Heparin-Eluting Tissue-Engineered Bioabsorbable Vascular Grafts

Round 1

Reviewer 1 Report

Matsuzaki et al in the manuscript titled “Heparin Eluting tissue engineered bioabsorbable vascular graft” the authors do a good job in highlighting the advancement in the field of TEVGs particularly involving heparin functionalized grafts.  However, the manuscript structure appears fair with regard to flow however there are many inconsistencies in the organization of the subsections. Also, the authors do not cite some recent works that has been highlighted in this review. Taking into account the suggestions made here, the quality of the manuscript could be improved. 

 

Comments:

1. Under the subsection “3-1-2. Mixing Heparin with sulfated biopolymers” the authors describe their research on heparin functionalized PCL-PLCL graft. Could the authors revise and use third person and passive-voice constructions (instead of “we”s), it almost appears as the authors are describing their results in a discussion format. 
2. The same paragraph above the authors describe “burst” release of heparin as a drawback and highlight their work as potentially an example that overcomes this drawback. But at the end of the paragraph the authors comment on the same drawback within their system also- i.e. burst release. This makes the flow very confusing.
3. In the design considerations section, the authors aptly highlight the effect of material properties, composition etc. For additional reading in this regard the authors may cite additional references by others here [PMID: 29194781, 32227919]
4. In Figure1, please make the font size of the text within the figure consistent and larger, some are hard to read. Also, please remove the spelling suggestion marker under “Kossa”.
5. In section 3-1-3, please remove the duplicate comma (,) after PGA.
6. Please re-write the following statement in a grammatically correct form “It can mix and match different materials in different ratios core and shell, fibers, and can cooperate to graft with different drugs, proteins or cells.”
7. In table 1, “Study model” column please specify the cell types used in the in vitro model. And separate the units from numbers with a space. Eg. 2 mm
8. In table 1, row 1, please specify the ELISA targets.
9. In table 1, please specify graft dimension for 7, 8 and 9.
10. Please correct all typos in the table for eg. “preendthelization”
11. Please provide full form of P(LLA-CL) where it appears first in the text.
12. Please also avoid statements like “we will talk” “we will discuss” that often appears in a textbook chapter style of writing.
13. Please make the correct the font and space inconsistencies in figure 2, 3   “scaf old”, “Nanof ber Stream”
14. Section 3-2-1 title “Non Electricstatic Interaction” has a typo in itself and follows the main sub section “Chemical conjugation” – non- electrostatic and electrostatic interactions is not regarded as methods of chemical conjugation. The authors must carefully use the terminologies and concepts so that it does not change the fundamental understanding of the well-known concepts. Please revise the section.
15. Please remove Figure 4, it is poor depiction of the grafts and microscopic architecture and is confusing and not informative.
16. Please change the title caption of Figure 5- the figure does not show manufacturing of the heparin eluting graft. Please also capitalize “heparin release profile” text and remove the local language text beside it.
17. The authors should know how to write ‘nanosponge’ and not write as “nano sponge”.
18. Section 4-3 ‘Material fabrication’ before the conclusion is out of place, please remove or incorporate in previous section.
19. In the review article, the authors should also highlight the examples of heparin grafts by Donghak et al [PMID: 31745143] Haizhu et al. [PMID:29782791].
20. The authors are also, encouraged to discuss the study by Larisa et al where they compare efficacy of graft made of PHBV-PCL with growth factors against Gore-Tex material in a sheep model. 

Author Response

Reviewer 1

1. Under the subsection “3-1-2. Mixing Heparin with sulfated biopolymers” the authors describe their research on heparin functionalized PCL-PLCL graft. Could the authors revise and use third person and passive-voice constructions (instead of “we”s), it almost appears as the authors are describing their results in a discussion format.

 

Thank you for your suggestion, we modified the sentence to be stated in a third person.

 

1. The same paragraph above the authors describe “burst” release of heparin as a drawback and highlight their work as potentially an example that overcomes this drawback. But at the end of the paragraph the authors comment on the same drawback within their system also- i.e. burst release. This makes the flow very confusing.

 

Sorry for the confusion. We have decided to leave out the last sentence.

 

1. In the design considerations section, the authors aptly highlight the effect of material properties, composition etc. For additional reading in this regard the authors may cite additional references by others here [PMID: 29194781, 32227919]

 

Thank you for your suggestion. We would like to cite these following two paper in the design consideration section.

[7] Thomas D, O’Brien T, Pandit A. Toward Customized Extracellular Niche Engineering: Progress in Cell-Entrapment Technologies. Adv Mater.2018:30(1)

[8] Nicolas J, Magli S, Rabbachin L, Sampaolesi S, Nicotra F, Russo L.3D Extracellular Matrix Mimics: Fundamental Concepts and Role of Materilas Chemistry to Influence Stem Cell Fate.Biomacromolecules.2020,21(6),1968-1994.

 

1. In Figure1, please make the font size of the text within the figure consistent and larger, some are hard to read. Also, please remove the spelling suggestion marker under “Kossa”.

 

Thank you for the heads up. We modified Figure 1’s word font size. Additionally , removed the underline.

1. In section 3-1-3, please remove the duplicate comma (,) after PGA.

 

Thank you for your suggestion, we modified this.

 

1. Please re-write the following statement in a grammatically correct form “It can mix and match different materials in different ratios core and shell, fibers, and can cooperate to graft with different drugs, proteins or cells.”

 

We rewrite this sentence as “It can mix different materials in different ratios to produce core and shell fibers. The grafts made by coaxial electrospinning also can cooperate with different drugs, proteins and cells.” Page6 Line 195-197

 

1. In table 1, “Study model” column please specify the cell types used in the in vitro model. And separate the units from numbers with a space. Eg. 2 mm
2. In table 1, row 1, please specify the ELISA targets.
3. In table 1, please specify graft dimension for 7, 8 and 9.
4. Please correct all typos in the table for eg. “preendthelization”

 

We modified this table following your directions. Page 3 line  102-103

 

1. Please provide full form of P(LLA-CL) where it appears first in the text.

 

We added the full form as Poly(l-lactide-co-ε-caprolactone) in Page 6 line206-207

 

1. Please also avoid statements like “we will talk” “we will discuss” that often appears in a textbook chapter style of writing.

 

We modified these sentence as per directions.

Page 7 Line 267-269

Page 10 line 352-3360, 365-367

 

1. Please make the correct the font and space inconsistencies in figure 2, 3   “scaf old”, “Nanof ber Stream”

Thank you for the heads up. We modified this.

 

1. Section 3-2-1 title “Non Electricstatic Interaction” has a typo in itself and follows the main sub section “Chemical conjugation” – non- electrostatic and electrostatic interactions is not regarded as methods of chemical conjugation. The authors must carefully use the terminologies and concepts so that it does not change the fundamental understanding of the well-known concepts. Please revise the section.

 

Thank you for your suggestion. Basically, this section describes the conjugation of heparin via covalent bonds, so we changed the title to Covalent interaction. Page 7 line 273

 

1. Please remove Figure 4, it is poor depiction of the grafts and microscopic architecture and is confusing and not informative.

 

Thank you for your suggestion. We will remove this figure.

 

1. Please change the title caption of Figure 5- the figure does not show manufacturing of the heparin eluting graft. Please also capitalize “heparin release profile” text and remove the local language text beside it.

 

Thank you for the heads up, we modified the figure title as “Subclinical CO2 technique for long-term sustained release of heparin”.

 

1. The authors should know how to write ‘nanosponge’ and not write as “nano sponge”.

 

Thank you for the heads up. We modified the sentence as per your suggestion.(Page 9 line 320 -327)

 

1. Section 4-3 ‘Material fabrication’ before the conclusion is out of place, please remove or incorporate in previous section.

 

Thank you for your suggestion. We've decided to add this to the previous section.

 

1. In the review article, the authors should also highlight the examples of heparin grafts by Donghak et al [PMID: 31745143] Haizhu et al. [PMID:29782791].

 

Thank you for your suggestions. We have added the following sentences to the Coaxial electrospinning section. -Furthermore, the functional inner layer using coaxial electrospinning technique is expected to prevent acute thrombosis of non-bioabsorbable materials such as ePTFE and promote rapid endothelialization of artificial vascular graft. Based on this finding, salvianolic acid B (SAB) promotes the proliferation and migration of endothelial cells. Kuang et al. fabricated the inner layer of an artificial graft using coaxial electrospinning and loaded it with heparin and SAB[41]  Kim et al. reported that in situ implantation of ePTFE grafts coated with Heparin/ substance P conjugated PLCL could ensure long-term patency by forming a proper endothelial layer and smooth muscle cells in the graft resembling a native artery[42].

 

[41] Kuang H, Wang Y,Hu J, Wang C, Lu S, Mo X. A Method for Preparation of an Internal Layer of Artificial Vascular Graft Co-Modified with Salvianolic Acid B and Heparin. ACS Appl Mater Interfaces.2018,10(23), 19365-19372.

 

[42] Kim D, Chung JJ ,Jung Y , Kim SH. The effect of Substance P/Heparin conjugated PLCL polymer coating of bioinert ePTFE vascular grafts on the recruitment of both ECs and SMCs for accelerated regeneration. Sci Rep.2019,9(1),17083    Page 7 line 249-257

 

1. The authors are also, encouraged to discuss the study by Larisa et al where they compare efficacy of graft made of PHBV-PCL with growth factors against Gore-Tex material in a sheep model.

 

Thank you for your suggestion. We have added the following sentences to the Physical conjugation section.

Antonova et al. had shown that polyhydroxybutyrate/valerate(PHBV)/ polycaprolactone (PCL) grafts modified with the growth factors such as VEGF, bFGF, and SDF-1α (PHBV/PCL-GFmix) performed well when implanted in the rat abdominal aortas[32].Subsequently,they used a sheep model, which is considered to be more suitable for an in vivo testing of cardiovascular implants[33]. Since they had expectations of a high incidence of thrombosis in a sheep model, attempted to reduce the thrombogenicity of the PHBV/PCL-GFmix grafts by combining heparin and iloprost on a surface of the grafts. At the end of one year, 50% of the PHBV/PCL-GFmixHep/Ilo grafts were completely patent. Indicating that modification with heparin and iloprost can greatly improve the performance of PHBV/PCL grafts in the experimental large animal models[34]

[32] Antonova, L.V.; Sevostyanova, V.V.; Kutikhin, A.G.; Velikanova, E.A.; Matveeva, V.G.; Glushkova, T.V.; Mironov, A.V.; Krivkina, A.V.; Barbarash, O.L.; Barbarash, L.S. Influence Of bFGF, SDF-1α, or VEGF incorporated into tubular polymer scaffolds on the formation of small-diameter tissue-engineered blood vessel in vivo. Vestnik Transplantologii i Iskusstvennykh Organov 2018, 20, 96–109.

 

[33] Yun, Y.R.; Won, J.E.; Jeon, E.; Lee, S.; Kang, W.; Jo, H.; Jang, J.H.; Shin, U.S.; Kim, H.W. Fibroblast growth factors: Biology, function, and application for tissue regeneration. J. Tissue Eng. 2010, 1, 218142.

 

[34]Antonova LV, Mironov AV, Yuzhalin AE,Krivkina EO,ShabaevAR, Rezvova MA, Tkachenko VO,

Khanova MY,Sergeeva TY, Krutitskiy SS, Barbarsh LS. A Brief Report on an Implantation of Small-Caliber Biodegradable Vascular Grafts in a Carotid Artery of the Sheep.Pharmaceuticals (Basel) 2020,13(5),101

Page 5 line 175- Page 6 line 187

Reviewer 2 Report

This is a narrative review suggested by a recognised and authoritative expert in the field of tissue-engineered vascular grafts (Toshiharu Shinoka). The aim of the review was to overview the studies on the development of heparin-eluting vascular grafts which have been tested in physiologically relevant large animal models (although authors also refer to a few studies on rabbit and rat models). To my opinion, such review is of major importance for the field and the personal opinion of Dr. Shinoka is highly valuable. Yet, it should be significantly improved before being accepted for publication.

My first concern is the choice of the models to be included into the review. Lessons from the experiments in my department indicated the meaninglessness of the rat model to assess the performance of TEVGs because of limited length of the grafts and very high polymer degradation rate which do not correspond to those in sheep and human organism. Albeit we did not carry out any experiments on rabbits, this is also a small animal model with a significantly faster metabolism and therefore higher rate of polymer degradation. In contrast, our experiments on the ovine model showed its significantly higher relevance to a putative clinical scenario. The degradation of the polymer scaffold was fairly low and heparin-eluting vascular TEVGs demonstrated an acceptable one-year primary patency rate. Therefore, I would suggest to consider only the studies on canine, porcine, and ovine models, as none of the small animal models have a translational meaning in TEVG field.

Second, some sections of the review seem to be redundant and duplicate each other (e.g., section 4 recapitulates section 3 to a considerable extent). My suggestion would be to remove section 4 and to re-distribute these studies across the subsections of section 3. In addition, every subsection within the section 3 should be wrapped up in a way to make a clear and critical conclusion from the findings described in the existing literature. However, section 4 might include the critical discussion on large animal models, as every of them has its advantages and shortcomings, and some of the authors' conclusions are of utmost importance for the field (e.g., the note on minimum graft length). Currently, the recommended length of TEVGs is limited to its 10-fold exceed of the diameter (4-6 cm for the ovine carotid artery grafts) but the authors suggest that 10-15 cm length is a mandatory requirement to properly assess graft endothelialisation, which is a key issue determining overall performace of TEVGs. I also think the field would benefit from a clear conclusion that small animal models are almost nonsensical in testing the efficiency of TEVG implantation, although they have certainly demonstrated the usefulness of some modification approaches and highlighted them for the future preclinical studies on large animals.

Third, I would suggest to shift the scope of the review from a pure narrative to a critical opinion, adding the discussion of the methodology to properly assess the performance of heparin-eluting TEVGs. Which methods are the best to assess the mechanical competence, hemocompatibility and biodegradation of such TEVGs? What histological and immunostainings are preferred? What should we change in our routine assessment of TEVGs in an implantation-to-excision period and upon the explantation? These issues are critical for the review and the personal experience of Dr. Shinoka is indispensable in this regard.

Author Response

1This is a narrative review suggested by a recognised and authoritative expert in the field of tissue-engineered vascular grafts (Toshiharu Shinoka). The aim of the review was to overview the studies on the development of heparin-eluting vascular grafts which have been tested in physiologically relevant large animal models (although authors also refer to a few studies on rabbit and rat models). To my opinion, such review is of major importance for the field and the personal opinion of Dr. Shinoka is highly valuable. Yet, it should be significantly improved before being accepted for publication.

My first concern is the choice of the models to be included into the review. Lessons from the experiments in my department indicated the meaninglessness of the rat model to assess the performance of TEVGs because of limited length of the grafts and very high polymer degradation rate which do not correspond to those in sheep and human organism. Albeit we did not carry out any experiments on rabbits, this is also a small animal model with a significantly faster metabolism and therefore higher rate of polymer degradation. In contrast, our experiments on the ovine model showed its significantly higher relevance to a putative clinical scenario. The degradation of the polymer scaffold was fairly low and heparin-eluting vascular TEVGs demonstrated an acceptable one-year primary patency rate. Therefore, I would suggest to consider only the studies on canine, porcine, and ovine models, as none of the small animal models have a translational meaning in TEVG field.

Thank you for pointing this out. We initially recognized that rat and rabbit studies were also necessary to illustrate the concept of heparin conjugation, but we agree with your point and have decided to omit citations for some rat and rabbit studies and cite new studies using sheep carotid artery.

2. Second, some sections of the review seem to be redundant and duplicate each other (e.g., section 4 recapitulates section 3 to a considerable extent). My suggestion would be to remove section 4 and to re-distribute these studies across the subsections of section 3. In addition, every subsection within the section 3 should be wrapped up in a way to make a clear and critical conclusion from the findings described in the existing literature. However, section 4 might include the critical discussion on large animal models, as every of them has its advantages and shortcomings, and some of the authors' conclusions are of utmost importance for the field (e.g., the note on minimum graft length). Currently, the recommended length of TEVGs is limited to its 10-fold exceed of the diameter (4-6 cm for the ovine carotid artery grafts) but the authors suggest that 10-15 cm length is a mandatory requirement to properly assess graft endothelialization, which is a key issue determining overall performance of TEVGs. I also think the field would benefit from a clear conclusion that small animal models are almost nonsensical in testing the efficiency of TEVG implantation, although they have certainly demonstrated the usefulness of some modification approaches and highlighted them for the future preclinical studies on large animals.

Thank you for your suggestion. I redistributed the content of Section 4 to Section 3. For this graft length, I incorporated this content as an Opinion in Discussion.

3. Third, I would suggest to shift the scope of the review from a pure narrative to a critical opinion, adding the discussion of the methodology to properly assess the performance of heparin-eluting TEVGs. Which methods are the best to assess the mechanical competence, hemocompatibility and biodegradation of such TEVGs? What histological and immunostainings are preferred? What should we change in our routine assessment of TEVGs in an implantation-to-excision period and upon the explanation? These issues are critical for the review and the personal experience of Dr. Shinoka is indispensable in this regard.

 

Thank you for your suggestion. In the Discussion section, I have added my suggestions for the future research on small diameter tissue engineered arterial grafts.　It would be appreciated if you could read and comment on this sentence.

Page 10 Line 365-page 11 line 420

 

This review describes two methods to improve small diameter arterial graft patency. One, heparin conjugation method to improve the patency of a grafts made from bioabsorbable materials. Second, a method of extending the suspended release period of heparin to maintain the graft patency rate for a longer time. Adequate and sustained heparin release is considered a means of increasing graft patency, while the long-term endothelialization process is completed. Heparin has been shown to prevent thrombosis associated with thrombin suppression, inhibition of vascular smooth muscle cell (VSMC) proliferation, and prevention of graft occlusion [60,61], but that is not all. Heparin also can stabilize physiologically unstable growth factors in the heparin binding domain [62,63]. Heparin has the potential to overcome the challenges associated with growth factor instability in solution, self-aggregation, low bioavailability, short half-life in vivo, and in vivo delivery [64,65,66]. This has led to many advances in the patency of the smallest diameter tissue-engineered arterial grafts.

In addition, to test the efficiency of TEVG grafting in these studies, small animal models are not suitable for preclinical testing due to the differences of metabolism, arterial pressure from humans. It is necessary to test the efficacy and safety of these materials in large animal models such as sheep or pigs prior to clinical trials. In fact, in this review, we have focused on experiments done on large animal models.

 

Although, we have no choice but to use animals, we must always consider the three R's of animal use (reduction, refinement, and replacement) in order to protect the animals. For instance, adequate mechanical strength should be verified before implantation in the animals.

Before implanting the grafts to the animals couple of in vitro tests needs to be performed. For example, the tensile strength test alone is not sufficient to ensure that the graft will not aneurysm in an arterial environment. Thus, beating tests simulating arterial pressure [67], and evaluation of heparin release using the toluidine blue method must be performed [26]. In vitro evaluation of the biocompatibility, such as platelet deposition on the luminal surface of the graft, the LDH assay[24] is necessary. For the biodegradability, although different reagents are used for different polymers, we should verify the degradation rates of a different biodegradable grafts using serial accelerated degradation studies [68].

Because of the nature of tissue engineering grafts, some studies expect neotissue formation using materials with fast degradation rates[69,70]. In the arterial environment, these grafts tend to dilate relatively quickly. Hence, materials with a longer degradation period are preferable at this point.  Animal studies should be performed with grafts that meet the above in vitro criterias.

 

 Many studies have shown that endothelialization is important to maintain the graft patency. However, these studies shown transanal endothelial growth at most 1 to 2 cm [43]. This might be the reason why TEVG has been relatively successful in experimental models such as sheep carotid or porcine femoral arteries, where 2-5 cm prostheses have been evaluated (currently, the recommended length of TEVG is limited to 10 times the diameter (4-6 cm for ovine carotid grafts)), but clinically, coronary and lower limb bypass graft prostheses are on the average of 15 cm in length[71].

Hematogenous or percutaneous endothelialization is a time-consuming process, and large-animal experimental designs need to use (>5 cm) grafts in length to validate clinical importance. After grafting, if patency can be maintained without graft dilatation, long-term observation for about a year is desirable. In regenerative medicine research, the assessment of tissue regeneration and inflammation typically requires animal sacrifice and graft harvesting for histological evaluation. There are existing imaging methods, such as echocardiography and angiography, that can assess the in vivo characteristics of implanted arterial grafts.  However, these methods are generally limited to anatomical evaluation of luminal diameter, neo-tissue formation and flow velocity. An imaging tools that can evaluate tissue regeneration in vivo, while the animal is alive will be helpful in the future. We are currently examining the use of nuclear medicine to evaluate the neotissue formation in grafts[72]. It would be ideal if we could prove safety by sacrificing the fewest number of large animals possible, use computational modeling to simulate optimal graft design and heparin release, and design a clinical trial.

In conclusion, although there are many obstacles to overcome, advances in heparin binding technology have further improved the patency of these experimental grafts. The impact of this research on the field of clinical cardiovascular surgery will be tremendous.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

The present article can be accepted in the present form.

Author Response

Thank you so much for checking this article. 

 

Round 2

Reviewer 2 Report

The authors have well addressed all my comments.