Microneedle Vascular Couplers with Heparin-Immobilized Surface

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Microneedle Vascular Couplers with Heparin -Immobilized Surface Improve Suture-Free Anastomosis Performance Dae-Hyun Kim, Jung Bok Lee, Mi-Lan Kang, Ji Hwan Park, Jin You, SeongMi Yu, Ju Young Park, Seung Bae Ryu, Gyeung Mi Seon, Jeong-Kee Yoon, Mi Hee Lee, Young Min Shin, Ki Dong Park, Jong-Chul Park, Woo Soon Jang, Won Shik Kim, and Hak-Joon Sung ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01097 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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ACS Biomaterials Science & Engineering

Microneedle Vascular Couplers with Heparin -Immobilized Surface Improve Suture-Free Anastomosis Performance Dae-Hyun Kim1, Jung Bok Lee1, Mi-Lan Kang1, Ji Hwan Park2, Jin You2, SeongMi Yu1, Ju Young Park1,2, Seung Bae Ryu3, Gyeung Mi Seon1,4, Jeong-Kee Yoon1, Mi Hee Lee1, Young Min Shin1, Ki Dong Park3, Jong‐Chul Park1, 4, Woo Soon Jang2, Won Shik Kim5*, and Hak-Joon Sung1* 1Department

of Medical Engineering, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea 2FutureBioWorks, 3Department

Seoul 08504, Republic of Korea

of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of

Korea 4Brain

Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea 5Department

of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul 03080, Republic of Korea *E-mail: [email protected] (W.S. Kim); [email protected] (H-J. Sung)

Abstract: To make up for the shortcomings of suture-based approach and current coupler devices including long suturing time, exhaustive training, additional mechanical setting, and narrow working windows for size and type of diverse vessel types, a new, suture-free microneedle coupler was developed in this study. The needle shape for improved anastomosis performance and the condition for anti-thrombotic surface immobilization were determined. In particular, the polymer materials help to maintain healthy phenotypes of main vascular cell types. The performance in rabbit and porcine models of end-to-end vascular anastomosis indicate that this device can serve as a potent alternative to the current approaches.

Keywords: microneedles, vascular anastomosis, tubular construct, cytocompatibility, heparin coating

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Due to surgical damages to organ and tissue, reconnecting blood vessels (“vascular anastomosis”) is essentially required for proper post-operative care1-2. Organ transplantation is also accompanied by complex anastomosis procedures between host and target organ vessels3. Poor anastomosis of vessels results in organ malfunction because of tissue necrosis and/or excessive bleeding. In particular, small blood vessels with a diameter range of 2-4 mm are mostly subject to the anastomosis procedure in a wide range of surgeries. Two major methods used currently are 1) suturing and 2) synthetic vascular coupler.

Suturing requires a high level of training and takes long time due to the complexity of procedure. The average surgical time is reported as 20-60 min for one case of anastomosis but often increases due to insufficient workmanship of surgeon. The average failure rate is ~ 10% but also increases drastically when the surgical skill of operator is not mature. When smaller vessels are anastomosed or organ transplantation is operated, the suture procedure gets more complex as a microscope or magnifying glass is used. Delaying an anastomosis procedure often results in poor prognosis in post-operative care.

Among coupler devices, the Synovis coupler (Synovis Micro Companies Alliance, Birmingham, AL) has been extensively used due to its easy, fast, safe performance2, 4. However, the device works for only vein-vein anastomosis, and its procedure requires an expensive mechanical setting. Jay Agarwal et al., has developed a new coupler with combination of endovascular part and external wall connector using a blend of polyethylene and polymethyl-methacrylate5. Its performance has been tested for artery anastomosis in a porcine model with intake of anti-thrombotic sodium heparin due to the placement of endo-vascular part. Another group has developed a similar concept of coupler with a diameter range of 4-7.5 mm and a length of 20 mm using a polymer material. Its 2 ACS Paragon Plus Environment

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performance has been tested in a lamb cadaver model. Both couplers have advantages in enhancing the anastomosis efficiency with quick and easy placement. Nonetheless, they work for limited size and type of vessels and thus are difficulty to customize depending the surgical situation. As a result, the suture-based method is mainly used for complex anastomosis cases such as organ transplantation. Together, there is an unmet need to develop a new coupler enabling i) customized designs in their size, structure and material properties; ii) easy and fast anastomosis procedure without suturing; and iii) anti-thrombotic coating without systemic intaking of anti-thrombotic medication.

In this study, microneedles were generated on the outer layer of tubular construct using a self-made manufacturing equipment (Figure S1). The design idea is that the microneedle tubular construct is coated with anti-thrombotic heparin and placed in an endovascular junction site of two vessels to anastomose, and the microneedles can hold the two connection ends of vessels by a stent strut-like placement into the intima layer of connected vessels (Figure 1A). In order to improve the efficiency of microneedle-based anastomosis, equilateral triangle and 45° angle inclined towards the anti-tension direction shapes were produced (Figure 1B). The procedure generating microneedles includes i) melting the surface of tube construct in contact with the heated metal pin and ii) moving the metal pin to generate microneedles through the moving direction. The different needle shapes were produced by controlling the pin movement and surface tension between the metal pin and melted polymer solution. The microneedle coupler was separated from the stainlesssteel wire by dissolving the inner coated alginate sacrificial layer in DI water.

The dimension of the coupler was measured to 400 µm wall thickness, 2.15 mm inner diameter, 2.45 mm outer diameter and 17.72 mm length. Since the coupler body was produced by dipping a 3 ACS Paragon Plus Environment


rod mold into a polymer solution, the diameter of rod mold determines the inner diameter of coupler body. Therefore, it is expected that the diameter of rod mold can be freely adjusted for the needle coupler to fit into small vessels with 200-500 µm in diameter. This type of size adjustment requires controlling the mechanical properties of both needle and coupler body to minimize damages to the thin and weak wall of small blood vessels as well as not to interrupt their compliance.

Due to temperature fluctuation resulting in inconsistent heating, the metal pin was heated between 260 and 310 degrees and left for 30 min, thereby stabilizing the temperature. The source polymer was not melted enough and often generated high viscosity when it was heated below the melting temperature. Hence, considering the melting temperatures of PCL (54 degrees) and PLA (163 degrees), the metal pin was heated up to the range. Moreover, when the pin was lifted fast, the end part of microneedle was bent and elongated inconsistently. Thus, the slow lifting speed was a critical factor to generate the consistent needle shape. An enough space among pins was kept to help pin-point heating, thereby minimizing the melting area. Depending on whether the lifting direction of pin was vertical or uniform tilting, the needle shape was determined to be equilateral triangle or 45° angle inclined towards the anti-tension direction.

Since well-qualified polyesters are used, the materials are biodegradable and compatible in maintaining healthy phenotypes of vascular cells (Figure 2). As test polymers are spin-coated on glass coverslips, the glass coverslip (GLASS) served as a substrate control. The test polymer type includes polycaprolactone (PCL), polylactic acid (PLA), and 1:1 mix of PCL and PLA (Mix). The test vascular cell type includes human umbilical vein endothelial cell (HUVEC) and human aortic smooth muscle cells (SMC). Cells were cultured on the test polymer substrates for 4 days. Very few cells were dead in both cell types when cell viability was assessed (Figure 2A). 4 ACS Paragon Plus Environment

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Vascular cell morphology indicates healthy and pathological stages of cell phenotype6-7. Healthy ECs demonstrate tight junction to maintain low cell-cell permeability and thus allow for blood perfusion. In this status, their morphology should be maintained to a cobble stone shape with a high range of circularity. Their morphology becomes spindle with decreases in the circularity as they undergo switching to pathological phenotypes. Post 4 day-culture on the test polymer substrates, HUVECs showed typical cobble stone shapes with high ranges of circularity though there were minutes variations in the distribution of circularity among the test groups (Figure 2B-C).

On the other hand, spindle shapes with a low range of circularity indicate a healthy (“contractile”) phenotype of vascular smooth muscle cells 8. In this status, they usually do not proliferate much with a low amount of protein production.

When their phenotype becomes pathological

(“synthetic”), their morphology becomes more circular with drastic increases in proliferation and protein production. When SMCs were cultured on the test polymer substrates, they showed typical spindle shapes with low ranges of circularity (Figure 2B-C). These results suggest that the test polymers are compatible in maintaining the healthy phenotypes of the major vascular cell types.

Endothelialization of endovascular constructs improves anti-thrombotic and anti-inflammatory responses for the past decades9. Therefore, endovascular coupler materials should allow for high levels of EC attachment and proliferation. Glass coverslips have been used as a reliable culture substrate in previous studies10-11. When HUVECs were seeded on the test polymer substrates, there was no significant difference in the attachment level compared to the glass control (Figure 2D), indicating the test materials are qualified to initiate endothelization of the construct. Proliferation



of HUVECs was also supported well in all the test groups as indicated by around or more than 300% increases from 1 to 4-day culture (Figure 2E).

In contrast, SMC attachment was not supported in the test polymer materials as much as the glass substrate (Figure 2D). The increased percentages of SMC proliferation from 1 to 4-day culture were ranged below 250%, which was less than those of HUVECs (Figure 2E). These results suggest that the test polyester types have promising potential to enhance endothelialization while mitigating the pathological behavior of SMCs. Interestingly, the proliferation levels of HUVECs in the test polymer groups were significantly lower than that of the glass control group. Moreover, SMC attachment in the test polymer groups was significantly lower than that of the glass control group while the proliferation levels of the test polymer groups were higher than that of the glass control group. However, the glass coverslip is not biodegradable and can’t be fabricated easily to produce an endovascular coupler, suggesting those polymer materials as a better choice for development of vascular anastomosis coupler.

Minimizing platelet adhesion is a key requirement for anti-thrombotic effects with successful performance of endovascular constructs. Hence, placement of endovascular anastomosis coupler or stent requires intake of anti-thrombotic drug. Although the dose and duration for systemic intaking of anti-thrombosis drugs have been successfully controlled, local setting to the endovascular implant serves as a better solution to reduce side effects such as stroke, indigestion, bleeding, diarrhea, etc. Among other methods tested so far12-13, bioactive heparin coating on the polymer surfaces is considered as a gold-standard treatment for the enhanced anti-thrombotic effect.


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Heparin inactivates blood clotting pathway14, because its negatively charged, hydrophilic property has non-fouling effects against protein and platelet adhesion15. Hence, anti-thrombotic heparin was immobilized to the surface of needle construct by the previously reported method16-18 (Figure 3A). A catecholic reaction has been widely employed19-20 because 3,4-dihydroxyphenylalanine (DOPA)-functionalized molecules can be immobilized on metals, ceramics, and polymers by a simple dipping method. A tyrosinase-triggered oxidative reaction is proven to overcome the limitations of DOPA system such as long reaction time (over 12 hours) and air oxidation-mediated loss of adhesiveness17-18. The catalyzation of tyrosinase enables the molecular conversion from phenol moiety to adhesive DOPA/DOPA-quinone within a short time (< 1 hour) while maintaining DOPA adhesiveness. Hence, heparin was immobilized to the coupler surface as shown previously17-18.

The amount of immobilized heparin increased as the heparin-tyramine (HT) concentration was increased from 0.1 to 0.25 and finally to 0.5 % in both concentration (left - 0.1 KU/mL and right0.4 KU/mL) of tyrosinase regardless of the reaction times (1 and 3 hours) (Figure 3B). These results indicate that the HT concentration is a major factor to increase the amount of immobilized heparin rather than the reaction time and tyrosinase concentration. The serial increases of immobilized heparin amount (0, 0.1, 0.2, 0.5 µg/cm2) results in significant reductions of platelet adhesion as shown in the SEM images (Figure 3C) and their quantified results (Figure 3D). Based on these results, 0.5 µg/cm2 of heparin was immobilized on the coupler to prepare samples for the follow-up studies.

The interaction between biomaterials and vascular cells has been studied widely. One study demonstrated that when heparin was coated onto the surface of allylamine by electrostatic 7 ACS Paragon Plus Environment


interaction, this surface significantly inhibited adhesion and proliferation of human umbilical artery smooth muscle cell (HUASMC), while enhancing adhesion, proliferation, migration and NO release of HUVECs21. Another study demonstrated that heparin-coated ePTFE vascular grafts supported attachment, growth and NO release of blood outgrowh ECs while reducing proliferation of vascular SMCs22. Interestingly, enhanced NO release of ECs was shown in both studies, indicating a potential mechanism related to the EC and SMC activities on the heparin-immobilized surface of needle coupler in the present study.

Since the needle shape is considered as one of the most important factors to determine the anastomosis efficiency, 45-degree angle towards an anti-tension direction and vertical equilateral triangle shape were generated and tested for the anastomosis efficiency in the end-to-end connection point of vessels. Microneedle constructs were produced to fit into the porcine carotid artery and then placed in a dynamic mechanical analyzer (DMA) as shown in Figure 4A. Continuous tension was loaded at a rate of 2 mm/min until slip happened between the anastomosed coupler and artery. As a result, the stress and strain curve exhibited that the needle shape with a 45-degree angle held the anastomosed vessels more tightly compared to the equilateral triangle shape (Figure 4B), as indicated by the higher stress value at any same strain point and longer break point. These results suggest that the 45 degree inclined needle shape as a promising design factor to improve the coupler performance.

The coupler with 45 degree inclined microneedles was inserted into the rabbit abdominal aorta as shown in Figure 4C. The anastomosis coupler was first inserted in the proximal vessel and then in the distal portion to complete the anastomosis. When the pulsed-waved Doppler ultrasonography (S22V, SonoScape Medical Corp.) was performed at the distal portion of the aorta, the vascular 8 ACS Paragon Plus Environment

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patency with pulsed blood flow was confirmed after anastomosis (Figure 4D). The microneedle traces of stent strut-like placement into the inner layer of rabbit abdominal aorta were observed from histological view of a cross-sectioned aorta (Figure 4E). When the anastomosis coupler was also applied to the anastomosis model of porcine carotid artery, the pulsing carotid artery with proper patency was observed (Movie S1 and S2). The average surgical time is reported as 20-60 min for one case of suture-based anastomosis but often increases when the workmanship of surgeon is not sufficient. When this needle coupler was used for these animal experiments, the average surgical time was significantly reduced to 5-10 min for one case, indicating a major advantage of using this suture-free coupler. These results suggest this device as a promising alternative to the current suture or coupler-based approaches.

In the future, since the coupler placement may affect a blood flow profile and result in inducing restenosis, the structure, wall thickness, and material properties of microneedle coupler will be optimized further. The high stiffness of needle coupler (Young’s modulus: 165 ± 37 MPa from N=3) is required for the needles to penetrate the vessel wall. Thus, deployment of stiff coupler may negatively influence blood vessel compliance due to interruption of vascular contraction, which is a weakness of this design to improve by further development. One idea currently considered is to generate different materials properties between the needle and coupler body through an ablation process. A soft material can be used to produce the coupler body so deployment of the coupler body does not interrupt vascular contraction while point ablation by exposing to femtosecond laser is used to generate stiff needles as shown in our previous studies 23-24. The microneedle shape will be also fine-tuned further to improve the coupler performance. Negative vascular remodeling may occur due to damages resulting from needle penetration into the vascular wall, which is difficult to determine in the current, short-term in vivo studies. Therefore, a long-term in vivo study of the 9 ACS Paragon Plus Environment


coupler after surgical implantation will be conducted to determine how long blood perfusion can be maintained in vivo and how the blood vessels will remodel around the coupler after the surgical procedure.

Conclusion: Forming microneedles on tubular constructs enables suture-free vascular anastomosis. The source polyesters are compatible in maintaining healthy phenotypes of vascular cells. Generating a 45-degree angle inclined towards the anti-tension direction in the needle shape more tightly anastomoses vessels compared to the equilateral triangle shape. The anti-thrombotic heparin coating significantly reduces attachment of platelets in vitro. In rabbit and porcine models, placement of needle constructs allows for blood perfusion, suggesting this device as a promising alternative to the current methods.


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Figure 1. Fabrication of micro-needled surface anastomosis coupler. (A) Schematic diagram of the anastomosis coupler: (i) placing micro-needles into the vascular tissue and (ii) anti-platelet adhesive surface modification. (B) Procedure to produce micro-needles with two different angles on the surface of anastomosis coupler: (i) equilateral triangle and (ii) 45° angle inclined towards the anti-tension direction shapes of micro-needle.



Figure 2. In vitro cellular responses to test polymeric substrates post 1 or 4 day-culture. Representative images of (A) cell viability and (B) morphology (F-actin) of human umbilical vein endothelial cell (HUVEC) and human aortic smooth muscle cell (SMC) on test material substrates (i.e. GLASS, PCL, PLA, and PCL/PLA mix) by live/dead and F-actin staining, respectively, with confocal imaging. Scale bar = 100 μm. (C) Circularities, (D) cell adhesion, and (E) cell proliferation of HUVEC and SMC cultured on the test polymer substrates. *p < 0.05.


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Figure 3. In vitro evaluation of heparin-immobilized coupler surface. (A) Schematic illustration of heparin-immobilization on the substrate surface by tyrosinase-catalyzed reaction. (B) Amounts of immobilized heparin with varying heparin-tyramine (HT) concentration (0.1-0.5%), tyrosinase concentration (left-0.1 kU/mL and right-0.4 kU/mL), and tyrosinase-reaction time (1 and 3 h). (C) Representative SEM images of adhered platelets on bare and heparinized substrates with (D) quantification. All results are shown as the average values ± S.D. (*p < 0.05 compared to bare, #p < 0.05 compared to 0.1 µg/cm2 heparin concentration group).



Figure 4. Ex vivo and in vivo evaluation of anastomosis efficiency. (A) Digital image of porcine carotid artery anastomosed by a microneedle coupler. Anastomosed porcine carotid artery with a coupler was loaded to DMA to measure tensile stress. (B) Strain-stress curves of arteries anastomosed by microneedle couplers with two different needle shapes (vertical- equilateral triangle and 45° angle inclined. The table results were obtained from 4 samples. Strain and stress were recorded until slip happened between loaded microneedle coupler and artery. (C) A gross image of microneedle anastomosis coupler placed into a rabbit abdominal aorta: i) The anastomosis coupler was first inserted in proximal ii) and then in the distal part of the vessel with iii) confirmation of vascular patency by pulsed-waved doppler ultrasonography. (D) Histological view of a cross-sectioned rabbit abdominal aorta post anastomosis by a microneedle coupler. Blue circles with magnified views (i and ii) indicate the representative sites of microneedle placement in the inner layer of rabbit abdominal aorta.


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Acknowledgements This study was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2016M3A9E9941743 and 2017M3A9E9087117).

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Table of contents Microneedle Vascular Couplers with Heparin -Immobilized Surface Improve Suture-Free Anastomosis Performance Dae-Hyun Kim, Jung Bok Lee, Mi-Lan Kang, Ji Hwan Park, Jin You, SeongMi Yu, Ju Young Park, Seung Bae Ryu, Gyeung Mi Seon, Jeong-Kee Yoon, Mi Hee Lee, Young Min Shin, Ki Dong Park, Jong‐Chul Park, Woo Soon Jang, Won Shik Kim, and Hak-Joon Sung


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Microneedle Vascular Couplers with Heparin-Immobilized Surface

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