Vascular Graft Infections: An Overview of Novel Treatments Using Nanoparticles and Nanofibers

Round 1

Reviewer 1 Report

This review focuses on an important and interesting area of vascular disease and its current treatment requiring vascular grafts to revascularize the lower/upper extremities, dialysis access creation, aortic aneurysms, and dissection repair. The issue of postoperative infection is highlighted resulting in the need for multiple surgeries to have complete or partial excision of the graft, vascular coverage, and extra anatomical revascularization. Although well written, below are a few recommendations:

1) The aim of the review needs to be made more specific rather than just a gathering of the latest information on nano-enabled techniques used for vascular graft infection control.

2) The authors should include a section on the synthesis techniques for the various nanostructures and relate the physicomechanical features to what an ideal system will constitute.

3) A section on the aspects of stability of the nano-enabled grafts to the host immune system needs to be included.

4) Aspects of cell-cell, cell-nanoparticle and nanoparticle-vessel interactions are critical for success of this technology and should be include as a sperate section\.

5) 3D-bioprinting should be included as one of the novel treatments gaining momentum. 
there is also a high risk of morbidity, mortality and limb loss. As a result, it is important to develop 14
a method to prevent or reduce the incidence of these infections. Numerous previous studies have 15
investigated the efficacy of antibiotic and antiseptic-impregnated grafts, such as rifampin-soaked 16
and silver-coated grafts, in reducing vascular graft infections. In comparison to these traditional 17
methods of creating antimicrobial grafts, nanotechnology confers several additional benefits, in- 18
cluding a higher active surface area and more controlled release of drugs. This review looks to 19
gather the latest information on nanoparticle and nanofiber techniques being used to reduce the 20
incidence of vascular graft infections.

Author Response

The authors greatly appreciate the reviewer’s comments. The updated version of the manuscript has been modified following the reviewer’s suggestions. In the revised manuscript, all changes have been highlighted in red, including additional references. Responses to the reviewer’s comments are detailed below.

Review 1

This review focuses on an important and interesting area of vascular disease and its current treatment requiring vascular grafts to revascularize the lower/upper extremities, dialysis access creation, aortic aneurysms, and dissection repair. The issue of postoperative infection is highlighted resulting in the need for multiple surgeries to have complete or partial excision of the graft, vascular coverage, and extra anatomical revascularization. Although well written, below are a few recommendations:

1) The aim of the review needs to be made more specific rather than just a gathering of the latest information on nano-enabled techniques used for vascular graft infection control.

We thank the reviewer for this feedback. As per the reviewer’s suggestion, we have more clearly defined the aim of the review. In particular, we have decided to analyze how novel nanotechnological approaches aim to solve the problem of vascular graft infections, highlighting promising aspects and limits of both nanofibers and nanoparticles.

2) The authors should include a section on the synthesis techniques for the various nanostructures and relate the physicomechanical features to what an ideal system will constitute.

We appreciate the suggestion of the reviewer and we have added a brief explanation of one of the major challenges from a mechanical standpoint in the fabrication of nanofibrous vascular grafts.

“Despite the positive features of nanostructured grafts, some limitations need to be considered. Long term patency of the engineered VG and risk of triggering negative immune response are drawbacks that still need to be overcome. Nanofibrous assemblies potentially tend to degrade over time deteriorating mechanical properties of the polymeric matrix, resulting in VG [59]”.

3) A section on the aspects of stability of the nano-enabled grafts to the host immune system needs to be included.

In consideration of the reviewer’s suggestion, we have added a section in which we describe the aspects related to nano-enabled grafts and host immune system.

“Moreover, host immune response is something that must be considered carefully, since it has been demonstrated how it can severely affect the patency of VG on the long run. In this regards, only autologous solutions do not trigger negative immunologic reaction. Allografts experience immunogenicity due to the interaction with the host body, and for this reason patients are subjected to immunosuppressive therapies [58]. Immune rejection is considered one of the main causes of allograft failure and rupture in the long term [59]. VG are subjected to the same fate, even exacerbating the outcomes. Aggressive reaction from the host immune system leads to stenosis, thrombosis and eventually failure of the implant. This aspect has been thoroughly studied by Hibino et al. using tissue engineered VG [60]. In a study performed on human, they observed major failure of tissue engineered VG due to stenosis [61]. In order to investigate the reason of the failure, they ad-dressed the role of host immune function for excess neo-tissue formation in tissue engineered VG using an immunodeficient mouse model. Interestingly, grafts implanted in immunodeficient mice showed greater patency over time compared to immunocompetent model. Sonography revealed stenosis of the grafts implanted in immunocompetent mice after 2 weeks, whereas the grafts of the immunodeficient group remained patent up to 10 weeks, suggesting a key role of the immune system in graft failure due to excessive formation of neo-intimal tissue.”

4) Aspects of cell-cell, cell-nanoparticle and nanoparticle-vessel interactions are critical for success of this technology and should be include as a sperate section\.

We thank the reviewer for this suggestion and following is lead we have included a section highlighting aspect of cell-cell, cell-nanoparticles and nanoparticles-vessel interaction.

 “This can be explained, because when nanoparticles reach the blood system, they come into direct contact with blood cells, endothelial cells and plasma proteins. The nanometric dimension of these nanoparticles can affect the intricate structure and critical functions of the blood components. In fact, plasma proteins tend to adsorb to the surface of nanoparticles to form a protein corona that significantly influences their interaction with blood components and may even lead to increased cellular activation [98].”

5) 3D-bioprinting should be included as one of the novel treatments gaining momentum.

We agree with the reviewer’s suggestion. A short section on 3D-bioprinting has been added.

“In this framework, 3D bioprinting is an attractive technology that has the potential to fab-ricate patient-specific grafts and could be very useful to overcome the challenges of growth potential, host-tissue integration, and anatomical differences [107]. 3D bioprinting could be an alternative to autologous or allogeneic tissue grafts for the replacement or treatment of damaged tissues [108]. Several categories of bioprinting methods have been developed in these years, but the most used is the extrusion bioprinting technique due to superior mechanical properties of the final products, as compared to other bioprinting methods [109]. 3D bioprinting allows the possibility to print constructs in layers, while controlling the spatial deposition of cell types. These unique features provide several advantages over other conventional processes. Although, bioprinted grafts made from hydrogels are very fragile and have insufficient strength to withstand hemodynamic pressures in vivo [110].”

Reviewer 2 Report

The authors present an interesting work with the title ‘Vascular Graft Infections: An Overview on Novel Treatments Using Nanoparticles and Nanofibers’. More specifically, the work should be of interest not only for basic researchers but also for vascular medicine clinicians. Generally, I think that the paper need a major revision before published. Here are some comments and suggestions from my side:

Introduction

General:

• I would like to see a deeper description on different graft infections per location (e.g. aorta, groin region, lower extremity etc.). I find the current description a bit superficial.
• I think what is interesting is to give some insights on the ‘complication’ of long-term antibiotic therapy that these patients need. This is missing from this review and it is indeed the most important for these patients.

38: Aortic graft infections have a mortality rate between 24-75%. Could you please provide references?

39-40: One aggressive approach in management of intracavitary graft infections consists of the excision of the graft, debridement of infected tissues, and extra-anatomic bypass along with antibiotic therapy. Could you please provide references?

43: Similarly, peripheral graft infections have a high morbidity and up to 40% requiring limb amputation. Please differentiate the graft infections per location as in my general comment.

59-60: …Dacron grafts containing collagen or gelatin have demonstrated excellent antibacterial activity with low rates of graft… Please provide refs: how low are these rates?

65-66: The fast release and clearance of impregnated drugs and inability to coat PTFE grafts are two major limitations of antibiotics coated grafts. How fast is the release and clearance? Please expand and describe the PTFE term.

84-85: …has also been increased interest in the use of nanotechnology to reduce the rates of vascular graft infections. The authors should better describe the novelty of their work? Why do we care about using nanotechnology? Why is this so important for the community.

Nanofibers

General:

• I would like to have a comprehensive introduction about what the nanofibers are? What are so important and exciting about them?
• After 122 I think that it would make sense to have a short and clear introduction to the topic ‘fabrication approaches’ and how these play a role.

102: A burst period of 2 days was shown initially which was followed by peaks between days 7 and 15. Ok, this is an interesting information. However, what is the level needed to be reached in blood in order to achieve therapy? This is the most interesting for the user.

121: …providing encouraging results for the composite’s potential use in vascular graft procedures. What do you exactly mean by ‘encouraging’? Please be specific.

127: …were also able to be incorporated into the medial layer in order to create antimicrobial inhibition. Why the medial layer? Why not the outer one? Is there a reason? This should be explained to the reader.

137: Antibacterial activity of eugenol was tested. How? Could you provide more information?

154-155: One study investigated the in vivo release characteristics of Vancomycin embedded within PLGA nanofibers. Reference is missing.

159: Another area of interest is the cytotoxicity of the nanofibers being studied. Then, if it is interesting, could you please provide some more insights?

Nanofibers

General:

• I would like to have a comprehensive introduction about what the nanoparticles are? What is for example the difference from nanofibers? Definition?

170: Several inorganic metals, including silver, copper, gold and zinc have excellent broad-spectrum antiseptic properties. References?

171-172: As compared to traditional metal-coated vascular grafts, metal nanocomposites have greater specific surface areas and thus enhanced antimicrobial properties. Why? How does this greater area lead to better antimicrobial properties? Please clarify for the reader.

179: However, other studies suggest that silver nanoparticles are cytotoxic to a range of mammalian cells, including coronary endothelial cells. Ok, this is interesting, but are there any other data on other endothelial/vascular wall cells (e.g., peripheral vasculature)?

Metal nanoparticles with antiseptic polymers

Figure 2: The resolution of the papers should be improved. It is difficult to have a thorough look.

Nanoparticles used against other biofilm infections

231-232: While much of this research was not done specifically on vascular grafts, it still provides possible future directions for vascular graft research, and will thus be described briefly here. Could you please provide a description on possible applications? This would be of interest for the reader.

Outlook

248: Metal and antibiotic-soaked grafts are currently already employed clinically to prevent vascular graft infections. References?

259: Li et. al’s work suggests that silver nanoparticle-impregnated polyurethane vascular scaffolds possess some cytotoxicity. Against what type of cells?

Conclusions

281: Vascular graft infections are serious complications that are associated with high morbidity and mortality rates. References?

Author Response

The authors greatly appreciate the reviewer’s comments. The updated version of the manuscript has been modified following the reviewer’s suggestions. In the revised manuscript, all changes have been highlighted in red, including additional references. Responses to the reviewer’s comments are detailed below.

The authors present an interesting work with the title ‘Vascular Graft Infections: An Overview on Novel Treatments Using Nanoparticles and Nanofibers’. More specifically, the work should be of interest not only for basic researchers but also for vascular medicine clinicians. Generally, I think that the paper need a major revision before published. Here are some comments and suggestions from my side:

Introduction

General:

• I would like to see a deeper description on different graft infections per location (e.g. aorta, groin region, lower extremity etc.). I find the current description a bit superficial.

We thank the reviewer for this suggestion. We have added a deeper description of different graft infections per location, including symptoms, prophylaxis and incidence rates.

“VGI can occur in different cardiovascular regions including intracavitary locations such as the supra-aortic trunk (SAT), thoracic aorta, and abdominal aorta, we well as extracavitary infections in peripheral arteries, with different incidence rates according to each region [6-9].

The incidence of SAT VGI is extremely low, with only 140 cases reported over the last three decades [10]. Due to its rare occurrence, it is challenging to identify the etiol-ogy and develop a possible general therapeutic approach [11, 12]. Thoracic aortic VGI incidence, however, can be up to 6%, with mortality rates that can reach up to 75% [13, 14]. In recent years, an increase in thoracic aortic VGI has been reported, highlighting the need for more effective solutions [15]. The thoracic aorta is anatomically poorly exposed and visible signs of infection are difficult to detect, resulting in a high mortali-ty rate [16]. The abdominal aortic VGI incidence rate is around 0.2% according to Vogel et al. [17], while Berger et al. reported a 1.6% incidence at 30 days, 3.6% at one year, and 4.5% after two years in a different study [18, 19].

“When the infection has been detected, the first step is to take control over the sepsis process, followed by removal of all the infected materials/devices and subsequent reconstruction in a clean environment, which can be complicated depending on the patient’s conditions and comorbidities (i.e. diabetes mellitus, chronic renal failure, chronic obstructive pulmonary disease and obesity) [5, 21]. Cryopreserved aortic allo-grafts have shown good results for the replacement of infected thoracic VGI in terms of resistance to infection [22]. However, they are prone to degeneration, rupture, and bleeding when attacked by necrotizing organisms such as P. aeruginosa or C. albicans [23]. Another approach relies on in polyethylene terephthalate (PET) rifampin-soaked and silver-coated synthetic VG, which have been used to decrease the risk of early in-fection. These treated PET VG have also shown promising results against the risk of re-infection. Moreover, the overall five-year survival is around 53% in comparison with 12% for the standard grafts [24, 25]. Among the treatment options, conservative ther-apy is considered only as a palliative strategy for patients who could not withstand open surgery. For all these individuals, long-term or lifelong antimicrobial therapy is the only possibility [26, 27].”

“In situ reconstruction with autologous material such as great saphenous vein and fem-oral vein is the main strategy used by surgeons [32, 33]. While treated prosthetic grafts have garnered a lot of interest due to their availability and reduced operation time, the associated risk of re-infection remains an open question, since the available data are not conclusive [34].”

• I think what is interesting is to give some insights on the ‘complication’ of long-term antibiotic therapy that these patients need. This is missing from this review and it is indeed the most important for these patients.

In consideration of the reviewer’s suggestion, we have added a section regarding long-term antibiotic therapy.

“When VGI occurs in a patient that would not withstand a re-operation, there are few strategies available. In these cases, the best way to proceed is a combination of percutaneous drains and recurrent antibiotic administration to control the infection level [35]. Broad spectrum antibiotics are administered in the acute phase to control infection and sepsis. Once the infecting organism is identified, more appropriate therapeutic approaches are chosen. Although antibiotic administration is of paramount importance, no specific guidelines are provided on the length of the therapy. Usually, intravenous antibiotics are administered for at least 2-4 weeks, with good debridement if possible [36]. When infection is under control, there is a general consensus that at least 4-6 weeks of parenteral antibiotic therapy is necessary [4]. In most cases requiring such graft preservation, patients are placed on lifelong suppressive therapy. While this is currently the only option to control infection, it can lead to development of antibiotic resistance or to antibiotic toxicity to the filtering organs (e.g., nephrotoxicity, ototoxicity) [20, 24, 37, 38].”

38: Aortic graft infections have a mortality rate between 24-75%. Could you please provide references?

Following reviewer’s suggestion, we have added the following reference.

Reference [37] - Legout, L., et al., Characteristics and prognosis in patients with prosthetic vascular graft infection: a prospective observational cohort study. 2012. 18(4): p. 352-358.

39-40: One aggressive approach in management of intracavitary graft infections consists of the excision of the graft, debridement of infected tissues, and extra-anatomic bypass along with antibiotic therapy. Could you please provide references?

Following reviewer’s suggestion, we have added the following references.

References have been added: [32,33,35]

43: Similarly, peripheral graft infections have a high morbidity and up to 40% requiring limb amputation. Please differentiate the graft infections per location as in my general comment.

We thank the reviewer for this feedback. To address this suggestion we have added a more detailed description and differentiation of peripheral vascular graft infections.

“PAD has an overall prevalence rate of 12% in adult Americans [28]. VGI incidence ranges from 2.5% in femorofemoral prosthetic bypasses [29] to 2.8% in femoropopliteal bypasses [30], with higher occurrence in patients with critical limb-threatening ischemia. The groin region is the most subject to VGI. Although the peripheral location could be perceived as less dangerous than thoracic or abdominal infections, conservative treatment is not an option for lower limb VGI. In fact, peripheral VGI is associated with high mortality, up to 45% at five years [31], as well as recurrent infection, anastomotic disruption, and active bleeding, with up to 40% of cases requiring limb amputation [4].

59-60: …Dacron grafts containing collagen or gelatin have demonstrated excellent antibacterial activity with low rates of graft… Please provide refs: how low are these rates?

Following reviewer’s suggestion, we have expanded this section bringing more data and a deeper description of the study.

“According to Colburn et al., tests performed on dogs showed how the reinfection rate decreased from 100% to 62.5% using Dacron grafts soaked with rifampin and sealed with gelatin or collagen instead of just Dacron grafts [42]. In a similar study, Almeida and colleagues investigated collagen im-plants impregnated with gentamicin (Collatamp) in the prevention of VGI. In 60 patients with lower limb ischemia who underwent femoropopliteal PTFE prosthetic by-pass, the control group had a surgical site infection rate of 20% (6 of 30), whereas the implant group, which had Collatamp applied next to the prosthesis, had a surgical site infection rate of 0% (0 of 30) [44].”

65-66: The fast release and clearance of impregnated drugs and inability to coat PTFE grafts are two major limitations of antibiotics coated grafts. How fast is the release and clearance? Please expand and describe the PTFE term.

We appreciate the suggestion of the reviewer. In light of this comment, we have included more data to better describe some of the limitations of antibiotic coated grafts and the meaning of the acronym ePTFE.

”expanded polytetrafluoroethylene (ePTFE)”

“Previous in vitro tests with this system demonstrated a duration of in vitro activity of 22 days, but in vivo experiments demonstrated that, at 10 and 12 days, the susceptibility of these rifam-pin/collagen-bonded grafts to a bacteremic challenge was similar to that of control grafts [41, 43].”

84-85: …has also been increased interest in the use of nanotechnology to reduce the rates of vascular graft infections. The authors should better describe the novelty of their work? Why do we care about using nanotechnology? Why is this so important for the community.

In light of the suggestion provided by the reviewer we have added a thorough description of the most promising characteristics of nanotechnology highlighting both positive aspects and current challenges to overcome.

“In addition to traditional antibiotic- and antiseptic-impregnation techniques, nanotechnology could help to reduce the rates of VGI. Nanotechnology can be exploited to develop a new generation of VG, where synthetic materials can be used together with endothelial cells, growth factors, and other active biomolecules to promote biocompatibility. Moreover, nanofabrication techniques allow the production of tailored solutions, recapitulating fundamental features of the extracellular matrix (ECM) such as interconnected porosity and architectural arrangement of the fibers [50-52]. Autologous grafts are considered the gold standard, due to their innate biocompatibility and mechanical properties. However, low availability, risk of comorbidities in the donor, and expensive costs for harvesting are critical limitations. Conversely, allografts are not limited in supply; however, they have the potential to cause an immune response and carry the risk of disease transfer, in addition to remaining an expensive solution due to harvesting and cryopreservation. Those limitations have pushed researchers towards the development of new strategies. In particular, nanofibers and nanoparticles, both nanotechnologies, are able to encapsulate and release different types of drugs. Moreover, they have shown promising results in terms of biocompatibility and infection prevention in different surgical approaches [51-54].

It has been broadly reported that nanofibrous mats, patches, and scaffolds, mainly fabricated by electrospinning, can imitate the nanostructure of natural ECM, thus im-proving cell adhesion and guiding phenotype differentiation as confirmed by more efficient vascular cell attachment and spreading [55-57]. Furthermore, nanofibers exhibit unique physicochemical properties that give them the ability to coat any surface area, even PTFE [46]. In nanoparticles, as compared to traditional metal-coated VG, the metal nanocomposites have greater specific surface area to volume ratios, due to their small size, and thus enhanced antimicrobial properties [58]. In particular, silver nanoparticles have drawn significant attention as a means to address implant-associated infections. Despite the positive features of nanostructured grafts, some limitations need to be considered. Long term patency of the engineered VG and risk of triggering negative immune responses are drawbacks that still need to be overcome. Nanofibrous assemblies can potentially degrade over time, deteriorating the mechanical properties of the polymeric matrix and resulting in VG failure [59]. Moreover, the host immune re-sponse is something that must be considered carefully, since it has been demonstrated how it can severely affect the patency of VG in the long run.

In this regard, only autologous solutions do not trigger negative immunologic re-action. Allografts experience immunogenicity due to the interaction with the host body, and for this reason patients must be subjected to immunosuppressive therapies [60]. Immune rejection is considered one of the main causes of allograft failure and rupture in the long term [61]. VG are subjected to the same fate, even exacerbating the outcomes. Aggressive reaction from the host immune system leads to stenosis, thrombosis, and eventually failure of the implant. This aspect has been thoroughly studied by Hibino et al. using tissue engineered VG [62]. In a study performed in humans, they observed major failure of tissue engineered VG due to stenosis [63]. In order to investigate the reason for the failure, they examined the role of host immune function in excess neo-tissue formation in tissue engineered VG using an immunodeficient mouse model. Interestingly, grafts implanted in immunodeficient mice showed greater patency over time compared to those in an immunocompetent model. Sonography revealed stenosis of the grafts implanted in immunocompetent mice after 2 weeks, whereas the grafts of the immunodeficient group remained patent up to 10 weeks, suggesting a key role of the immune system in graft failure due to excessive formation of neo-intimal tissue. Despite the listed limitations, it is clear the enormous potential that nanocomposite materials bring in engineering novel solutions for the fabrication of more efficient VG.

This review aims to summarize the improvements achieved by exploiting nanofiber and nanoparticle technologies for VGI treatment, highlighting aspects of novelty and current challenges towards broader use in the clinical field.”

Nanofibers

General:

• I would like to have a comprehensive introduction about what the nanofibers are? What are so important and exciting about them?

We thank the reviewer for this suggestion. To address this point we have added a description of what nanofibers are giving a thorough description of the fabrication process.

“Nanofibers are an example of nanomaterials that are typically produced by the electrospinning technique. Electrospinning is a simple and cost-effective method that enables control over the fiber diameter and allows formation of fibers composed of a selected polymeric solution [54]. Nanofibers can be obtained from a wide range of both natural and synthetic polymers, according to the specific application [54, 64-66]. Porosity as well as alignment of the nanofibers can be controlled with unprecedented detail [67]. Due to their nanometric dimensions, it is possible to exploit a greater surface area compared to bulk materials, allowing enhanced adhesion of cells or surface engrafting of proteins and drugs [68,69]. Moreover, mechanical properties of the nanofibrous constructs can be customized to great extent, tuning key features like flexibility and stiffness.”

• After 122 I think that it would make sense to have a short and clear introduction to the topic ‘fabrication approaches’ and how these play a role.

In consideration of reviewer’s suggestion we have added a brief introduction on the fabrication technique to obtain nanofibrous deposition.

“Briefly, the polymer solution is squeezed from a syringe into a drop in a high electrostatic field; this process allows for the formation of a jet, and while the solvent evaporates, the nano-sized fibers form (Figure 1).”

102: A burst period of 2 days was shown initially which was followed by peaks between days 7 and 15. Ok, this is an interesting information. However, what is the level needed to be reached in blood in order to achieve therapy? This is the most interesting for the user.

We thank the reviewer for this suggestion. In this regard we have added more data on antibiotic concentration necessary to ensure minimum inhibitory concentration.

“In this study, the in vitro release curves showed a continuous release of Vancomycin at a concentration of 2 µg/ml. Concentration levels above the 90% minimum inhibitory concentration (MIC₉₀) were maintained for over 30 days. A burst period of 2 days was shown initially which was followed by peaks between days 7 and 15.”

121: …providing encouraging results for the composite’s potential use in vascular graft procedures. What do you exactly mean by ‘encouraging’? Please be specific.

Following reviewer’s suggestion we have added one of the main reasons why such results could be encouraging and beneficial for future application to treat vascular graft infections.

“This study shows an interesting approach that could be used to develop viable alternatives for the treatment of VGI, using a combination of nanofibers and nanoparticles, especially for small diameter VG. These results clearly prove the feasibility of these techniques, but the efficacy is still far from that of the current standard of care.”

127: …were also able to be incorporated into the medial layer in order to create antimicrobial inhibition. Why the medial layer? Why not the outer one? Is there a reason? This should be explained to the reader.

In light of the reviewer’s suggestion, we have expanded this section, better explaining the main reason to coat the medial layer of the vascular graft instead of the outer one.

“Antimicrobial products were incorporated within the medial layer to obtain a slower release of drugs within the body. Shielding the medial layer with an inner and an outer nanofibrous level ensured a slower and prolonged release, due to reduced contact with biological fluids [69,76].”

137: Antibacterial activity of eugenol was tested. How? Could you provide more information?

We thank the reviewer for this feedback. We have provided more information regarding this study including eugenol as antimicrobial agent.

“Different eugenol concentrations were used, namely 5, 10, 20 and 30 wt%, and all were found to have similar release patterns in vitro. Antibacterial activity was evaluated with an “immersion” method, where bacterial solutions of E. coli and S. aureus were added to wells containing discs of the electrospun membrane with different eugenol content.”

154-155: One study investigated the in vivo release characteristics of Vancomycin embedded within PLGA nanofibers. Reference is missing.

Following reviewer’s suggestion, we have added the following reference.

Reference [70]: Liu, K.-S., et al., Sustained release of vancomycin from novel biodegradable nanofiber-loaded vascular prosthetic grafts: in vitro and in vivo study. 2015. 10: p. 885.

159: Another area of interest is the cytotoxicity of the nanofibers being studied. Then, if it is interesting, could you please provide some more insights?

We agree on the suggestion provided by the reviewer and we have added more information regarding this important aspect.

“Cytotoxicity has been proposed as one of the reasons for VG rupture in the medium to long term. In particular, aggressive strategies of infection prevention, involving high concentrations of drugs in impregnated VG, have led to necrosis at the anastomotic sites, eventually leading to graft failure [78]. For this reason, it is of vital im-portance to evaluate possible cytotoxic effects due to not only the polymer but also drug concentration. Nanofibers and nanoparticles could solve the problem of drug-related cytotoxicity because a lower drug content can be used to avoid infection and drugs can be slowly released over time. A common way to evaluate cytotoxicity of electrospun constructs involves cell lines (e.g., murine fibroblasts, breast cancer cells, kidney epithelial cells) cultured and then exposed to different extracting solutions composed of dissolved electrospun matrix and antimicrobial agents such as vancomycin, eugenol, and gentamicin [2, 70, 71].”

Nanoparticles

General:

• I would like to have a comprehensive introduction about what the nanoparticles are? What is for example the difference from nanofibers? Definition?

Considering the reviewer’s suggestion, we have added a description of what nanoparticles are and their applications in the biomedical field.

“Nanoparticles are small particles that range from 1-100 nm in diameter [82,83]. Due to their nanoscale sizes and high surface to volume ratios, nanoparticles have attracted significant attention for their potential in drug delivery, imaging, and other stimuli-responsive applications [84-86]. For example, liposomal formulations are already used in the delivery of chemotherapeutics such as doxorubicin [84,87] and vincristine [84,88], while gold and iron oxide nanoparticles are used in computed tomography, magnetic resonance imaging, and positron emission tomography as contrast agents [86].”

170: Several inorganic metals, including silver, copper, gold and zinc have excellent broad-spectrum antiseptic properties. References?

Following reviewer’s suggestion, we have added the following references.

References [89-91]:

89. Schrand, A.M., et al., Metal‐based nanoparticles and their toxicity assessment. 2010. 2(5): p. 544-568.
90. Sánchez-López, E., et al., Metal-based nanoparticles as antimicrobial agents: an overview. 2020. 10(2): p. 292.
91. Fernando, S., T. Gunasekara, and J. Holton, Antimicrobial Nanoparticles: applications and mechanisms of action. 2018.

171-172: As compared to traditional metal-coated vascular grafts, metal nanocomposites have greater specific surface areas and thus enhanced antimicrobial properties. Why? How does this greater area lead to better antimicrobial properties? Please clarify for the reader.

We thank the reviewer for this feedback. In consideration of this suggestion we have added a brief explanation to clarify for the reader.

“As compared to traditional metal-coated VG, metal nanocomposites have larger specific surface areas; these larger exposed surfaces enable them to have greater antimicrobial activity per unit mass [92].”

179: However, other studies suggest that silver nanoparticles are cytotoxic to a range of mammalian cells, including coronary endothelial cells. Ok, this is interesting, but are there any other data on other endothelial/vascular wall cells (e.g., peripheral vasculature)?

In consideration of the reviewer’s feedback, we have provided a deeper description of the risk of toxicity that can arise with the use of nanoparticles, giving a clear explanation of the underlying mechanisms. Moreover, we have added two studies performed on endothelial cells.

“Silver nanoparticles, together with other types of nanoparticles such as silica and tricalcium phosphate have been found to cause significant hemolysis [95, 96]. Gliga et al. have observed a size-dependent toxicity for silver nanoparticles. Using epithelial cells isolated from normal human bronchial epithelium (BEAS-2B) they denoted how toxicity increased with nanoparticles with a diameter of 10 nm compared to 20 and 50 nm [97]. This can be explained, because when nanoparticles reach the blood system, they come into direct contact with blood cells, endothelial cells and plasma proteins. The nanometric dimension of these nanoparticles can affect the intricate structure and critical functions of the blood components. In fact, plasma proteins tends to adsorb to the surface of nanoparticles to form a protein corona that significantly influences their interaction with blood components and may even lead to increased cellular activation [98]. A pilot study by Sun et al. showed that 24 h exposure to ZnO NPs (primary size 45.3 nm) was associated with significantly decreased mitochondrial activity in human cardiac microvascular endothelial cells (HCMECs), with a threshold as low as 5 μg/mL [99]. Liang et al. showed that 24 h exposure to ZnO NPs (primary size 70 nm) at the concentrations ≥15 μg/mL significantly induced cytotoxicity in human aortic endothe-lial cells (HAECs) as decreased mitochondrial activity, lactate dehydrogenase (LDH) release and apoptosis [100].”

Metal nanoparticles with antiseptic polymers

Figure 2: The resolution of the papers should be improved. It is difficult to have a thorough look.

We thank the reviewer for this suggestion, but the figure’s resolution could not be enhanced. The figure was taken directly from the original paper. We have tried to enlarge the image so it could be easier to view all the main elements of the figure.

Nanoparticles used against other biofilm infections

231-232: While much of this research was not done specifically on vascular grafts, it still provides possible future directions for vascular graft research, and will thus be described briefly here. Could you please provide a description on possible applications? This would be of interest for the reader.

We thank the reviewer for this suggestion. We think in the following lines (449 - 467) we have gathered some possible applications that could be applied to vascular grafts in general.

Outlook

248: Metal and antibiotic-soaked grafts are currently already employed clinically to prevent vascular graft infections. References?

Following reviewer’s suggestion, we have added the following references.

References [11,14,25]:

11. Kahlberg, A., et al. How to best treat infectious complications of open and endovascular thoracic aortic repairs. in Seminars in vascular surgery. 2017. Elsevier.
12. Spiliotopoulos, K., et al., Open descending thoracic or thoracoabdominal aortic approaches for complications of endovascular aortic procedures: 19-year experience. 2018. 155(1): p. 10-18.
13. Girdauskas, E., et al., Secondary surgical procedures after endovascular stent grafting of the thoracic aorta: successful approaches to a challenging clinical problem. 2008. 136(5): p. 1289-1294.

259: Li et. al’s work suggests that silver nanoparticle-impregnated polyurethane vascular scaffolds possess some cytotoxicity. Against what type of cells?

We thank the reviewer for this feedback. We have added the cell type as suggested.

“endothelial progenitor cells [99].”

Conclusions

281: Vascular graft infections are serious complications that are associated with high morbidity and mortality rates. References?

Following reviewer’s suggestion, we have added the following references.

References added [20,35,117]:

20. Hasse, B., et al., Vascular graft infections. 2013. 143: p. w13754.
21. Wilson, W.R., et al., Vascular graft infections, mycotic aneurysms, and endovascular infections: a scientific statement from the American Heart Association. 2016. 134(20): p. e412-e460.
22. Pashneh-Tala, S., S. MacNeil, and F.J.T.E.P.B.R. Claeyssens, The tissue-engineered vascular graft—past, present, and future. 2016. 22(1): p. 68-100.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed the review comments satisfactory 

Author Response

We thank the reviewer for the precious advice that improved our manuscript.

Reviewer 2 Report

I think that the reviewers addressed my comments adequately. I would suggest a thorough revision of the english languange before publication.

Author Response

We thank the reviewer for the precious advice, we revised the manuscript and have and an English editor helped to finalize and improve the grammar of our manuscript.