula A, Parikh KS, Pitha I, Ensign LM. Engineering biomaterials to prevent post- operative infection and fibrosis. Drug delivery and translational research. Aug 2021;11(4):1675- 1688. doi:10.1007 / s13346-021-00955-0 25. Kariduraganavar MY, Kittur AA, Kamble RR. Chapter 1 - Polymer Synthesis and Processing. In: Kumbar SG, Laurencin CT, Deng M, eds. Natural and Synthetic Biomedical Polymers. Elsevier; 2014:1-31. 26. Palani N, Vijayakumar P, Monisha P, Ayyadurai S, Rajadesingu S. Electrospun nanofibers synthesized from polymers incorporated with bioactive compounds for wound healing. J Nanobiotechnol. Apr 272024;22(1)doi:ARTN 211 10.1186 / s12951-024-02491-8 27. Augustine R, Gezek M, Bostanci NS, Nguyen A, Camci-Unal G. Oxygen-generating scaffolds: One step closer to the clinical translation of tissue engineered products. Chemical Engineering Journal. 2023 / 01 / 01 / 2023;455:140783. doi:https: / / doi.org / 10.1016 / j.cej.2022.140783 28. Thabut G, Mal H. Outcomes after lung transplantation. Journal of Thoracic Disease. 2017 / 08;9(8)doi:10.21037 / jtd.2017.07.85 29. Crespo MM. Airway complications in lung transplantation. Journal of Thoracic Disease. 2021 / 11;13(11)doi:10.21037 / jtd-20-2696 30. Anand N, Arora S, Clowes M. Mitomycin C augmented glaucoma surgery: evolution of filtering bleb avascularity, transconjunctival oozing, and leaks. Br J Ophthalmol. Feb 2006;90(2):175-80. doi:10.1136 / bjo.2005.077800 31. Ayyala RS, Michelini-Norris B, Flores A, Haller E, Margo CE. Comparison of different biomaterials for glaucoma drainage devices: part 2. Arch Ophthalmol. Aug 2000;118(8):1081-4. 32. Doloff JC, Veiseh O, de Mezerville R, et al. The surface topography of silicone breast implants mediates the foreign body response in mice, rabbits and humans. Nature Biomedical Engineering.2021 / 10 / 012021;5(10):1115-1130. doi:10.1038 / s41551-021-00739-4 33. Grover DS, Flynn WJ, Bashford KP, et al. Performance and Safety of a New Ab Interno Gelatin Stent in Refractory Glaucoma at 12 Months. Am J Ophthalmol. Aug 042017;183:25-36. doi:10.1016 / j.ajo.2017.07.023 34. Kam KR, Walsh LA, Bock SM, Ollerenshaw JD, Ross RF, Desai TA. The effect of nanotopography on modulating protein adsorption and the fibrotic response. Tissue Eng Part A. Jan 2014;20(1-2):130-8. doi:10.1089 / ten.TEA.2012.0772 35. Harvey AG, Hill EW, Bayat A. Designing implant surface topography for improved biocompatibility. Expert Review of Medical Devices. 2013 / 03 / 01 2013;10(2):257-267. doi:10.1586 / erd.12.82 36. Harawaza K, Cousins B, Roach P, Fernandez A. Modification of the surface nanotopography of implant devices: A translational perspective. Materials Today Bio. 2021 / 09 / 01 / 2021;12:100152. doi:https: / / doi.org / 10.1016 / j.mtbio.2021.100152 37. Nazarnezhad S, Kargozar S, Ramakrishna S. Chapter 18 - Electrospun nanofibers for angiogenesis strategies. In: Kargozar S, Mozafari M, eds. Biomaterials for Vasculogenesis and Angiogenesis. Woodhead Publishing; 2022:383-414.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 38. Nazarnezhad S, Baino F, Kim HW, Webster TJ, Kargozar S. Electrospun Nanofibers for Improved Angiogenesis: Promises for Tissue Engineering Applications. Nanomaterials (Basel, Switzerland). Aug 172020;10(8)doi:10.3390 / nano10081609 39. Kaur G, Sharma D, Bisen S, et al. Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8 / CXCL1 expression and pathological neovascularization. Communications Biology 20236:1.2023-05-13;6(1)doi:10.1038 / s42003-023-04905-z 40. Kong D-H, Kim YK, Kim MR, Jang JH, Lee S. Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer. International Journal of Molecular Sciences.2018 / 04;19(4)doi:10.3390 / ijms19041057 41. Kaur G, Sharma D, Bisen S, Mukhopadhyay CS, Gurdziel K, Singh NK. Vascular cell- adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8 / CXCL1 expression and pathological neovascularization. Communications Biology. 2023 / 05 / 13 2023;6(1):516. doi:10.1038 / s42003-023-04905-z 42. Martin D, Galisteo R, Gutkind JS. CXCL8 / IL8 Stimulates Vascular Endothelial Growth Factor (VEGF) Expression and the Autocrine Activation of VEGFR2 in Endothelial Cells by Activating NFκB through the CBM (Carma3 / Bcl10 / Malt1) Complex. The Journal of Biological Chemistry. 2009 / 03 / 03;284(10)doi:10.1074 / jbc.C800207200 43. Cook-Mills JM, Marchese ME, Abdala-Valencia H. Vascular Cell Adhesion Molecule-1 Expression and Signaling During Disease: Regulation by Reactive Oxygen Species and Antioxidants. Antioxidants & Redox Signaling.2011 / 09 / 09;15(6)doi:10.1089 / ars.2010.3522 44. Andreasson ASI, Borthwick LA, Gillespie C, et al. The role of interleukin-1β as a predictive biomarker and potential therapeutic target during clinical ex vivo lung perfusion. The Journal of Heart and Lung Transplantation.2017 / 09;36(9)doi:10.1016 / j.healun.2017.05.012 45. Borthwick LA. The IL-1 cytokine family and its role in inflammation and fibrosis in the lung. Seminars in Immunopathology.2016;38(4)doi:10.1007 / s00281-016-0559-z 46. Pandolfi L, Bozzini S, Morosini M, et al. Significant Upregulation of BAL-f IL-1β in Lung Transplant Recipients During Stability and CLAD. The Journal of Heart and Lung Transplantation.2022 / 04 / 01;41(4)doi:10.1016 / j.healun.2022.01.758 47. The role of interleukin-10 in lung transplantation - PubMed. Transplant immunology.2002 May;9(2-4)doi:10.1016 / s0966-3274(02)00045-x 48. Eelen G, Treps L, Li X, Carmeliet P. Basic and Therapeutic Aspects of Angiogenesis Updated. Circulation Research.2020-07-03;127(2)doi:10.1161 / CIRCRESAHA.120.316851 EXAMPLE 2 Introduction

[0106] Lung transplantation is a life-saving therapy for end-stage pulmonary disease, but its long-term success is limited, with a median survival of 5.8 years which is significantly lower than other conventional solid organ transplants.1-4While chronic lung allograft dysfunctionAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 (CLAD) is the leading cause of mortality in lung transplantation, one in three lung transplant recipients will face airway complications comprised of stenosis, dehiscence, or infection of the bronchial anastomosis.5-11These complications are likely driven by ischemia at the bronchial anastomosis, resulting from the routine division of the bronchial arteries during donor lung procurement. Mounting evidence suggests that airway ischemia may be an early contributor to eventual development of CLAD, particularly bronchiolitis obliterans syndrome (BOS).12-15

[0107] Few effective interventions currently exist to address post-transplant airway ischemia. Bronchial artery revascularization (BAR), performed at the time of transplant by connecting the left internal mammary artery to a patch of donor aorta comprising the origin of the bronchial arteries, is performed only by a minority of select centers due to technical complexity of the operation.15-17There is an unmet need for therapies that promote revascularization of the transplanted airways in a manner compatible with routine lung transplantation.

[0108] Local delivery of deferoxamine (DFO), an iron chelating agent, has shown promise in initiating angiogenesis in transplanted airways.18We hypothesized that sustained, controlled release of DFO locally at the airway anastomosis would promote durable revascularization of the ischemic airways. Previously, our group has shown the therapeutic potential of using nanofiber thin films to serve as a scaffold for addressing airway ischemia in a rodent model.19

[0109] Here, we report the development and testing of a DFO-eluting nanofiber thin film for improving airway perfusion after transplant. We first evaluated the material properties, drug release profile, and biocompatibility of the thin film. We then tested its therapeutic efficacy in a rat tracheal auto-transplantation model and a swine tracheal patch allotransplantation model that replicates airway ischemia present in lung transplantation.19,20Results Fabrication of Nano-Structured, DFO-Eluting Thin Films

[0110] Electrospinning produced uniform PGS-based, randomly-aligned nanofiber films with or without deferoxamine. We also produced smooth, randomly oriented microfiber, and randomly aligned nanofiber thin films (FIGS. 15A-15F). Select thin film characteristics are shown in Table 1.2, demonstrating modification of porosity, fiber diameter and directionality,Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 strength, and contact angle (n=4). DFO loading into randomly-aligned nanofiber thin films significantly reduced fiber diameter, and doubling loading enabled tuning of the release profile from sustained release over 7 days at 10% loading to burst release in the first 24 h at 20% loading (n=3), which also correlated with a loss in strength (FIGS.16A-16C). Drug loading for random nano PGS / 10% DFO and PGS / 20% DFO was 17 ± 1 and 35 ± 1 µg per mg of film, respectively (n=3).50 µm thick films were evaluated in a rat auto-transplantation model (below) to determine the effect of architecture and / or local delivery of an iron-chelator, DFO on airway perfusion.

[0111] Table 1.2: Summary of the structural and mechanical properties of PGS-based films. The table presents the average fiber diameter, porosity, tensile strength, modulus, and fibers directionality. Data are reported as mean ± SD. (N=3).DFO-Eluting Thin Films Significantly Improve Airway Perfusion in Rats

[0112] Tracheal devascularization and autotransplantation (FIGS. 17A-17B) was performed in n=54 rats, with treatment groups (n=6-9 per group) consisting of operated control (without thin film placement), smooth film placement, random micro, aligned nano, random nano, random-nano-DFO 10%, and random-nano-DFO 20%. All rats survived to the endpoint of 10 days. At the terminal end point, all thin films had fully dissolved and incorporated into the tissue. Results of LSCI perfusion imaging at the terminal end point are shown in FIGS.18A-18B. Lower percent perfusion loss in the autograft indicated that there was improved perfusion in the autograft. There was a stepwise improvement in perfusion in the autograft as fibers decreased in size and were oriented randomly. Rats receiving nano-DFO 10% films had the least loss of perfusion (1.3%±6.2%) between native trachea and autograft, significantly improved compared to operatedAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 control (28.4%±11.7%) (p<0.001). Rats in the nano-DFO 20% (perfusion loss 7.0%±11.4%) group did not have improved perfusion compared to nano-DFO 10% (p>0.05).

[0113] Lectin Perfusion Confirmed Functional Perfusion of Tracheal Autografts

[0114] Lectin perfusion staining (n=3-5 per treatment group) corroborated the LSCI findings by highlighting functional microvessels in the grafts (FIGS. 19A-19B). Given that the lectin was injected remotely in the rats, positive endothelial staining in the explanted tracheal grafts represented functional perfusion. Healthy, unoperated controls are shown to demonstrate native patterns of lectin perfusion. In control grafts at day 10, lectin staining revealed sparse perfused microvasculature: only a few thin, discontinuous vessels were stained in the submucosa, reflecting incomplete revascularization. Similarly to the LSCI perfusion data, improvements in lectin staining were seen with random topography and nanofiber composition of the thin film. Rats treated with the DFO 10% thin film experienced the greatest improvement in lectin staining and microvascular perfusion with a 32% increase in perfused vessel density compared to controls (p<0.0001). Additionally, DFO 10% thin films produced an average 18.7% increase in lectin staining compared to DFO 20% thin films (p=0.01). Notably, the lateral polarity of lectin staining seen in healthy controls, corresponding to the lateral tracheal arteries, was not seen in the treated groups. Rather, stained blood vessels followed a more widespread distribution. HIF1a is Downregulated at the Terminal End Point in the DFO-10% Group

[0115] Quantitative PCR was performed on unoperated healthy control tissue as a reference group, operated control tissue, and rats that received random nanofiber thin films (no- drug PGS) and nano-DFO 10% (n=3-5 per treatment group) (FIG.20). Hypoxia inducible factor 1a (HIF1a) was expressed in operated controls at over 15 mean fold increase compared to healthy controls (p<0.0001). Expression of HIF1a was also significantly higher in operated controls compared to the no-drug PGS, and the DFO 10% groups (p<0.0001). Rats receiving DFO 10% thin films expressed the lowest HIF1a at the terminal end point compared to operated controls and no-drug PGS (p<0.0001). IL-10 was significantly elevated in all three treatment arms compared to healthy controls, with the highest expression in no-drug PGS (>50 mean fold increase compared to 35x and 30x mean fold increase in operated controls, and DFO 10% respectively, p<0.0001). While PTGS2 was significantly increased compared to healthy controls in all three treatmentAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 groups, it was expressed at a mean fold increase of over 40x compared to less than 5x in no-drug PGS and DFO 10% (p<0.0001). VEGF was only significantly increased in operated control and DFO 10%, both at approximately 5x mean fold increase (p<0.0001). DFO-Eluting Thin Films Significantly Improve Airway Perfusion in Swine

[0116] Given the favorable properties and treatment response of the random nanofiber DFO 10%-eluting thin film in the rat autotransplant model, treatment groups for the swine tracheal patch allotransplant model (FIGS. 21A-21B) were chosen to be operated control, randomly oriented nanofiber thin film without drug, and randomly oriented nanofiber thin film with DFO 10% (n=4 per treatment group). All 12 pigs underwent successful tracheal patch allotransplantation from sex- and blood-type-matched donors and survived to the 14-day endpoint.

[0117] LSCI of the tracheal allografts at the terminal end points demonstrated the most favorable perfusion of the tracheal allografts in the pigs receiving DFO-10% thin films (FIG.22). Pigs receiving a DFO-10% thin film had an average perfusion loss of 9.96% ± 2.36% compared to pigs receiving a no-drug PGS thin film (41.60% ± 12.94%) (p<0.01) and operated control (34.46% ± 11.03%) (p<0.05). Like rats, the thin films were totally incorporated in the entirety of treatment groups. DFO-10% Treated Swine have Improved Healing on Bronchoscopy at 2 Weeks

[0118] Bronchoscopy performed prior to sacrifice demonstrated worse signs of necrosis and increased presence of granulation tissue in the operated control and PGS treated pigs, compared to those receiving random nano PGS DFO-10% thin films (FIG. 23). Notably, there were more visible vessels and even hyperemia in the DFO-10% group compared to dusky / pallorous tracheal tissue at the patch site in the PGS and operated control groups. No swine had an air leak after completion of the index anastomosis upon air leak testing.

[0119] Discussion

[0120] Airway ischemia prompting bronchial anastomotic complications including stenosis, dehiscence, and most commonly infection cause significant morbidity and mortality after lung transplantation, and there is little widespread uptake of treatment strategies to mitigate theseAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 issues.15,17,21In this study, we used a rat model of syngeneic tracheal autotransplantation and a swine model of allogeneic tracheal patch transplantation to study the efficacy of sustained delivery of deferoxamine on airway revascularization and anastomotic healing.

[0121] We produced thin films comprised of textured and non-textured (smooth) micro- and nanofibers and tested the various topographies and fiber sizes for handleability, degradation profile, drug loading and unloading capabilities. We implanted a range of thin films without drug in the rat model to explore the healing properties of the thin films alone but loaded drug onto only the randomly aligned PGS nanofiber thin film given favorable properties most notably in drug unloading. Previously, we showed that a polydioxanone thin film without drug had some angiogenic capability in work that validated the use of a rat tracheal autotransplant model to study airway devascularization.19We hypothesized that nano-structured thin films provide architecture for endothelial progenitor cell engraftment in areas of ischemia, which may assist in anastomotic healing, validated by upregulation of vascular cell adhesion molecule-1 (VCAM-1) on proteomic analysis. In the present study we opted to use PGS instead as it has a faster degradation profile, had been used in FDA approved therapies, and had improved handleability while retaining the ability to load and provide sustained release of DFO.

[0122] In our LSCI imaging, there was a clear stepwise progression of improved perfusion with randomly aligned topography, and nanofibers over microfibers. This helped inform the choice to load drug onto randomly aligned nano PGS thin films. The drug release profile indicated that 10% and 20% concentrations of DFO were worth testing, though 10% DFO had improved drug release kinetics, while 20% had an element of burst release in the first 24 hours. We hypothesized that sustained release over at least 3 days could be beneficial for neovascularization.

[0123] The study from Jiang et al. helped inform the choice of deferoxamine as the drug of choice to load onto our thin films.18In their study, mouse tracheas were dipped into a solution containing DFO bound to nanoparticles and then transplanted into a recipient mouse trachea, and subsequently had greater rates of microvascular regeneration though questions remained whether these were functional microvessels, and how long they last given the transient application of DFO.18Nevertheless, the angiogenic properties of DFO appear to be promising with a host of mechanisms proposed from early upregulation of HIF-1α, to persistent stromal cell derived factor- 1 (SDF-1) and placental growth factor-1 (PLGF) upregulation.18,22As such, we hypothesized thatAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 sustained delivery would lead to long-term vascular regeneration and last through the effects of complement-mediated vascular destruction as well.23

[0124] In our study, rats undergoing tracheal autotransplantation wrapped with a nanofiber thin film loaded with DFO-10% experienced the greatest improvement in perfusion in the tracheal autografts compared to operated controls and trended towards significantly improved perfusion compared to the DFO-20%. Visually, the treated tracheas had pink, healthy appearing tissue, with reconstitution of horizontal tracheal artery perfusion. This was further corroborated by lectin staining, which represented functional perfusion. By remotely injecting the lectin, the only way that endothelial cells within the autograft could be stained positive would be through intact, regenerated vasculature. The stark difference between the DFO-10% group and the operated control suggests a degree of neovasculogenesis, and that the perfusion through these new blood vessels was meaningful and penetrated the entire circumferential trachea. Furthermore, the contrast between the polar pattern of the healthy control compared to the dispersed pattern of the treatment groups suggests that the vasculature was not just reconstitution of existing blood vessels but truly de novo blood vessel formation. The increased lectin positivity between the DFO-10% versus the DFO-20% group also suggests that sustained release achieved by certain embodiments is important in maintaining long-term perfusion. This also addresses a limitation to the methodology proposed by Jiang et al., in that the entirety of DFO was delivered at the index surgery in their model.18

[0125] Mechanistically, the DFO-10%-eluting film appears to exert its effects through HIF-1α mediated angiogenesis. DFO is established to stabilize HIF-1α and thereby induce a host of pro-angiogenic genes.9,18,22,24-27Although we did not capture the transient early spike in HIF- 1α or VEGF in the tissue (due to the day-10 analysis), our results show a clear proxy of HIF-1 activity: the control grafts had high HIF-1α expression at day 10, whereas DFO-treated grafts did not. This inverse relationship suggests that DFO had triggered angiogenesis early, leading to reperfusion that alleviated hypoxia by day 10 (hence less HIF-1α needed). In essence, DFO activity may have set up a positive feedback loop – it induced vessel growth which then satisfied the oxygen demand and turned off the hypoxia signal. We view the lowered HIF-1α at day 10 in the DFO-10% treatment group as an indicator of successful resolution of ischemia.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0126] Consistent with these findings is the significant upregulation of PTGS-2 in the operated control group compared to the thin film groups; PTGS-2 is associated with ischemic injury and particularly ischemia reperfusion injury in the lungs post-transplant.28The near- resolution of PTGS-2 expression in the treatment groups further suggests that ischemic signaling was downregulated as ischemic conditions continued to improve, which may not have happened in the operated control group. Furthermore, the slight but significant reduction in RNA expression of PTGS-2 in the DFO-10% treatment group is consistent with the LSCI and lectin improvements seen in this treatment group compared to no-drug PGS application. Qian et al. investigated the role of PTGS-2 among other immune related genes via a rat lung ischemia reperfusion injury model and demonstrated a relationship between PTGS-2 and infiltration of various immune cells including T cells, macrophages, and neutrophils, encompassing both adaptive and innate immunity.

[0127] Interestingly, VEGF was significantly elevated in both the DFO-10% and the operated control groups. This may suggest that in hypoxic conditions (operated control) VEGF remains persistently elevated29as well as may be upregulated by the actions of deferoxamine (DFO-10%).

[0128] These results justified translation of the randomly aligned nanofiber PGS thin film loaded with DFO-10% in a pig model of large airway allotransplantation. We relied on the tracheal patch allotransplantation model for its technical feasibility, excellent survival, and the ability to allow for some degree of rejection without immediately compromising the health of the animal.20,30A swine model was furthermore relevant given the similar circulatory characteristics the pig airway has compared to human bronchial circulation, as well as the similar dimensions of the pig trachea to the human bronchus. Finally, it allowed us to utilize bronchoscopy to assess airway healing, and grade airway healing to predict future airway complications.

[0129] Like rats, pigs treated with random nano PGS DFO-10% thin films experienced the greatest perfusion in the patch allograft and had the healthiest appearing tissue externally and on bronchoscopy. Unlike the rat model however, swine receiving no-drug PGS thin films did not have a significant improvement in perfusion compared to operated control and had similar bronchoscopic findings. We hypothesize that PGS thin films in an allogeneic model may provide activation of an inflammatory response that then drives complement mediated vascular destructionAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 as seen in allograft rejection.31On bronchoscopy, both operated control and no-drug PGS groups demonstrated ischemic changes, with increased granulation tissue, and hypo-perfused plaques on the endoluminal surface, whereas pigs receiving DFO-10% thin films appeared healthy, with even regions of hyperemia across all pigs within the study group.

[0130] A key insight from the swine model was the feasibility of utilizing a thin film in clinical lung transplantation. A potential barrier to adoption of bronchial artery revascularization is the significant time investment and operative complexity added to the procedure, despite its improved outcomes.17However, wrapping a thin film around the anastomosis with the assurance that it will naturally, adhere, incorporate into the tissue, and then dissolve represents a straightforward addition to lung transplantation, without additional technical challenge or significant time, as the film was easy to apply in the surgical field, adding only an additional minute to the procedure.

[0131] There are several related approaches worth noting. Systemic therapies aimed at improving post-transplant outcomes have been tried – for example, systemic recombinant VEGF or angiogenic growth factors have been contemplated, but systemic VEGF could cause hypotension or off-target angiogenesis (e.g. in tumors) and thus is risky.25,32Another approach is enhancing the graft’s resilience by genetic modulation: delivering IL-10 to donor lungs ex vivo was shown to reduce inflammation and improve graft function.33-35While promising, gene therapy is logistically complex. Our method instead provides a straightforward in vivo adjunct. Furthermore, gene therapy is not mutually exclusive from application of a thin film—in the future these therapies may work in conjunction to protect graft function. Additionally, antithrombotic strategies like clopidogrel have been used in experimental lung transplant to preserve microcirculation; interestingly, clopidogrel reduced obliterative bronchiolitis in a mouse model, presumably by preventing microvascular occlusions.36-38This underscores that maintaining microvascular flow is beneficial. A local DFO film tackles the same issue from an angiogenesis angle – potentially complementary to antithrombotics. Systemic mTOR inhibitors (rapamycin) can also influence graft vascularization (rapamycin tends to inhibit VEGF-driven angiogenesis while reducing fibrosis)37, but systemic therapy has side effects and inconsistent impact on airway healing. A localized combined approach (e.g. a film releasing both an angiogenic factor and an anti-fibrotic or immunomodulatory agent) could be envisioned in the future.19Indeed, ourAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 platform is flexible – in principle, other pro-angiogenic small molecules or biologics could be incorporated, and the polymer could be adjusted for different release profiles. Conclusion

[0132] In conclusion, our work may help address a critical unmet need in lung transplantation by presenting a method to therapeutically revascularize the transplanted airway. By leveraging a bioengineered film for drug delivery, we achieved significant improvements in airway perfusion and structural integrity in both rodent and swine models. This strategy has the potential for clinical translation to reduce the risk of airway complications and improve airway health. With further validation, the DFO-eluting random nanofiber film could be translated into a clinically deployed therapy that surgeons can easily apply to safeguard the bronchial anastomosis, thereby enhancing healing and ultimately improving the longevity of lung transplant patients. Methods Thin Film Fabrication and Characterization Materials

[0133] Polyglycolide (PGS) with an inherent viscosity of 1.4 dL / g was purchased from Corbion (Lenexa, KS). Dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), formic acid (FA), acetonitrile (CAN) water (HPLC grade), hexafluoroisopropanol (HFIP) were purchased from Sigma Aldrich (St. Louis, MO) and used as received. Deferoxamine (DFO) was purchased from Cayman (Ann Arbor, MI). Preparation of the films using electrospinning and solvent casting

[0134] Electrospinning solutions were prepared by dissolving 12% and 18% (w / v) of PGS with varying concentrations of DFO (0%, 10%, and 20% w / w) in HFIP, followed by overnight shaking at 70 °C. Electrospinning was conducted using a needle-based system with controlled humidity and temperature. The solution was loaded into a horizontally positioned syringe (8.7 mm diameter, BD). A Gamma high-voltage research power supply was used to charge the solution, with the positive electrode connected to a 20 G blunt-tip needle (0.9 mm diameter) and the ground electrode attached to the collector. A programmable syringe pump regulated the flowAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 rate. Random fibers were collected on a flat, non-stick aluminum foil-covered target. The electrospinning parameters were: Voltage: 12–13 kV, Tip-to-collector distance: 13–15 cm, Flow rate: 550 and 2000 μL / h, Deposition time: 45–60 min, Humidity: 30%, Temperature: 22 °C. Smooth PGS films were made via the solvent casting of 5% w / v PGS solution at room temperature. Morphological analysis

[0135] Scanning electron microscope (SEM) imaging was performed using the JSM- IT700HR InTouchScope™ Field Emission SEM to analyze sample morphology. Prior to imaging, all samples were sputter-coated with a 10 nm thin film of Au / Pd using a Desk II sputter coater (Denton Vacuum, Moorestown, NJ). Fiber diameter, porosity, and directionality were quantified using ImageJ 1.52a. Mechanical Testing

[0136] PGS films were cut into 3 cm × 0.5 cm strips, clamped vertically, and subjected to uniaxial tensile testing at a strain rate of 16 mm / min using a 5966 Dual Column Tabletop Testing System (Instron, Norwood, MA). Drug Loading

[0137] DFO loading was determined by dissolving 2 cm × 2 cm film samples in HFIP, followed by solvent evaporation using a Rotary Evaporator (Heidolph™ Hei-VAP Core) for 10 minutes. The dried residue was then reconstituted in 1 mL of PBS and filtered through a 20 µm PVDF membrane before analysis. High-performance liquid chromatography (HPLC) was performed using a Waters Corporation system equipped with a polar C18 (5 µm) column. The mobile phase consisted of 0.1% (v / v) TFA in water:acetonitrile (85:15 v / v), with a flow rate of 1 mL / min. DFO elution was detected at 202 nm, with a retention time of 5.8 minutes. Drug release

[0138] The release profile of DFO from the films was evaluated by incubating 2 cm × 2 cm samples in 1.5 mL microvials containing 1 mL of PBS at 37 °C. At designated intervals (1 hour, 6 hours, and days 1, 2, 3, 4, 5, 6, and 7), the PBS was collected and replaced with fresh PBSAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 to maintain sink conditions. The concentration of released DFO was measured using HPLC, following the same analytical parameters as described for DFO loading. Pharmacokinetic Study

[0139] A standard solution containing the target analyte, DFO (1 mg / mL) was prepared in DMSO. This stock standard solution was diluted with 0.2% Formic Acid in ACN:Water(75:25) to prepare a mixed working solution with different concentrations. calibration curve was prepared in plasma and used for plasma and homogenized tissue samples in plasma. Deferoxamine-d7 (5 ng / mL in 0.2% FA in ACN: Water (75:25)) was used as the internal standard (I.S Stock and working solutions were stored at 4°C). Plasma samples were ready for analysis and did not need homogenization. The tissue samples were homogenized in blank plasma. Based on the results of preliminary experiments 0.2% FA in ACN: Water (75:25) was selected as extraction solvent since it yielded good extraction recoveries for DFO.50 µL of the tissue mixture or plasma samples with 250 µL of the internal standard solution were transferred to microtubes (1.5 mL), vortexed, and centrifuged at high speed (~2,600 rcfs) for about 10 minutes. After centrifugation, 150 µL of the supernatant was transferred into autosampler vials for LCMS / MS analysis. Separation was achieved with a BEH C18, 2.1 x 50 mm, 1.7 µm, column. Solvent A was purified, deionized water containing 0.2% FA plus 0.04 mM EDTA, and acetonitrile containing 0.2% FA was used as solvent B. The addition of a proper concentration of EDTA in the mobile phase competitively inhibited DFO from complexing with ferric ion and prevented the decrease of DFO concentration. A gradient method was used, and solvent B was kept at 5% with a flow rate of 0.5mL / min for the initial step (0.5 minutes) and held for 2 minutes. It was then increased to 95% B over 0.5 minutes and held for 5 minutes, then returned to 5% B over 0.5 minute and kept for 0.5 minutes. The run time was 8 minutes. The column effluent was monitored using an AB Sciex Triple Quad 5500+ LC-MS / MS System (QTRAP® Ready) operating in positive electrospray ionization and multiple reaction monitoring (MRM) mode The MRM m / z transitions for each compound of interest were: 561.3 → 201.1 for DFO, and 568.4 → 201.1 for the internal standard, Deferoxamine-d7. Each experiment was repeated three times. For fiber diameter measurements, a total of N = 100 fibers were analyzed. Animal Models and Surgical ProceduresAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0140] All animal procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care and complied with the NIH Guide for the Care and Use of Laboratory Animals. Male and female Wistar rats (300–500 g; Charles River Labs, Wilmington, MA) were used for rodent experiments. Animals were housed in temperature- and humidity- controlled conditions with a 12-hour light / dark cycle and given food and water ad libitum. For the swine model, outbred domestic pigs (30-40 kg, both sexes) were obtained from a USDA-approved breeder. Donor / recipient pairs were sex- and blood type-matched to minimize acute immune reactions. All surgical procedures were performed under general anesthesia with appropriate analgesia. Treatment groups were performed in random order to eliminate learning curve bias.41Rat Tracheal Autotransplantation

[0141] We employed a syngeneic tracheal auto-transplant model to simulate bronchial devascularization.19,42-44Rats were anesthetized with an intraperitoneal cocktail of ketamine (70 mg / kg), xylazine (10 mg / kg), and atropine (0.5 mg / kg). Depth of anesthesia was confirmed by loss of pedal reflex. The animal was placed supine on a heating pad and the neck was sterilely prepped. A midline cervical incision was made, and the strap muscles were separated to expose the trachea. The tracheal adventitia was gently cleared using cotton applicators to remove small vessels. Segmental devascularization was achieved by bilaterally ligating the lateral tracheal vessels and circumferentially dissecting a 4–5 ring tracheal segment free from surrounding tissues. This segment was then resected (full cross-sectional transection) and re-anastomosed end-to-end using eight interrupted 8-0 Prolene sutures (Ethicon, Raritan, NJ) placed circumferentially. This procedure produces an in situ tracheal autograft with all peri-tracheal blood supply severed, relying on the anastomosis for any revascularization. In treatment group rats, a 2 cm × 2 cm electrospun thin film (sterile) was wrapped around the anastomosis after suturing. The film was placed circumferentially to cover the entire graft and overlapping by ~5 mm onto adjacent native trachea. For rats receiving DFO-loaded films, care was taken to orient the film such that the more fibrous (non-foil contact) surface faced the trachea for optimal integration. Control animals received no film (operated control). To determine the effects of topography of the scaffold alone in the absence of drugs, thin film groups included a non-textured PGS film (smooth), PGS thin film comprised of microfibers in a random orientation (random micro), and PGS thin films composed of nanofibers in an organized aligned formation (aligned nano), and another in a random orientation (random nano). Deferoxamine was loaded onto randomly aligned nanofiber thin filmsAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 (Nano-DFO10% / 20%). After film application, the overlying strap muscles were reapproximated with a 5-0 absorbable suture and the skin was closed with 4-0 chromic sutures. Rats were recovered on supplemental oxygen and given buprenorphine for analgesia. The pre-specified terminal point was 10 days. Swine Tracheal Patch Transplant

[0142] A porcine allotransplant model was used to assess translational feasibility of the DFO-eluting film in a large airway as well as the effects of the immune system. Donor pigs underwent anesthesia with telazol (4–6 mg / kg IM) induction and isoflurane maintenance. Through a midline neck incision, a 4 cm × 2 cm elliptical full-thickness section of trachea was harvested from the donor (including cartilage and mucosa). The recipient pig was anesthetized similarly and intubated. Via cervical incision and partial sternotomy, the upper thoracic trachea was exposed. A matching 4×2 cm defect was created in the anterior tracheal wall of the recipient. The donor tracheal patch was sutured into the defect of the recipient’s trachea using interrupted 4-0 Prolene (Ethicon) sutures placed circumferentially around the patch. This resulted in a “patch tracheoplasty” wherein the periphery of the patch forms an anastomosis with the recipient trachea (an allogeneic airway anastomosis). In the treatment group (n=4), a 10% DFO-loaded PGS nanofiber film (4×4 cm) was laid over the patch anastomosis and sutured at the corners to secure it in place (covering the suture line). Control pigs (n=4) underwent the patch transplant without any film. An additional treatment group (n=4) comprised pigs who received a random-nano film without drug. The strap muscles and subcutaneous tissues were closed in layers, and the skin was closed with staples. Pigs were recovered and monitored in a dedicated large-animal facility. Analgesia (intramuscular sustained release buprenorphine 0.1mg / kg [Fidelis, North Brunswick, NJ]) was given every three days as needed. Immunosuppression was a daily dose of oral methylprednisolone (1mg / kg) (Pharmaceutical Associates Inc, Greenville, SC), to model a robust immune response and isolate the angiogenic intervention effect in the presence of rejection. All pigs survived the procedure and remained healthy until the study endpoint of 14 days. Perfusion Assessment by Laser Speckle Imaging

[0143] Regional blood flow in the trachea was noninvasively measured using laser speckle contrast imaging (LSCI). We used the RFLSI-ZW Laser Speckle Imaging system (RWD LifeAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 Sciences, Sugar Land, TX) which captures real-time perfusion maps at high resolution. The principle of LSCI relies on the speckle pattern formed by coherent laser light scattering from moving blood cells: perfused tissue appears as blurred speckles, the extent of blur correlating with blood flow velocity.45-47This technique does not require exogenous contrast agents and can detect flow in vessels down to ~10 µm diameter.45-47

[0144] Speckle imaging was obtained at the index (immediately post-procedure with the neck open) and terminal end points (upon re-entry into the neck) in rats. In swine specifically tracheal perfusion was assessed by LSCI at two time points: immediately after patch transplantation (baseline) and at the 14-day endpoint. For the baseline measurement, following patch suturing (and film placement if applicable) the surgical field was temporarily exposed before closure, and the speckle imaging camera was positioned 30 cm above the trachea. A perfusion map of the patch and surrounding trachea was recorded. The chest and neck were then closed as described. At post-operative day 14, the pigs were anesthetized, and the tracheal transplant site was re-exposed via the previous incision. LSCI was performed on the patch area to measure perfusion in the transplanted patch versus adjacent native trachea. Percent perfusion loss in the patch was calculated as above. Because movement can affect speckle measurements, care was taken to suspend ventilation briefly (~10 s) during image capture to minimize motion artifact.

[0145] For imaging, the surgical field was illuminated with the instrument’s 780 nm laser and serial frames were recorded (speckle frame rate ~50 fps). We acquired 2-second video streams (≈100 frames) and generated perfusion maps using the manufacturer’s software, which computes a dimensionless perfusion unit (PU) for each pixel based on speckle contrast. Bright regions on the perfusion maps correspond with increased perfusion, and dark / black areas represent lack of perfusion. Regions of interest (ROI) encompassing the native trachea (non-transplanted area above the anastomosis) and the transplanted segment were defined, and mean perfusion units in each ROI were quantified from averaged speckle images. The percent perfusion loss in the graftwas calculated as:100 ∗^^ಿೌ^^ೡ^ି^^ಸ^ೌ^^.Lectin Patency

[0146] In rats, to corroborate the perfusion imaging and directly visualize perfused microvessels, we performed lectin perfusion staining before sacrifice. Lectins are proteins thatAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 bind endothelial glycoconjugates, allowing in vivo labeling of the vasculature.18,48At the terminal time point, rats were injected intravenously with fluorescein-conjugated Lycopersicon esculentum (tomato) lectin (Vector Laboratories, Burlingame, CA). Rats received 0.5 mg / kg via internal jugular vein injection. After five minutes of perfusion, the rat was euthanized with potassium chloride, and 10mL of 1% paraformaldehyde (Sigma Aldrich) diluted in phosphate buffered solution (PBS) (Simga Aldrich) was perfused through the internal jugular vein to flush unbound lectin and preserve the tissue. Tissues were embedded in optimal cutting temperature compound (OCT) and snap-frozen in liquid nitrogen. Cryosections (8 µm) were prepared and fixed in cold acetone. Sections were imaged by fluorescence microscopy (Keyence, Kansas City, MO) to detect lectin bound to endothelium of perfused microvessels. Regions around the suture line were chosen for analysis. Perfused microvessel density (lectin-positive vessels per mm²) was quantified using ImageJ in a blinded manner. Histological and Immunohistochemical Analysis

[0147] For general histology, harvested tracheal grafts from rats and swine were fixed in 10% neutral buffered formalin and paraffin embedded. Cross-sections (5 µm) were stained with hematoxylin and eosin (H&E; ThermoFisher Scientific). Slides were examined for characteristic features of airway ischemic injury, including epithelial sloughing, loss of ciliated cells and goblet cells, submucosal edema or inflammation, cartilage necrosis (loss of basophilic chondrocyte nuclei), and luminal occlusion by granulation tissue. A semi-quantitative histologic injury score was assigned to each graft as described by Hyytinen et al.43Briefly, injury in three compartments (epithelium, subepithelium, cartilage / peritracheal tissue) was graded 0 (normal) to 3 (severe diffuse injury). Two observers scored each sample in a blinded fashion; any scoring discrepancies were reviewed jointly to reach consensus. Mean composite scores (sum of compartment scores, max 9) were compared between groups.

[0148] For immunohistochemistry (IHC), frozen sections were used to detect specific markers of angiogenesis and inflammation. Sections were fixed in cold acetone, blocked with 5% goat serum, and incubated with primary antibodies against von Willebrand factor (vWF, 1:200, Dako Agilent) to identify blood vessels, CD31 (PECAM-1, 1:100, Bio-Rad) for endothelial cells, and α-smooth muscle actin (α-SMA, 1:200, Sigma) for smooth muscle (to distinguish mature vessels). After washing, slides were incubated with AlexaFluor-conjugated secondary antibodiesAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 (ThermoFisher) and counterstained with DAPI. Imaging was performed on a Zeiss fluorescence microscope. Capillary density and vessel size distribution in the graft were assessed from vWF / CD31-stained sections using ImageJ. Separately, immunostaining for HIF-1α (1:100, Novus Biologicals) was done on nuclear protein-fixed sections (using the manufacturer’s antigen retrieval protocol) to qualitatively evaluate HIF-1α localization in graft tissues.

[0149] Quantitative PCR for Gene Expression

[0150] Real-time quantitative PCR was employed to measure expression of angiogenesis- related genes in transplanted tracheas. Approximately 50 mg of frozen tracheal tissue (from graft region) was pulverized and total RNA was extracted using TRIzol reagent (ThermoFisher) following the manufacturer’s instructions. Any contaminating DNA was removed by DNase I treatment (Invitrogen). cDNA was synthesized from 1 µg RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qPCR was performed on a QuantStudio 6 PCR system (Applied Biosystems) using SYBR Green PCR Master Mix (ThermoFisher). Primers for Hif1a, Vegfa, PTGS2, Il10, and housekeeping gene GAPDH were designed based on published sequences (rat and pig primers were validated separately). Each sample was run in triplicate. Thermal cycling conditions were: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. No-template and no-reverse-transcriptase controls confirmed absence of contamination. Melting curve analysis ensured specific amplification. The 2^–ΔΔCt method was used to calculate relative gene expression in treated vs control groups, normalized to GAPDH. Unoperated control tracheas were used as the control group. Bronchoscopic Evaluation (Swine Model)

[0151] At 14 days post-surgery, prior to sacrifice, pigs were examined with bronchoscopy to inspect the transplanted patch airway lumen. Under anesthesia, a flexible video bronchoscope (5 mm outer diameter) was introduced through the endotracheal tube and advanced to the patch site. Video images were recorded to document the mucosal appearance, healing of the anastomotic suture line, and any evidence of complications (such as granulation tissue, stricture, or dehiscence). An airway grading was assigned according to established criteria for bronchial anastomoses.49After bronchoscopy, the pigs were euthanized for tissue harvesting as described above.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0152] Statistical Analysis

[0153] Data are presented as mean ± standard deviation unless otherwise specified. Data were analyzed in Excel (Microsoft, Seattle, WA), OriginPro 1.52a (Microsoft), and Prism 10 (GraphPad Software, Boston, MA). Group comparisons were conducted using Prism 10 with significance set at p < 0.05. For two-group comparisons, an unpaired Student’s t-test was used (two-tailed). For experiments with more than two groups, one-way analysis of variance (ANOVA) was performed followed by Tukey’s post hoc test for multiple comparisons. Non-parametric scoring data were compared using the Mann–Whitney U test. Outliers were assessed and removed using Grubb’s test.

[0154] References for Example 2 1. Iyengar A, Kwon OJ, Sanaiha Y, et al. Lung transplantation in the Lung Allocation Score era: Medium-term analysis from a single center. Clinical transplantation. 2018;32(8):e13298- e13298. doi:10.1111 / ctr.13298 2. Rana A, Gruessner A, Agopian VG, et al. Survival benefit of solid-organ transplant in the United States. JAMA surgery.2015;150(3):252-9. doi:10.1001 / jamasurg.2014.2038 3. Singh TP, Cherikh WS, Hsich E, et al. Graft survival in primary thoracic organ transplant recipients: A special report from the International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation. The Journal of Heart and Lung Transplantation.2023 / 10 / 01;42(10)doi:10.1016 / j.healun.2023.07.017 4. Valapour M, Lehr CJ, Schladt DP, et al. OPTN / SRTR 2021 Annual Data Report: Lung. American Journal of Transplantation.2023;23(2):S379-S379. doi:10.1016 / J.AJT.2023.02.009 5. Crespo MM. Airway complications in lung transplantation. Journal of Thoracic Disease. 2021 / 11;13(11)doi:10.21037 / jtd-20-2696 6. Herrera JM, McNeil KD, Higgins RSD, et al. Airway complications after lung transplantation: treatment and long-term outcome. The Annals of Thoracic Surgery. 2001;71(3):989-993. doi:10.1016 / s0003-4975(00)02127-5 7. Higgins R, McNeil K, Dennis C, et al. Airway stenoses after lung transplantation: management with expanding metal stents. J Heart Lung Transplant. Sep-Oct 1994;13(5):774-8.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 8. Muñoz-Fos A, Moreno P, González FJ, et al. Airway Complications after Lung Transplantation-A Contemporary Series of 400 Bronchial Anastomoses from a Single Center. J Clin Med. Apr 232023;12(9)doi:10.3390 / jcm12093061 9. Pasnupneti S, Nicolls MR. Airway hypoxia in lung transplantation. Curr Opin Physiol. Feb 2019;7:21-26. doi:10.1016 / j.cophys.2018.12.002 10. Subasi M, Duger M. Preoperative risk factors of airway complications in adult lung transplant recipients: A systematic review and meta-analysis. Turkish Journal of Thoracic and Cardiovascular Surgery.2023;31(4):517-529. doi:10.5606 / TGKDC.DERGISI.2023.25399 11. Van De Wauwer C, Van Raemdonck D, Verleden GM, et al. Risk factors for airway complications within the first year after lung transplantation☆. European Journal of Cardio- Thoracic Surgery.2007 / 04 / 01;31(4)doi:10.1016 / j.ejcts.2007.01.025 12. AN B, T M, JM T, et al. Microvascular destruction identifies murine allografts that cannot be rescued from airway fibrosis - PubMed. The Journal of clinical investigation. 2007 Dec;117(12)doi:10.1172 / JCI32311 13. Conrad CK, Hedlin H, Chin H, et al. Auto-inflammation and auto-immunity pathways are associated with emergence of BOS in pediatric lung transplantation. Pediatric Transplantation. 2022;26(4)doi:10.1111 / PETR.14247 14. Luckraz H, Goddard M, McNeil K, et al. Microvascular changes in small airways predispose to obliterative bronchiolitis after lung transplantation. Journal of Heart and Lung Transplantation.2004;23(5):527-531. doi:10.1016 / j.healun.2003.07.003 15. Nicolls MR, Zamora MR. Bronchial blood supply after lung transplantation without bronchial artery revascularization. Current Opinion in Organ Transplantation. 2010;15(5):563- 567. doi:10.1097 / MOT.0b013e32833deca9 16. Guthaner DF, Wexler L, Sadeghi AM, Blank NE, Reitz BA. Revascularization of tracheal anastomosis following heart-lung transplantation. Investigative Radiology. 1983;18(6):500-503. doi:10.1097 / 00004424-198311000-00003 17. Unai S, Yun J, Pettersson GB. Bronchial Artery Revascularization: Surgical Technique. Operative Techniques in Thoracic and Cardiovascular Surgery. 2023;28(2):139-153. doi:10.1053 / j.optechstcvs.2022.11.004 18. Jiang X, Malkovskiy AV, Tian W, et al. Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles. Biomaterials. 2014;35(2):803-813. doi:10.1016 / j.biomaterials.2013.09.092Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 19. Krishnan A, Forouharshad M, Heng E, et al. Application of a degradable thin film to modulate perfusion to post-autotransplantation airways in rats. JTCVS Open. 2025 / 01 / 20;doi:10.1016 / j.xjon.2025.01.008 20. Taniguchi D, Kamata S, Rostami S, et al. Evaluation of a decellularized bronchial patch transplant in a porcine model. Scientific Reports 2023 13:1. 2023-12- 08;13(1)doi:10.1038 / s41598-023-48643-y 21. Crespo MM. Airway complications in lung transplantation. J Thorac Dis. Nov 2021;13(11):6717-6724. doi:10.21037 / jtd-20-2696 22. Shen H, Ma Y, Qiao Y, Zhang C, Chen J, Zhang R. Application of Deferoxamine in Tissue Regeneration Attributed to Promoted Angiogenesis. Molecules. 2024 Apr 29;29(9)doi:10.3390 / molecules29092050 23. Jiang X, Hsu JL, Tian W, et al. Tie2-dependent VHL knockdown promotes airway microvascular regeneration and attenuates invasive growth of Aspergillus fumigatus. J Mol Med (Berl). Sep 2013;91(9):1081-93. doi:10.1007 / s00109-013-1063-8 24. Dongiovanni P, Valenti L, Fracanzani AL, Gatti S, Cairo G, Fargion S. Iron depletion by deferoxamine up-regulates glucose uptake and insulin signaling in hepatoma cells and in rat liver. American Journal of Pathology.2008;172(3):738-747. doi:10.2353 / ajpath.2008.070097 25. Heim C, Motsch B, Jalilova S, et al. Reduction of obliterative bronchiolitis (OB) by prolyl- hydroxylase-inhibitors activating hypoxia-inducible transcription factors in an experimental mouse model. Transplant Immunology.2016;39:66-73. doi:10.1016 / j.trim.2016.08.007 26. Jiang X, Hsu JL, Tian W, et al. Tie2-dependent VHL knockdown promotes airway microvascular regeneration and attenuates invasive growth of Aspergillus fumigatus. Journal of Molecular Medicine.2013;91(9):1081-1093. doi:10.1007 / S00109-013-1063-8 27. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix- loop-helix-PAS heterodimer regulated by cellular O2 tension. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(12):5510-5514. doi:10.1073 / PNAS.92.12.5510 28. Qian J, Xu Z, Yin M, Qin Z, Pinhu L. Bioinformatics analyses of immune-related genes and immune infiltration associated with lung ischemia-reperfusion injury. Transplant Immunology.2023 / 12 / 01;81doi:10.1016 / j.trim.2023.101926Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 29. Ramakrishnan S, Anand V, Roy S. Vascular Endothelial growth factor signaling in hypoxia and Inflammation. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology.2014 Mar 9;9(2)doi:10.1007 / s11481-014-9531-7 30. Lama VN, Belperio JA, Christie JD, et al. Models of Lung Transplant Research: a consensus statement from the National Heart, Lung, and Blood Institute workshop. JCI Insight. 05 / 10 / 2017;2(9)doi:10.1172 / jci.insight.93121 31. Khan MA, Nicolls MR. Complement-mediated microvascular injury leads to chronic rejection. Adv Exp Med Biol.2013;735:233-46. doi:10.1007 / 978-1-4614-4118-2_16 32. Brizzi MF, Tarone G, Defilippi P. Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Current Opinion in Cell Biology. 2012;24(5):645-651. doi:10.1016 / J.CEB.2012.07.001 33. The role of interleukin-10 in lung transplantation - PubMed. Transplant immunology.2002 May;9(2-4)doi:10.1016 / s0966-3274(02)00045-x 34. Functional repair of human donor lungs by IL-10 gene therapy - PubMed. Science translational medicine.10 / 28 / 2009;1(4)doi:10.1126 / scitranslmed.3000266 35. Immunomodulation of the donor lung with CRISPR-mediated activation of IL-10 expression - PubMed. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2023 Oct;42(10)doi:10.1016 / j.healun.2023.06.001 36. Preservation of Microvascular Integrity in Murine Orthotopic Tracheal Allografts by Clopidogrel - PubMed. Transplantation.2019 May;103(5)doi:10.1097 / TP.0000000000002571 37. Gräbner C, Ramsperger-Gleixner M, Kuckhahn A, Weyand M, Heim C. Chronic rejection after lung transplantation: Overview and experimental approaches for long-term organ protection. Zeitschrift fur Herz-, Thorax- und Gefasschirurgie. 2023;37(3-4):157-163. doi:10.1007 / S00398- 023-00562-W 38. Preidl RHM, Eckl S, Ramsperger-Gleixner M, et al. Clopidogrel reduces post-transplant obliterative bronchiolitis. Transplant International. 2013;26(10):1038-1048. doi:10.1111 / TRI.12163 39. Pasupneti S, Nicolls MR. Airway hypoxia in lung transplantation. Current Opinion in Physiology.2019;7:21-26. doi:10.1016 / J.COPHYS.2018.12.002Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 40. Hsu JL, Manouvakhova OV, Clemons KV, et al. Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation. Science translational medicine.2018 Feb 21;10(429)doi:10.1126 / scitranslmed.aag2616 41. JA C, CR R, P F. Statistical evaluation of learning curve effects in surgical trials - PubMed. Clinical trials (London, England).2004;1(5)doi:10.1191 / 1740774504cn042oa 42. Behrend M, Wasielewski Rv, Klempnauer J. Failure of airway healing in an ovine autotransplantation model that includes basic fibroblast growth factor. The Journal of Thoracic and Cardiovascular Surgery.2002 / 08 / 01;124(2)doi:10.1067 / mtc.2002.120335 43. Hyytinen T, Paavonen T, Inkinen K, Ahonen J, Mattila S. Airway anastomotic healing in the rat tracheal autograft. Eur Surg Res.1999;31(2):155-61. doi:10.1159 / 000008634 44. R N, M H, K Y. Improved airway healing using basic fibroblast growth factor in a canine tracheal autotransplantation model - PubMed. Annals of surgery. 1998 Mar;227(3)doi:10.1097 / 00000658-199803000-00018 45. Laser Speckle Contrast Imaging: theory, instrumentation and applications - PubMed. IEEE reviews in biomedical engineering.2013;6doi:10.1109 / RBME.2013.2243140 46. Li D-Y, Xia Q, Yu T-T, et al. Transmissive-detected laser speckle contrast imaging for blood flow monitoring in thick tissue: from Monte Carlo simulation to experimental demonstration. Light: Science & Applications 202110:1. 2021-12-03;10(1)doi:10.1038 / s41377- 021-00682-8 47. Senarathna J, Rege A, Li N, Thakor NV. Laser Speckle Contrast Imaging: Theory, Instrumentation and Applications | IEEE Journals & Magazine | IEEE Xplore. doi:10.1002 / jor.20178"> 48. Grazul-Bilska AT, Borowicz PP, Reynolds LP, Redmer DA. Vascular perfusion with fluorescent labeled lectin to study ovarian functions. Acta Histochemica. 2013 / 10 / 01;115(8)doi:10.1016 / j.acthis.2013.03.006 49. Dutau H, Vandemoortele T, Laroumagne S, et al. A new endoscopic standardized grading system for macroscopic central airway complications following lung transplantation: the MDS classification. European Journal of Cardio-Thoracic Surgery. 2014 / 02 / 01;45(2)doi:10.1093 / ejcts / ezt499.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02

[0155] While various embodiments of the present invention have been described above, they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described illustrative embodiments but should instead be defined only in accordance with the following claims and their equivalents.

[0156] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the disclosure, specific terminology is employed for the sake of clarity. However, the disclosure is not intended to be limited to the specific terminology so selected. The above-described embodiments of the disclosure may be modified or varied, without departing from the invention, as appreciated by those skilled in the art considering the above insights. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. For example, it is to be understood that the present disclosure contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.

Claims

Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 WE CLAIM:

1. A film for integrating with tissue and promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization, comprising a plurality of fibers arranged in an overlapping configuration to form a porous, thin film with an average pore size of at least 0.01 µm2and less than 60 µm2, wherein said plurality of fibers has an average diameter of at least 10 nm and less than 1,000 nm, wherein each of said plurality of fibers have an aspect ratio of length to width of at least 10, and each of said plurality of fibers is a biocompatible material.

2. The film according to claim 1, wherein said plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.

71.

3. The film according to claim 1, wherein said plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.

51.

4. The film according to claim 1, wherein said plurality of fibers are arranged in a random pattern such that directional coherence is at least 0.00 and less than 0.

36.

5. The film according to any one of claims 1-4, wherein said average pore size is at least 0.01 µm2and less than 40 µm2.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 6. The film according to any one of claims 1-4, wherein said average pore size is at least 0.01 µm2and less than 30 µm2.

7. The film according to any one of claims 1-4, wherein said average pore size is at least 0.01 µm2and less than 10 µm2.

8. The film according to any one of claims 1-4, wherein said average pore size is at least 0.02 µm2and less than 2.2 µm2.

9. The film according to any one of claims 1-8, wherein said plurality of fibers have an average diameter of at least 50 nm and less than 900 nm.

10. The film according to any one of claims 1-9, wherein said film has a thickness greater than 25 µm and less than 1 mm.

11. The film according to any one of claims 1-10, wherein said film has an average Young’s modulus of at least 20 kPa and less than 1 GPa.

12. The film according to any one of claims 1-11, wherein said film has an average breaking strength greater than 0.2 N.

13. The film according to any one of claims 1-12, wherein said plurality of fibers are synthetic degradable fibers that are composed of one or more polymer or copolymer of at least one of Polylactic Acid (PLA), Polyglycolic Acid (PGA), Polycaprolactone (PCL), Poly(lactic-Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 co-glycolic Acid) (PLGA), Polydioxanone (PDO), Poly(ε-caprolactone), Polyorthoesters (POEs), Polyanhydrides, Polyhydroxyalkanoates (PHAs), Poly(β-amino esters), and biodegradable Polyurethanes with molecular weights of at least 1 kDa and less than 2,000 kDa.

14. The film according to claim 13, wherein said molecular weights of the polymers or copolymers are at least 25 kDa and less than 500 kDa.

15. The film according to any one of claims 1-14, further comprising at least one proangiogenic moiety loaded into at least some of said plurality of fibers.

16. The film according to claim 15, wherein said proangiogenic moiety has a pattern that directionally stimulates vascularization.

17. The film according to claim 15 or 16, wherein said proangiogenic moiety is a small molecule drug consisting of Deferoxamine, CEP03, SC-3-149, Rocaglamide, Fasudil, Deforolimus, Statins, Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II Receptor Blockers (ARBs), Sildenafil, Tadalafil, Metformin, Nitrates, and any combination thereof.

18. The film according to claim 15 or 16, wherein said proangiogenic moiety is vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor, angiopoietin-1, heparin, oxygen, oxygen releasing moiety, or any combination thereof.Att’y Ref.: 2240-616360 Applicant Ref.: P18388-02 19. A device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization, comprising: a film according to any one of claims 1-18; and an oxygen sensor incorporated into said film, wherein said oxygen sensor is configured to monitor status of vascularization and perfusion at the treatment site and transmit data for remote monitoring.

20. The device according to claim 19, wherein said sensor transmits said data wirelessly.

21. The device according to claim 19 or 20, further comprising a second film according to any one of claims 1-18.

22. The device according to claim 21, wherein said second film has at least one physical parameter that is different from said first film.

23. The device according to any one of claims 19 to 22, further comprising at least one of a coating, a protective layer, an adhesive layer or any combination thereof in contact with at least one of said first and second films.

24. A device for promoting at least one of endothelial cell adhesion, migration, proliferation, ingrowth, infiltration, angiogenesis, sprouting angiogenesis, neovascularization, and vascularization, comprising: a first film according to any one of claims 1-18; andAtt’y Ref.: 2240-616360 Applicant Ref.: P18388-02 a second film according to any one of claims 1-18, wherein said second film has at least one physical parameter that is different from said first film.

25. The device according to claim 24, further comprising at least one of a coating, a protective layer, an adhesive layer or any combination thereof in contact with at least one of said first and second films.

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