Electronic Blood Vessel

Abstract

•Electronic blood vessel consists of biodegradable polymer and metal-polymer conductor•Electrical stimulation promotes HUVEC proliferation and migration in situ•Electroporation enables controlled gene delivery in different blood-vessel cell layers•Excellent compatibility in the vascular system and great patency in a rabbit model Existing small-diameter (<6 mm) tissue engineered blood vessels (TEBVs) rely mainly on the host remodeling progress and cannot provide further diagnostics or therapeutics in response to various pathological issues after implantation. Here we report an electronic blood vessel, with excellent biocompatibility, flexibility, mechanical strength, and degradability, that could enable in situ electrical stimulation (to facilitate the endothelialization process) and electroporation (to deliver genes in specific layers of blood-vessel cells) and exhibit excellent patency in a rabbit model. It endows electrical function on conventional biodegradable TEBVs and provides us with a new platform for tackling problems threatening the small-diameter blood vessel. When combined with emerging technologies such as artificial intelligence, it can greatly boost future personalized medicine by bridging the vascular tissue-machine interface and empowering health data collection/storage and early intervention. Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface. Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface. Cardiovascular diseases remain the number one cause of mortality worldwide.1WHOWorld Health Statistics 2019: Monitoring Health for the SDGs. World Heal. Organ., 2019Google Scholar In the treatment of cardiovascular diseases via coronary artery bypass grafting surgery, no existing small-diameter (<6 mm) tissue engineered blood vessel (TEBV) has met clinical demands.2Seifu D.G. Purnama A. Mequanint K. Mantovani D. Small-diameter vascular tissue engineering.Nat. Rev. Cardiol. 2013; 10: 410-421Crossref PubMed Scopus (312) Google Scholar To fabricate the TEBV, a range of approaches, such as decellularized matrix,3Quint C. Kondo Y. Manson R.J. Lawson J.H. Dardik A. Niklason L.E. Decellularized tissue-engineered blood vessel as an arterial conduit.Proc. Natl. Acad. Sci. U S A. 2011; 108: 9214-9219Crossref PubMed Scopus (262) Google Scholar, 4Dahl S.L. Kypson A.P. Lawson J.H. Blum J.L. Strader J.T. Li Y. Manson R.J. Tente W.E. DiBernardo L. Hensley M.T. et al.Readily available tissue-engineered vascular grafts.Sci. Transl. Med. 2011; 3: 68ra9Crossref PubMed Scopus (376) Google Scholar, 5Lawson J.H. Glickman M.H. Ilzecki M. Jakimowicz T. Jaroszynski A. Peden E.K. Pilgrim A.J. Prichard H.L. Guziewicz M. 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Specifically, a complex interplay between the blood flow and the TEBV can often cause inflammatory responses, resulting in thrombosis, neointimal hyperplasia, or smooth muscle cell accumulation near the scaffold,2Seifu D.G. Purnama A. Mequanint K. Mantovani D. Small-diameter vascular tissue engineering.Nat. Rev. Cardiol. 2013; 10: 410-421Crossref PubMed Scopus (312) Google Scholar,15Hoenig M.R. Campbell G.R. Rolfe B.E. Campbell J.H. Tissue-engineered blood vessels: alternative to autologous grafts?.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1128-1134Crossref PubMed Scopus (136) Google Scholar in different pathological stages. To address these issues, next-generation TEBVs should not only function as scaffolds to provide the mechanical support and facilitate host cell recruitments, but also have the capability to actively respond to and couple with the native remodeling process in order to provide adaptive treatments after implantation. Combining living tissues with flexible electronics16Yan J. Lu Y. Chen G. Yang M. Gu Z. Advances in liquid metals for biomedical applications.Chem. Soc. Rev. 2018; 47: 2518-2533Crossref PubMed Google Scholar,17Choi S. Han S.I. Kim D. Hyeon T. Kim D.H. High-performance stretchable conductive nanocomposites: materials, processes, and device applications.Chem. Soc. Rev. 2019; 48: 1566-1595Crossref PubMed Google Scholar could endow the conventional TEBV with more functionalities and capabilities to overcome existing biomedical problems, such as precision diagnostics, by sensing the blood flow and temperature in situ, and treatments by therapeutic drug or gene delivery.18Son D. Lee J. Lee D.J. Ghaffari R. Yun S. Kim S.J. Lee J.E. Cho H.R. Yoon S. Yang S. et al.Bioresorbable electronic stent integrated with therapeutic nanoparticles for endovascular diseases.ACS Nano. 2015; 9: 5937-5946Crossref PubMed Scopus (150) Google Scholar,19Du Z.J. Bi G.Q. Cui X.T. Electrically controlled neurochemical release from dual-layer conducting polymer films for precise modulation of neural network activity in rat barrel cortex.Adv. Funct. Mater. 2018; 28: 1-12Crossref Scopus (16) Google Scholar In previous work, we have developed many approaches to fabricate structures that mimic natural blood vessels with multiple types of blood-vessel cells in different layers, including the stress-induced self-rolling membrane13Cheng S. Jin Y. Wang N. Cao F. Zhang W. Bai W. Zheng W. Jiang X. Self-Adjusting, polymeric multilayered roll that can keep the shapes of the blood vessel scaffolds during biodegradation.Adv. Mater. 2017; 29: 1700171Crossref Scopus (74) Google Scholar,20Gong P. et al.A strategy for the construction of controlled, three-dimensional, multilayered, tissue-like structures.Adv. Funct. Mater. 2013; 23: 42-46Crossref Scopus (59) Google Scholar, 21Jin Y. Wang N. Yuan B. Sun J. Li M. Zheng W. Zhang W. Jiang X. Stress-induced self-assembly of complex three dimensional structures by elastic membranes.Small. 2013; 9: 2410-2414Crossref PubMed Scopus (18) Google Scholar, 22Yuan B. Jin Y. Sun Y. Wang D. Sun J. Wang Z. Zhang W. Jiang X. A strategy for depositing different types of cells in three dimensions to mimic tubular structures in tissues.Adv. Mater. 2012; 24: 890-896Crossref PubMed Scopus (189) Google Scholar and layer-by-layer techniques.23Wang N. Tang L. Zheng W. Peng Y. Cheng S. Lei Y. Zhang L. Hu B. Liu S. Zhang W. et al.A strategy for rapid and facile fabrication of controlled, layered blood vessel-like structures.RSC Adv. 2016; 6: 55054-55063Crossref Google Scholar Recently, we have developed a printable metal-polymer conductor (MPC), which exhibited excellent compatibility and high stretchability.24Tang L. Mou L. Zhang W. Jiang X. Large-scale fabrication of highly elastic conductors on a broad range of surfaces.ACS Appl. Mater. Inter. 2019; 11: 7138-7147Crossref PubMed Scopus (34) Google Scholar,25Tang L. Cheng S. Zhang L. Mi H. Mou L. Yang S. Huang Z. Shi X. Jiang X. Printable metal-polymer conductors for highly stretchable bio-devices.iScience. 2018; 4: 302-311Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Here, we develop an electronic blood vessel that integrates flexible electrodes into the biodegradable scaffold by combining liquid metal with poly(L-lactide-co-ϵ-caprolactone) (PLC) into an MPC. As a proof of concept, we used the electronic blood vessel to carry out in vitro electrical stimulation and electroporation. By electrical stimulation, the electronic blood vessel can effectively promote cell proliferation and migration in a wound healing model. It can also deliver a green fluorescent protein (GFP) DNA plasmid in situ into three kinds of blood-vessel cells via electroporation. We evaluated the efficacy and biosafety of the electronic blood vessel in the vascular system through a 3-month in vivo study by using a rabbit carotid artery replacement model and confirmed its patency through ultrasound imaging and arteriography. Our results pave the way to integrating flexible, degradable bioelectronics into the vascular system, which can serve as a platform to carry out further treatments, such as gene therapies, electrical stimulation, and electronically controlled drug release. We fabricated the electronic blood vessel (Figure 1A) by rolling up a PLC-based MPC (MPC-PLC) membrane (Figure 1B) with the assistance of a polytetrafluoroethylene (PTFE) mandrel. The MPC circuit was well distributed in the three-dimensional (3D) multilayered tubular structure. The inner diameter of the electronic blood vessel was around 2 mm (Figure 1A) and the minimum diameter could be around 0.5 mm (Figure S1A). The MPC-PLC membrane is flexible and degradable, and the MPC circuit is conductive (Figures 1B–1D). The conductivity of the MPC circuit is about 8 × 103 S cm−1 and the ΔR/R of the circuit remained constant after around 1,000 cycles of bending and rubbing (Figure 1C). The PLC is projected to be entirely degraded by around 1–2 years by the manufacturer. The MPC-PLC membrane underwent a mass loss of around 10% during an 8-week incubation in PBS (37°C) (Figure 1D). We observed a relatively quick degradation in the first week (Figure S2). To fabricate the MPC-PLC membrane, we screen-printed conductive ink on a polyethylene terephthalate (PET) membrane (Figure 1E). The electrode design was optimized for electroporation and electrical stimulation. By preparing different electrode designs, we could either target individual blood-vessel layers (tunica intima/media/adventitia) with the electrodes distributed in specific areas or target all three layers with the full electrode (Figures S1B and S1C). We prepared the liquid metal conductive ink by sonicating a mixture of gallium-indium alloy (EGAIn, ≥99.99%, Sigma, USA) and a volatile solvent (1-decanol, Macklin, Shanghai) (Figures 1F and 1H). The liquid metal particles (LMPs) exhibit a core-shell structure, where the core is the Ga-In alloy and the shell is the Ga-In oxide (Figure 1G). The diameter of the LMPs is around 2 μm (Figure 1J). We embedded the LMP-based circuit in the PLC solution (5 wt% in CH2Cl2) and peeled the MPC-PLC membrane off the PET substrate after evaporation of the solvent in a chemical hood (Figure 1E). The thickness of the MPC-PLC membrane is about 50 μm and it is tunable by changing the volume of the PLC solution. The LMPs could be broken during the process of peeling and release the Ga-In alloy to make the circuit conductive.25Tang L. Cheng S. Zhang L. Mi H. Mou L. Yang S. Huang Z. Shi X. Jiang X. Printable metal-polymer conductors for highly stretchable bio-devices.iScience. 2018; 4: 302-311Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar We confirmed the structure of the conductive circuit via corrosion of the Ga-In alloy by adding excessive hydrochloric acid (Beijing Chemical Works, China). The LMPs were evenly distributed in the cellular PLC host (Figures 1K and 1L). The liquid metal could form a coherent conductive pathway in the polymeric host. To evaluate the biocompatibility of the electronic blood vessel, we used microfluidic technology to realize an accurate 3D pattern of three kinds of blood-vessel cells in a natural blood-vessel mimicking fashion. By employing a multichannel microfluidic chip, we delivered human umbilical vein endothelial cells (HUVECs, blue), human aortic smooth muscle cells (SMCs, green), and human aortic fibroblasts (HAFs, red) sequentially on the MPC-PLC membrane (Figures 2A–2E). We designed the width of each channel to match the circumference of each layer of the tube based on the thickness of the MPC-PLC membrane and the diameter of the tube (Figure 2B). To distinguish different cell types, we stained the HUVECs, SMCs, and HAFs with different fluorescent dyes (HUVECs, CellTracker violet; SMCs, CellTracker green; HAFs, CellTracker deep red) before seeding them into the microfluidic chip. After 1-day incubation in culture medium (DMEM, 10% fetal bovine serum FBS, 37°C, 5% CO2), cells were attached to the MPC-PLC membrane (Figures 2D and 2H). We peeled the microfluidic chip off the cell-laden MPC-PLC membrane and rolled it up with a PTFE mandrel, forming a 3D multilayered tubular structure (Figures 2E–2G) with HUVECs, SMCs, and HAFs evenly distributed in the inner layer, middle layer, and outer layer, respectively (Figures 2I and 2J). This structure mimics well the structure of the natural blood vessel. To better understand the blood-vessel cells distributed in the different layers of the electronic blood vessel, we stained HUVECs, SMCs, HAFs, and the MPC-PLC layer with CellTracker DiO, CellTracker DiI, CellTracker DiD, and CellTracker blue to show the relative distribution of the different layers (Figure S3). We used a biomedical fibrin glue to facilitate the combination of the different layers.18Son D. Lee J. Lee D.J. Ghaffari R. Yun S. Kim S.J. Lee J.E. Cho H.R. Yoon S. Yang S. et al.Bioresorbable electronic stent integrated with therapeutic nanoparticles for endovascular diseases.ACS Nano. 2015; 9: 5937-5946Crossref PubMed Scopus (150) Google Scholar We incubated the cell-laden electronic blood vessel for 14 days and stained the cells with calcein-AM green. The evenly distributed green color on the MPC-PLC membrane indicated that the cells exhibited high viability after a 14-day culture (Figure 2K). We measured the transport of ions through the MPC-PLC membrane to further evaluate and quantify the permeability of the electronic blood vessel (Figure S4). Ca2+, Fe3+, and Mg2+ could permeate the electronic blood vessel over time. We also conducted a hemolysis test, which showed that the electronic blood vessel exhibited very good blood biocompatibility (Figure 2L). The in vitro characterization demonstrated that the electronic blood vessel exhibited excellent biocompatibility, and we then conducted further functional tests of the embedded MPC circuits, i.e., electrical stimulation and electroporation. To prove the functionality of the electronic blood vessel, we carried out in vitro electrical stimulation to improve proliferation and migration of HUVECs. A direct current (DC) electric field has been shown to effectively increase angiogenesis in vitro and in vivo.26Bai H. McCaig C.D. Forrester J.V. Zhao M. DC electric fields induce distinct preangiogenic responses in microvascular and macrovascular cells.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1234-1239Crossref PubMed Scopus (93) Google Scholar We patterned HUVECs on the MPC-PLC membrane (DMEM, 10% FBS, 37°C, 5% CO2) by using a multichamber polydimethylsiloxane (PDMS) chip. The initial number of cells in each chamber was the same (3 × 104). After 12-h incubation, we applied different DC voltages to yield different electrical field strengths: 25, 50, 75, 100, 200, and 400 mV mm−1 (Figures 3A and 3B ). After 2-day incubation and electrical stimulation, we randomly selected six different domains on each sample and analyzed them by using laser scanning confocal microscopy (LSM 710, Zeiss, Germany). We stained nuclei (blue) with Hoechst 33342 (Invitrogen, USA) and stained the living and dead cells with calcein-AM (green) and propidium iodide (PI) (red), respectively. The green cells occupied the whole domain, indicating that electrical stimulation did not hurt the proliferation of HUVECs (Figure 3C). We counted the nuclei using ImageJ. The cell number under 50 mV mm−1 was highest, about 2.4 times that of the control (Figure 3E). We used the CCK-8 kit to confirm this conclusion (Figure 3F). We speculated that the DC electric field had selectively regulated the production of certain growth factors and cytokines important for angiogenesis. We explored the migration of HUVECs under different DC electric field strengths. We made a scratch on a PDMS substrate using a 10-μL tip. After application of an electric field of 50 mV mm−1, HUVECs migrated 750 μm and the wound completely healed after 24 h (Figure 3D). Different strengths enhanced migration differently compared with the control group without electrical stimulation (Figure 3G). The in vitro DC electrical stimulation thus proved to have effectively promoted the proliferation and migration of HUVECs. We further evaluated the effectiveness of electrical stimulation in a 3D model of endothelialization. We patterned the HUVECs on the MPC-PLC membrane and made a scratch on the cell-laden membrane using a 10-μL tip. We transformed the 2D cell-laden membrane into a 3D cell-laden structure and connected it to the electrochemical station to test the proliferation and migration. We applied an electric field of 50 mV mm−1 on multiple samples and we observed the proliferation and migration at different time points. We stained the living and dead cells with calcein-AM (green) and PI (red). We counted the cells and the cell density was higher than that of the control group (Figure 3H). The HUVECs formed a complete endothelial layer after 24 h (Figure 3I). To better evaluate the biocompatibility of the MPC circuit under electrical stimulation, we extended the electrical stimulation time to 10 days, and the live/dead staining showed that cells exhibited excellent viability (Figure S5). To further prove the functionality of the electronic blood vessel, we designed multiple circuit patterns for electroporation, being able to target different pathological issues in different layers of blood-vessel cells (Figure S1B). We conducted the electroporation with a circuit pattern that could target all three layers. We seeded the cells onto the MPC-PLC membrane and transformed it into a 3D tubular structure for electroporation. We immersed the 3D cell-laden electronic blood vessel in the GFP plasmid DNA solution for 10 min before electroporation. The GFP plasmid DNA could also be lyophilized onto the MPC-PLC membrane before seeding cells (Figure S7A) and transforming to the 3D structure for electroporation. We connected the 3D cell-laden electronic blood vessel to an electroporator (BTX, CM630, US) to generate DC pulses (Figure 4A) and achieved delivery of the GFP DNA plasmid in the three kinds of blood-vessel cells (Figure 4B). To optimize the performance of the electronic blood vessel, we found two major parameters determining the efficacy of electroporation, including voltage and pulse duration. We conducted the electroporation with different voltages (40/60/80 V) and pulse durations (100 μs/1 ms). If the voltage was too low, it would cause low efficacy or no transfection; if the voltage was too high or if the pulse duration was too long, it would cause low efficacy and cell death (Figure S6). To realize the optimal efficacy, we exerted a square wave with a voltage of 60 V, pulse duration of 100 μs, and pulse interval of 1 s for five pulses. We delivered the GFP plasmid DNA into three kinds of blood-vessel cells and the GFP DNA realized expression with more than 95% of cells showing green fluorescence (Figure 4B). We observed successful expression of GFP among all three layers of the blood-vessel cells and they exhibited a uniform 3D distribution in the electronic blood vessel (Figure 4C). To evaluate the potential of the electronic blood vessel for in vivo electroporation, we lyophilized the GFP plasmid DNA on the MPC-PLC membrane to test its effectiveness (Figure S7A). We carried out electroporation by attaching the plasmid-laden MPC-PLC membrane to an isolated rabbit vascular tissue with a voltage of 60 V, pulse duration of 100 μs, and pulse interval of 1 s for five pulses. We observed successful expression of GFP in the isolated rabbit vascular tissue (Figure S7B) after a 2-day incubation. These promising in vitro results encouraged us to carry out in vivo tests on the electronic blood vessel. To find out whether the electronic blood vessel is suitable for in vivo implantation, we measured the mechanical properties, including stress-strain curve, compliance, and burst pressure, of the electronic blood vessel with a diameter of 2 mm prior to implantation (Figures 5A–5F and S8). The elastic modulus of the electronic blood vessel is about 130 MPa; this value is much higher than that of the native carotid artery. The ultimate tensile strength of the electronic blood vessel is about 27 MPa; this value is also much higher than that of the native carotid artery. The initial compliance of the electronic blood vessel (n = 5) is about 5% per 100 mm Hg in the range of 80–120 mm Hg; this value is apparently below that of the native carotid artery (n = 3). The burst pressure of the electronic blood vessel (n = 5) is about 2,800 mm Hg; this value is similar to that of the native carotid artery (n = 3). The elongation at break of the electronic blood vessel is about 650% (n = 5), which is twice the value of the native carotid artery (n = 3). The mechanical properties of the electronic blood vessel were considered robust enough for implantation. To investigate the electronic blood vessel as a vascular implant, we chose the New Zealand rabbit (age, 200–300 days; body weight, 3–4 kg) as the animal model and replaced the native carotid artery with the electronic blood vessel (Figures 6A–6C ). To avoid possible immunological response of the host tissue, we used the acellular electronic blood vessel in the preliminary in vivo study. We monitored the implanted electronic blood vessel in situ by Doppler ultrasound imaging (Figures 6D–6I) and arteriography (Figures 6J and 6K). Doppler ultrasound imaging showed that the electronic blood vessel allowed for good blood flow 3 months post-implantation (Figures 6D–6G and Video S1). The asymmetric velocity curve synchronized with the ultrasonic pulses indicated that the signal is from the carotid artery rather than the vein (Figure 6D). The diameter of the electronic blood vessel remained at a relatively constant value, about 2.3 mm, during the half-month to 3 months post-implantation (Figures 6G and 6H). The mean velocity of the blood flow in different samples at different time points was about 0.47 m s−1, which was in the range of the normal value (Figure 6I). As the gold standard of the blood-vessel patency, arteriography showed that the electronic blood vessel matched the native carotid artery very well and allowed for excellent blood flow (Figures 6J, 6K, and S9, and Video S2). There was no sign of narrowing. The electronic blood vessel allows straightforward visualization under arteriography because the liquid metal-based circuitry has sufficiently high contrast over host tissues (Figure 6J). The red frame in the figure outlines the electronic blood vessel with an alternate strip structure from the MPC circuit. https://www.cell.com/cms/asset/dd90f582-ce4c-4e88-bdab-3fa9cd2c8282/mmc2.mp4Loading ... Download .mp4 (2.31 MB) Help with .mp4 files Video S1. Doppler Ultrasound Imaging 3 Months Post-implantationThe electronic blood vessel allowed for good blood flow. https://www.cell.com/cms/asset/2df4a978-5dba-4007-b896-07305db7ce00/mmc3.mp4Loading ... Download .mp4 (1.9 MB) Help with .mp4 files Video S2. Arteriography 3 Months Post-implantationThe electronic blood vessel matched with the native carotid artery very well and allowed for excellent blood flow. We dissected all the implanted electronic blood vessels 3 months post-implantation for characterization. The lumen and the outer surfaces of the explanted electronic blood vessel were smooth, covered by the remodeling tissues (Figures 7A and 7B ). The diameter of the native blood vessel was significantly reduced due to the lack of blood pressure, whereas the electronic blood vessel remained the same as before (Figure 7C). We observed the microstructure of the circuit by scanning electron microscopy (SEM) after explanting the electronic blood vessel from the rabbit. The MPC-PLC membrane still maintained interdigitated structure with the MPC circuit and PLC host (Figure 7E). There was a layer of neo-tissue formed, which well covered the MPC-PLC membrane (Figures 7F–7H). We could also see some red blood cells on the top of the circuits, which were similar in number to those on native blood vessels (Figure 7D). We tested the conductivity of the circuit in the electronic blood vessel. The MPC circuit was still conductive and the conductivity was around 7.2 × 103 S cm−1. To study the histological changes in the implanted electronic blood vessels, we performed histological staining of the electronic blood vessels, setting the native carotid blood vessel as a positive control. H&E staining (Figure 7I) of the cross section of the electronic blood vessel showed it as round shaped, continuous, and red, which is similar to the native blood vessel. The three-layered MPC-PLC membrane merged into one intact layer with secretion of a substantial extracellular matrix between the different layers of electronic blood vessel. We could clearly see dark blue nuclei (red arrows in Figure 7I) in all the layers, which indicated successful migration and infiltration of host cells into the electronic blood vessel. We compared the cell density in the electronic blood vessel with the native carotid. The cell density of the electronic blood vessel was around 400 cells mm−2, whereas that of the native carotid was around 535 cells mm−2. More importantly, a dense layer of cells with curved structure was formed in the lumen of the electronic blood vessel, which indicated the excellent endothelialization and thus ensured good blood flow. To further confirm the components in the implanted electronic blood vessel, we performed Masson's trichrome (Figure 7J) and Verhoeff's staining (Figure 7K). Masson's trichrome can stain and assess keratin and muscle fibers (red) or collagen (blue) and Verhoeff's can stain and assess the presence of elastin fibers. Masson's trichrome and Verhoeff's staining showed well-distributed collagen and elastin fibers both inside the layers and between different layers, indicating appropriate remodeling. Compared with the abundance level of extracellular matrix in the native blood vessel, the electronic blood vessel still requires more time for material degradation and tissue remodeling. These results indicated that the electronic blood vessel might still be functioning in the presence of conductive materials and host remodeling 3 months post-implantation. To investigate the influence of the electronic blood vessel on the host, we performed cross-sectional and histological staining of the major organs, including heart, liver, spleen, lung, and kidney, together with dissection of the implanted electronic blood vessel 3 months post-implantation. The H&E staining and Masson's Chrome staining showed that there were no significant pathological changes or inflammatory responses in these organs (Figure S10). To evaluate whether there was chronic inflammation or infection, we conducted ELISA on three important proteins in the blood, comprising interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) (Figure S11). The concentrations of IL-6 and PCT were not higher than the normal value of healthy rabbits (red dotted lines in the figure). The concentration of CRP was higher than the normal value of healthy rabbits (red dotted line). All three indexes decreased over time, a tendency that was expected. The results showed that most of the indexes were in the normal range and there were no significant pathological changes or inflammatory responses. We also conducted a complete blood count of the rabbit, including white blood cell count, absolute neutrophil count, and absolute lymphocyte count, most of which were in the normal range over time (Figure S12). These results confirmed that as an implant for vascular system, the electronic blood vessel had no significant detriment to the host. None of the existing small-diameter TEBVs has met the demands of treating cardiovascular diseases. Conventional TEBVs can be greatly improved to provide next-generation treatment by integrating with flexible bioelectronics. In this work, we report an electronic blood vessel with excellent biocompatibility, flexibility, mechanical strength, and degradability by combining MPC with a US FDA-approved biodegradable polymer. As a proof of concept, we verified that the electronic blood vessel can accelerate HUVEC proliferation and migration by electrical stimulation, thus facilitating the endothelialization process, which is important to an engineered vascular conduit in preventing early thrombosis. Because most TEBVs occluded by 2 weeks post-implantation, our electronic blood vessel, with a patency of at least 12 weeks, is of great promise for clinical application. We also showcase that it could be used to perform in situ gene delivery via electroporation, which laid the foundation for future design and optimization such that we can carry out further gene therapies targeting different pathological problems after implantation. The electronic blood vessel has a high level of safety. The liquid metal16Yan J. Lu Y. Chen G. Yang M. Gu Z. Advances in liquid metals for biomedical applications.Chem. Soc. Rev. 2018; 47: 2518-2533Crossref PubMed Google Scholar,27Lu Y. Hu Q. Lin Y. Pacardo D.B. Wang C. Sun W. Ligler F.S. Dickey M.D. Gu Z. Transformable liquid-metal nanomedicine.Nat. Commun. 2015; 6: 10066Crossref PubMed Scopus (306) Google Scholar has been proven to be highly biocompatible and the PLC has been approved by the US FDA for implants.28Kenar H. Ozdogan C.Y. Dumlu C. Doger E. Kose G.T. Hasirci V. Microfibrous scaffolds from poly(L-lactide-co-ϵ-caprolactone) blended with xeno-free collagen/hyaluronic acid for improvement of vascularization in tissue engineering applications.Mater. Sci. Eng. C. 2019; 97: 31-44Crossref PubMed Scopus (29) Google Scholar We validated its biosafety in the vascular system by a 3-month implantation in a rabbit carotid artery model. Both the in situ monitoring, including Doppler ultrasound imaging and arteriography, and the ex vivo study demonstrated that it was safe as an electronic implant in the vascular system and at the body level. The electronic blood vessel exhibited higher strength than the native blood vessel. The potential harm of a rigid synthetic blood vessel is the mismatch with host tissue after implantation. However, from the in vivo results, these discrepancies did not bring about any major issues, and the electronic blood vessel matched very well with the host carotid artery during the time of in situ monitoring (Figures 6J and 6K, Video S2). The reasons we chose liquid metal in the electronic blood vessel are as follows: (1) compared with gold or platinum, the Ga-In liquid metal allows superior flexibility and stretchability while maintaining good conductivity, which is critical for an artificial blood vessel to adapt to the deformation due to rhythmic beating; (2) it exhibits excellent cytocompatibility and blood compatibility according to our results; and (3) compared with other sophisticated microfabrication techniques, using the screen-printing technique is much more straightforward and could enable industrial-scale mass production in a cost-effective manner. As a vascular substitute, the electronic blood vessel breaks through the limitations of the existing vascular scaffold by endowing the electrical function on a conventional biodegradable TEBV and provides us a new platform for tackling the problems threatening the small-diameter blood vessel. By integrating with other electronic devices, the electronic blood vessel can provide various treatments, such as electrical stimulation, electroporation, electrically controlled drug release, and so forth. When combined with emerging technologies such as artificial intelligence, it can greatly boost future personalized medicine by bridging the vascular tissue-machine interface and empowering health data collection and storage, such as blood velocity, blood pressure, and blood glucose level. In the future, optimizing its function and creating a multifunctional electronic blood vessel can greatly benefit human cardiovascular health. In this work, by integrating liquid metal-based conducting circuitry with a biodegradable polymer, we develop an electronic blood vessel, with excellent biocompatibility, flexibility, conductivity, mechanical strength, and degradability, that enables in situ electrical stimulation to facilitate the endothelialization process and electroporation to deliver genes into specific layers of blood-vessel cells. It exhibited excellent patency and biosafety 3 months post-implantation in the vascular system of a rabbit model. In the future, the electronic blood vessel can be integrated with other electronic components and devices to enable diagnostic and therapeutic functions and greatly empower personalized medicine by creating a direct link in the vascular tissue-machine interface.