In this study, we attempted the direct construction of biomimetic TEBVs composed of compartmentalized endothelial and muscular layers using vascular-tissue-specific bioinks and a reservoir-assisted triple-coaxial cell printing (RTCCP) technique [and]. We designed a remodeling process to simultaneously modulate the cellular activities of ECs and VSMCs localized in fabricated TEBVs, leading to the formation of a matured endothelium and dense smooth muscles. We also investigated the performance of prematured TEBVsusing a proof-of-concept rat model. Lumen patency, tissue maturation, and host tissue integration were monitored over a period of 3 weeks []. Our directly cell-printed TEBVs show the potential to become an appealing alternative option for small-diameter vascular graft construction.

To successfully cell-print a functional TEBV, it is crucial to select cell-favorable bioinks that could both promote the cell functionality and enable the direct fabrication of vessels. While the hydrogels applied to engineer vascular grafts (e.g., fibrin,elastin,collagen,silk fibroin) generally have poor printability,the materials suitable for coaxial printing (e.g., alginate, gelatin methacryloyl, photocurable hyaluronic) usually result in cell behaviors that are limited in generating functional tissues due to the low cellular affinity or inappropriate moduli.In our previous report, we developed a vascular-tissue-specific bioink composed of vascular-tissue-derived extracellular matrix (VdECM) and alginate that not only stimulate the cellular functions of ECs by providing a tissue-specific microenvironment but also facilitate the fabrication of endothelialized tubes.However, its potential for constructing small-diameter vascular substitutes with both endothelial and muscular tissues has not yet been demonstrated.

An important advantage of 3D cell printing is its extraordinary ability to construct tissue/organ equivalents. By precisely positioning cell-laden bioinks to emulate the anatomical characteristics of the target tissue/organ, biomimetic constructs can be created as the localized cells proliferate, migrate, and differentiate for tissue morphogenesis.In particular, the coaxial-extrusion technique can print multiple materials through a concentrically assembled nozzle, resulting in the vessel-like structures. This distinct advantage has provoked extensive interest in applying this method to fabricate perfusable tubular filaments to better understand cell-cell interactions and vascular pathophysiology.However, no study has successfully constructed vascular equivalents to small-diameter blood vessel grafts.

Tissue engineering has emerged as a promising approach to viable alternative small-diameter arterial grafts. Diverse strategies, including decellularized tissue,synthetic polymer scaffolds,self-assembling cell-sheets,and hydrogel mold-castinghave been utilized to create living conduits to replace native blood vessels; these strategies have achieved varying levels of success. However, these approaches generally produce vessel analogs by constructing a robust tubular matrix containing vascular smooth muscle cells (VSMCs) or fibroblasts followed by the seeding and cultivation of endothelial cells (ECs) to achieve endothelialization. This slow and multistep construction process requires complicated procedures and long fabrication times.In addition, the seeding efficacy and cell retention rate of ECs are highly influenced by the seeding approach, surface properties, cell density, and culture conditions,and thus current methods can hardly be considered reliable for the generation of intact functional endothelial tissues. Therefore, the reconstruction of biomimetic multiple-layered tissue-engineered blood vessels (TEBVs) remains a challenging task that demands new construction techniques.

Cardiovascular disease is one of the leading causes of mortality worldwide and requires over one million vascular bypass/replacement surgeries annually in the United States alone.Although autologous vessels are the gold standard for clinical use, they are unsuitable for a large number of patients because of vascular disease, amputation, or previous harvest.Despite the clear clinical need for vascular substitutes, currently available synthetic grafts (e.g., Dacron and Teflon) have only proved successful in large-caliber vessel implantations; in contrast, small-diameter (i.e., <6 mm) vascular substitutes have yielded disappointing results overall, with major negative side effects such as acute thrombosis, hyperplasia, and aneurysm.Graft failures are generally attributed to the relatively low blood flow velocity in these small-caliber vessels, which increases the rate of irregular interactions between the blood and the polymeric implants.Therefore, there are stringent requirements regarding the biofunctionality of designed small-diameter grafts. To produce grafts with regular vascular functions, reconstruction of two critical constituents must be achieved, namely: (1) a confluent and quiescent endothelium offering a nonthrombogenic interface to inhibit thrombosis, and (2) contractile smooth muscle tissues that can withstand hemodynamic stress, exhibit physiological compliance, and adapt to local blood pressure changes via constriction and relaxation.

Staining against human α-SMA (H-α-SMA) showed that the HAoSMCs maintained a circumferential orientation and elongated morphologies []. In addition, a large proportion of cells were found to be positive to Ki67 (37.79 ± 6.91%) [], a cell proliferation marker, indicating that the cells actively remodeled the implanted TEBVs. Smooth muscle tissue maturation was evaluated by Masson's Trichrome staining. Compared to native rat aorta [], although matured muscle fibers were absent, abundant consecutively stratified collagen was observed in the 3-week-old implants []. To investigate whether ECM remodeling was due to the cellular activity of HAoSMCs, immunofluorescence staining against several human proteins was conducted. The results of elastin staining clearly visualized elastin fibers in the native rat aorta []. However, although abundant human-derived elastin was found in the explanted TEBVs [], no assembled fibrous structures were generated, consistent with the results of Masson's Trichrome staining. On the other hand, the observed collagen fibers in TEBVs were analogous to those of the native rat aorta [] and positive to H-COL-I, demonstrating that HAoSMCs participated in ECM synthesis and remodeling duringconditioning []. Moreover, the presence of rat-cell-positive collagen (negative to human cells) at the adventitial side suggested the migration of host cells, implying the initiation of tissue integration. The staining of longitudinally sectioned samples further indicated that the host cells migrating toward the TEBVs were positive to vimentin but negative to α-SMA, which is identical to the observed staining of native rat fibroblasts (Fig. S8). This suggested that the early integration of TEBV with host tissues was led by the migration of rat fibroblasts, which formed a layer of connective tissue on the external surface of the implants. Collectively, these outcomes demonstrated the remarkableperformance of the TEBVs as a small-diameter vascular graft. They not only provided a matured endothelium that could avoid acute thrombosis but were also actively remodeled to facilitate tissue maturation and integration. Hence, we anticipate that the developed TEBVs have the potential to transform to natural functional blood vessels.

The harvested TEBVs were stained via immunohistochemistry to evaluate theirmaturation. Hematoxylin and eosin (H&E) staining revealed that the lumens of the TEBVs explanted at both 1 and 3 weeks postimplantation were similar to that of the native abdominal aorta []. This result was consistent with the ultrasonographic data and further confirmed the patency of the TEBVs. The absence of acute thrombosis may have been due to the presence of a matured endothelium in the implanted TEBVs. To investigate the preservation of endothelium, the samples explanted after 3 weeks were sectioned at their proximal, middle, and distal regions for staining against human CD31 (H-CD31). The results showed an intact single layer of HUVECs lining the lumen of the TEBVs [], which suggested that the endothelium of the TEBVs could resist the deprivation induced by the shearing forces of blood flow.

Ultrasonography was used to monitor the condition of TEBVs and predict the incidence of thrombosis. B-mode images identified a clear lumen in all the implanted TEBVs, indicating the great patency rate of the implants (6/6) during 3 weeks []. In addition, color Doppler flow mapping showed that blood was flowing homogeneously at a constant velocity and filled the lumen of the TEBVs [and supplementary material , Video 6]. Moreover, the blood flow spectrum, detected by pulse-Doppler wave examination, indicated periodic pulses of a tall-sharp systolic peak followed by a low-flat end diastolic velocity, consistent with the regular velocity spectrum of arterial flow []. Furthermore, duringexamination, the measured peak systolic velocity, which is an indicator of stenosis diagnosis,showed no significant differences between the implanted TEBVs and native abdominal aorta (sham control, n = 3) []. These outcomes implied the absence of blood clotting or significant stenosis during the observation period.

To investigate theperformance of the cell-printed TEBVs, a proof-of-concept study was conducted using a Sprague Dawley rat model. Prematured TEBVs (ID: 2 mm, length: 10 mm) with similar dimensions to those of the rat abdominal aorta (ID: 1.5–1.8 mm) were implanted as interposition grafts []. Although the mechanical strength of the TEBVs was inferior to that of native arteries, the end-to-end anastomosis was successfully achieved using from 6 to 8 circumferential stitches. In addition, the implanted vessel was able to withstand arterial blood pressure after the removal of the clamps []. However, accidental ruptures in the TEBVs were observed within 24 h postsurgery, caused by suboptimal elastic modulus and BP values. To prevent these unexpected ruptures, a polycaprolactone (PCL) sheath was wrapped around the TEBVs [and supplementary material , Video 5]. Using this approach, the TEBVs were successfully implanted into six of the rat models and harvested at 1 or 3 weeks (n = 3) for subsequent investigations. To avoid the immune response, immunosuppressants were administrated daily by intraperitoneal injection as a standard regimen of immunosuppression.

To evaluate the effects of dynamic conditioning on increasing the strength of the TEBVs, we assessed two essential mechanical properties of blood vessels: ultimate tensile strength (UTS) and burst pressure (BP). The TEBVs that were statically cultured (no pulsatile perfusion) for an identical remodeling period were used as a control. The UTS of TEBVs that underwent dynamic stimulation was increased (195 ± 43 kPa, n = 5) by approximately 4-fold compared to that of the statically cultivated samples (47 ± 19 kPa, n = 5) []. Similarly, the static culture resulted in a low BP of 63 ± 11 mmHg (n = 5) apparently lower than the healthy diastolic pressure in the human body (80 mmHg);in contrast, the pulsatile conditioning substantially increased the BP of TEBV to 174 ± 55 mmHg (n = 5) []. Although the prematured TEBVs showed a lower strength than the native vessels (Sprague Dawley rat abdominal aorta; UTS: 1212 ± 348 kPa, BP: 897 ± 235 mmHg), the currentremodeling process markedly enhanced the strength of the cell-printed vessels. Collectively, we demonstrated that the designed remodeling process not only improved cell alignment, ECM deposition, and tissue generation but also significantly promoted the mechanical properties of the TEBVs.

In addition to its biological responses, the TEBVs exhibited significant physical changes in terms of dimensions and mechanical strength during the remodeling process. Owing to the spreading and migration of cells, the hydrogel matrix underwent severe contraction. The WT of vessels drastically dropped at day 4 (62.81 ± 7.34%) followed by slight reductions at day 18 (53.98 ± 3.29%) compared to their initial values after fabrication, resulting in dimensionally stabilized TEBVs with ID of 1.98 ± 0.05 mm and WT of 0.53 ± 0.16 mm (n = 5) []. The length of the vessels showed a relatively mild reduction from 39.88 ± 2.57 mm to 32.76 ± 1.59 mm at day 4 (82.15 ± 3.98%) []. These structural compactions are beneficial to produce dense robust tissues in hydrogel-based constructs.

We further evaluated the changes in ECM secretion and tissue generation in the prematured TEBVs during the remodeling process. In the endothelial layer, the expression of VE-cadherin was detected on the luminal wall of the TEBVs [], representing the formation of adhesion junctions between HUVECs [inset of], a hallmark of endothelium maturation.In addition, the deposition of human laminin (H-Laminin) on the basolateral side of the endothelium was detected [], indicating that the HUVECs actively synthesized and localized structural protein components of the basement membrane.On the other hand, the staining against human elastin (H-Elastin) and human type I collagen (H-COL-I) demonstrated thesecretion and deposition of human ECM components from HAoSMCs [], which is essential for enhancing the mechanical properties of TEBVs.

Despite the reorganization of the cell alignments, the compartmentalization of the endothelial and muscular layer was clearly retained over the cultivation period. These results could be attributed to the design of the bioink formulations and the remodeling process. Due to their limited cellular activities in the 3V2A bioink matrix, the HAoSMCs could not migrate into the endothelial layer; rather, they became circumferentially oriented in the muscular layer as a response to the given cyclical radial strain. Moreover, because of the medium supplied through the perforations on the mounting needles, the HUVECs were protected against hypoxia during the mounting stage, resulting in regular cell spreading. Through the perfusion of EGM-2 medium in the subsequent dynamic conditioning process, these cells migrated toward the luminal surface, where essential growth factors for ECs (e.g., vascular endothelial growth factor) were located, leading to the formation of endothelium.

On the other hand, the α-SMA staining results revealed that the HAoSMCs exhibited a random stretch after 4 days of static culture []. However, over the following 2 weeks of pulsatile stimulation, circumferentially oriented cells with an elongated morphology were observed to be aligned following the curve of the vessel wall [], analogous to its native counterparts []. Moreover, the contractile function of HAoSMCs was examined by increasing the cytosolic free Caconcentration using the stimulation of a high Kconcentration, a widely used approach for testing the contractile ability of VSMCs.The samples (segments of prematured TEBV) activated by Kshowed size changes (76.05 ± 6.43% of the original) in width as a result of HAoSMCs contraction ( supplementary material , Video 4), while those were treated with K-free physiological salt solution maintained the dimension at 97.68 ± 1.64% of the original (Fig. S7). These results demonstrated that the cell-printed HAoSMCs exhibited cellular function to support the contractile property of the constructed TEBVs.

The cell-printed TEBVs underwent significant biological and physical changes during the course ofremodeling. First of all, the dynamic conditioning effectively regulated cellular alignments of both HUVECs and HAoSMCs, leading them to resemble their native counterparts. Immunofluorescence staining of CD31 visualized the transition of HUVECs from local spread in the printed EC layer [] to the formation of an intact monolayer on the luminal wall of the TEBVs [], which is comparable to that of native blood vessels (Sprague Dawley rat abdominal aorta) []. In addition, the detected expression of ZO-1 (Fig. S5), a marker of tight junction, indicated the maturation of endothelium on the cell-printed TEBV. Moreover, based on the given pulsatile perfusion signals, the SEM images indicated that the endothelial cells showed elongated morphology and were oriented in the direction of flow (Fig. S6). These representative responses toward shear stress demonstrated the regular activity and response of endothelium generated on the TEBVs.

Although the dual-layered vessels were successfully constructed using RTCCP, cell-laden structures in this form would be unsuitable for use as vascular grafts because the tissues are immature tissues and the mechanical strength is very low. Therefore, we designed anremodeling process to organize the alignment of the cells, stimulate deposition of ECM, and reinforce the mechanical properties of the vessels []. To achieve this, the fabricated vessels were maintained at 37 °C for 24 h after printing to completely crosslink the collagen components in VdECM [Fig. S4(a)]. To prevent changes in ID and tube deformation caused by hydrogel contraction, the vessels were subsequently mounted on 12G needles (50 mm in length). Due to the large WT of the printed vessels (approximately 1 mm), the mounting needles were manufactured with perforated holes [50 holes (600m) per needle] so that the vessel can be fed the medium through the luminal side to avoid hypoxia in the HUVECs [Fig. S4(b)]. After a static culture period of 3 days, hydrogel shrinkage was stabilized, and the vessels became sufficiently strong enough to be handled ( supplementary material , Video 2). It allowed us to apply a self-designed bioreactor to stimulate the vessels with radial strain through pulsatile perfusion (frequency: 0.5 Hz; flow rate: 200 ml/h) for 2 weeks [ supplementary material , Video 3 and Fig. S4(c)]. During the dynamic culture stage, endothelial growth medium-2 (EGM-2) was pumped in the vessels, and the chamber of the bioreactor was filled with smooth muscle cell growth medium-2 (SMCGM-2) to separately support the growth of HUVECs and HAoSMCs.

To overcome this obstacle, we applied a reservoir containing 100 mM CaClsolution to additionally treat the bioink, which is difficult for the Careleased from CPF127 to crosslink, thus increasing the WT of printed vessels []. In this study, a printed blood vessel with the ID of 2 mm and the WT of 1 mm [], dimensions similar to those of the human coronary artery, was selected for subsequent experiments. To fabricate this construct, a 12G/9G/7G triple coaxial nozzle was applied to print CPF127, HUVEC-laden, and HAoSMC-laden bioinks at a pneumatic pressure of 70 kPa, 15 kPa, and 30 kPa, respectively. The resultant tube possessed a thin layer (approximately 50m) of HUVECs surrounded by a thick layer (800m–1000m) of HAoSMCs; it was visualized by staining against CD31 and α-SMA []. More importantly, this fabrication strategy allowed us to print vascular equivalents with tunable WT and ID by adjusting the bioink flow rate and nozzle gauge (Table S1). For instance, due to the presence of the CaClreservoir, the WT of vessels printed by an 18G/14G/10G triple coaxial nozzle was increased from 200m to 800m as the flow rate of 3V0.5A bioinks in the shell needle increased by raising the extrusion pressure from 20 kPa to 40 kPa []. On the other hand, enlarging the gauge of the core needle helped to effectively increase the ID of the printed vessels []. The 18G/14G/10G, 15G/12G/9G, and 12G/9G/7G nozzles produced dual-layered conduits with similar WT (600–800m) but varied ID (approximately 900m, 1500m, and 2000m, respectively).

Despite the successful printing of the dual-layered tubes, the control of essential dimensions, such as inner diameter (ID) and wall thickness (WT), should be considered to fully mimic the structure of native blood vessels. However, because the bioinks were crosslinked by diffused Caions, a dense alginate fiber network was formed, limiting the further penetration of Caions and resulting in a maximum WT of approximately 200m. In contrast, native human vessels with small diameters (e.g., coronary artery, internal mammary artery) usually possess a greater WT (from 500m to 1000m) with sufficient strength to withstand blood pressure.Therefore, it is critical to fabricate the vascular equivalents with a thick muscular layer.

To print a biomimetic vascular construct with multiple concentric layers, the HUVEC- and HAoSMC-laden bioinks were added to the middle and shell needles, respectively, of a triple-coaxial-nozzle (18G/14G/10G), while Pluronic F127 containing Ca(CPF127) was extruded through the core nozzle to support vessel fabrication [, and supplementary material , Video 1]. The staining of vascular endothelial (VE)-cadherin (an intercellular junction protein between ECs) and α-smooth muscle actin (α-SMA; an actin isoform represent contractility of VSMCs) revealed that the coaxially printed conduits underwent successful endothelialization (96.42 ± 2.13% luminal surface coverage) [] and muscularization [] within 7 days. The codispensation of these two cell-laden bioinks enabled the construction of a dual-layered vessel composed of an endothelial and a muscular layer []. More importantly, its compartmentalized architecture was well-preserved and the related tissues, including endothelium and musclelike stratum, were locally generated after a culture period of 7 days.

We modified the vascular tissue-specific bioink, an essential element for the construction of a functional vascular substitute, to promote the activity of VSMCs based on a previously developed bioink recipe [3% (w/v) VdECM mixed with 2% (w/v) alginate; 3V2A] for endothelial progenitor/primary cells.We found that the cell spread and viability of the encapsulated human aortic smooth muscle cells (HAoSMCs) were impaired by the presence of alginate, which lacks cell-friendly moieties to support cell adhesion (Fig. S1). Accordingly, the concentration of alginate was reduced to 0.5% (w/v), the minimum concentration required for coaxial extrusion of tubular structures, to promote the activity of HAoSMCs. To evaluate the biological performance of this bioink, type I collagen, a widely used hydrogel for tissue-engineered vascular constructs,at an identical concentration to that of VdECM in the bioink [3% (w/v)] was selected as a control. The resultant 3V0.5A bioink exhibited superior promotion of cell proliferation [Fig. S2(a)], upregulated expression levels of differentiation markers of VSMCs [α-SMA, SM22-α, and smoothelin (SMTN)] [Figs. S2(b) and S2(c)],and extracellular matrix (ECM) synthesis of HAoSMCs [Fig. S2(d)]. In addition, this bioink supported the coaxial-printing of HAoSMCs-laden tubes without impairing cell viability (Fig. S3). Therefore, the 3V2A and 3V0.5A bioinks were used to carry human umbilical vein endothelial cells (HUVECs) and HAoSMCs, respectively, for the fabrication of biomimetic vascular constructs.

III. DISCUSSION Section: Choose Top of page ABSTRACT I. INTRODUCTION II. RESULTS III. DISCUSSION << IV. CONCLUSIONS V. MATERIALS AND METHODS SUPPLEMENTARY MATERIAL CITING ARTICLES

The findings of this study suggested several advantages of the constructed TEBVs for vascular tissue engineering. First, we demonstrated the strong potential of VdECM as a useful source of bioink for generating vascular grafts. The contents of several dominant ECM proteins known to be present in native blood vessels were quantified, as shown in Table S2. Notably, other than these proteins, there might be numerous biochemical molecules such as growth factors and cytokines preserved after the decellularization process, which needs to be identified in the future. Due to the extensive preservation of the complexity of natural ECM observed in native blood vessels, this material can provide vascular cells with intrinsically favorable microenvironments that are difficult to replicate by reconstituting soluble ECM components. As a result, the cellular activity of HUVECs and HAoSMCs were strikingly promoted, leading to the formation of matured endothelium and dense smooth muscle tissues. Previous efforts have used decellularized xenogeneic vascular tissues, often from a pig, as implantable scaffolds. However, these grafts usually face the challenge of recellularization, and thus have difficulty with tissue remodeling and host integration after implantation. In addition, technical limitations make it impossible to control the dimensions of these decellularized grafts. Therefore, converting VdECM to printable bioinks could pave the way to the successful engineering of cell-laden vascular graft with tunable geometries. Through supplementation with alginate, the formulated vascular tissue-specific bioink not only facilitated the coaxial printing of vessel-like structures but also preserved the performance of VdECM to promote the cellular activity. With these advantages, biomimetic cell-laden TEBVs that showed remarkable in vivo performances were engineered from dECM material using a cell-printing technique for the first time.

13 16(3), 341 (2010). 13. G. A. Villalona, B. Udelsman, D. R. Duncan, E. McGillicuddy, R. F. Sawh-Martinez, N. Hibino, C. Painter, T. Mirensky, B. Erickson, and T. Shinoka, Tissue Eng., Part B(3), 341 (2010). https://doi.org/10.1089/ten.teb.2009.0527 in vitro conditioning, the encapsulated ECs proliferated, migrated, and eventually evolved to mature endothelium without requiring additional steps. 43 7(23), 1801102 (2018). 43. G. Gao, J. Y. Park, B. S. Kim, J. Jang, and D.-W. Cho, Adv. Healthcare Mater.(23), 1801102 (2018). https://doi.org/10.1002/adhm.201801102 Moreover, the developed triple-coaxial-cell-printing technique enabled the direct construction of vascular substitutes that contain both the endothelial and muscular layer. Traditional methods for preparing such dual-layered vessels usually relied on the seeding of endothelial cells onto a preprocessed tubular matrix. However, since the efficiency of seeding endothelial cells is highly dependent on the surface roughness, biocompatibility of the matrix material, cell density, seeding time, and interventions (e.g., structure inversion or rotation).Hence, there is a high risk that matured endothelium will not be successfully generated on the TEBVs. Moreover, the additional seeding and culture techniques inevitably lead to longer production time for matured constructs. In contrast, the fabrication technique presented here can homogeneously embed ECs in a vascular tissue-specific bioink as the interior layer of the fabricated vessels. Duringconditioning, the encapsulated ECs proliferated, migrated, and eventually evolved to mature endothelium without requiring additional steps.Moreover, due to the incorporation of both EC and VSMC layers in the fabricated conduits, the remodeling of endothelial and muscular tissues could be implemented simultaneously. Hence, we have developed an expedient and stable strategy for the construction of TEBVs.

44–46 115(10), 1285 (2007). 44. J. E. Deanfield, J. P. Halcox, and T. J. Rabelink, Circulation(10), 1285 (2007). https://doi.org/10.1161/CIRCULATIONAHA.106.652859 34(12), 1508 (2002). 45. B. E. Sumpio, J. T. Riley, and A. Dardik, Int. J. Biochem. Cell Biol.(12), 1508 (2002). https://doi.org/10.1016/S1357-2725(02)00075-4 130(26), 2819 (2017). 46. D. M. Coenen, T. G. Mastenbroek, and J. M. E. M. Cosemans, Blood(26), 2819 (2017). https://doi.org/10.1182/blood-2017-04-780825 in vivo proof-of-concept evaluation confirmed that all the implants (6/6) retained clearly patent lumens over an observation period of 3 weeks, suggesting that acute thrombosis was prevented. This positive result could be ascribed to the role of the intact mature endothelium in the implanted TEBVs. A previous study, in which endothelium-free TEBVs were implanted in rat abdominal models, demonstrated bulk thrombus generation at only 2 weeks postimplantation, emphasizing the importance of endothelium in vascular grafts. 47 102, 120 (2016). 47. L. Gui, B. C. Dash, J. Luo, L. Qin, L. Zhao, K. Yamamoto, T. Hashimoto, H. Wu, A. Dardik, and G. Tellides, Biomaterials, 120 (2016). https://doi.org/10.1016/j.biomaterials.2016.06.010 in vivo. The presence of endothelium is one of the key prerequisites for successful blood vessel grafting, especially in terms of blood vessels with small diameters (<6 mm). The endothelium physiologically provides an antithrombogenic surface to regulate platelets adhesion/activation and thrombus formation while minimizing the risk of graft implantation failure caused by blockage.Ourproof-of-concept evaluation confirmed that all the implants (6/6) retained clearly patent lumens over an observation period of 3 weeks, suggesting that acute thrombosis was prevented. This positive result could be ascribed to the role of the intact mature endothelium in the implanted TEBVs. A previous study, in which endothelium-free TEBVs were implanted in rat abdominal models, demonstrated bulk thrombus generation at only 2 weeks postimplantation, emphasizing the importance of endothelium in vascular grafts.An important concern is the efficacy of the endothelium in preventing thrombosis over a prolonged period; this is an obstacle to the clinical implementation of small-diameter blood vessel grafts. Hence, future investigations will focus on long-term patency and endothelium remodeling

17,48 3(1), 134 (2015). 17. Y. Zhang, Y. Yu, A. Akkouch, A. Dababneh, F. Dolati, and I. T. Ozbolat, Biomater. Sci.(1), 134 (2015). https://doi.org/10.1039/C4BM00234B 30(43), 1706913 (2018). 48. Q. Pi, S. Maharjan, X. Yan, X. Liu, B. Singh, A. M. van Genderen, F. Robledo-Padilla, R. Parra-Saldivar, N. Hu, and W. Jia, J. Adv. Mater.(43), 1706913 (2018). https://doi.org/10.1002/adma.201706913 in vitro remodeling process, abundant de novo human elastin and collagen were detected in the prematured TEBVs, which substantially enhanced their mechanical strength. In addition, through pulsatile stimulation, the HAoSMCs developed into a circumferential orientation that emulated their natural counterparts. As a result, for the first time, biomimetic TEBVs consisting of a mature endothelium and dense smooth muscles were achieved using the cell-printing technique. More importantly, under in vivo conditions, abundant collagen fibers and elastic ECM components were observed, indicating the potential of TEBVs to develop into natural blood vessels. The coprinted smooth muscle layer of the constructed TEBVs contributed to a major increase in mechanical strength, demonstrating the promise of this technique for developing small-diameter vascular grafts. Although several pioneer works have attempted to print vascular equivalents using the coaxial-extrusion or microfluidic-fabrication method, previous efforts usually focused on the fabrication of concentrically layered microtubes and the investigation of fundamental cell behaviors (e.g., viability and proliferation),rather than exploring its potentials for constructing vascular grafts. The resultant microtubes, however, usually possessed a thin wall, erratically aligned cells, and limited ECM synthesis, which led to delicate constructs that could not be used as vascular grafts. In this report, the advanced RTCCP technique allowed us to easily construct dual-layered TEBVs with tunable dimensions containing sufficient VSMCs. Therefore, it can be a flexible fabrication technique for constructing the blood vessel substitutes. More importantly, the use of vascular tissue-specific bioinks significantly promoted the cellular activity of HAoSMCs. During theremodeling process, abundanthuman elastin and collagen were detected in the prematured TEBVs, which substantially enhanced their mechanical strength. In addition, through pulsatile stimulation, the HAoSMCs developed into a circumferential orientation that emulated their natural counterparts. As a result, for the first time, biomimetic TEBVs consisting of a mature endothelium and dense smooth muscles were achieved using the cell-printing technique. More importantly, underconditions, abundant collagen fibers and elastic ECM components were observed, indicating the potential of TEBVs to develop into natural blood vessels.

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52 3(68), 68ra9 (2011). 52. S. L. M. Dahl, A. P. Kypson, J. H. Lawson, J. L. Blum, J. T. Strader, Y. Li, R. J. Manson, W. E. Tente, L. DiBernardo, and M. T. Hensley, Sci. Transl. Med.(68), 68ra9 (2011). https://doi.org/10.1126/scitranslmed.3001426 in vitro remodeling has not yet been optimized. It has been demonstrated that numerous factors can affect the outcomes of remodeling, including bioreactor definition (e.g., cyclic frequency, strain amplitude, flow rate, and stimulation period 53 20(9–10), 1499 (2014). 53. L. Gui, M. J. Boyle, Y. M. Kamin, A. H. Huang, B. C. Starcher, C. A. Miller, M. J. Vishnevetsky, and L. E. Niklason, Tissue Eng., Part A(9–10), 1499 (2014). https://doi.org/10.1089/ten.tea.2013.0263 54 22(4), 339 (2003). 54. J. L. Long and R. T. Tranquillo, Matrix Biol.(4), 339 (2003). https://doi.org/10.1016/S0945-053X(03)00052-0 A current limitation of the developed TEBVs is their suboptimal mechanical properties compared to the clinical gold-standard (e.g., human coronary artery or saphenous vein). However, our findings represent a fundamental step toward the achievement of cell-printing a biomimetic vascular graft, as theremodeling has not yet been optimized. It has been demonstrated that numerous factors can affect the outcomes of remodeling, including bioreactor definition (e.g., cyclic frequency, strain amplitude, flow rate, and stimulation period) and the choice of supplemented biomolecules (e.g., serum, TGF-β, insulin, and ascorbic acid). Therefore, future works will particularly aim at the optimization of the remodeling process to further reinforce the mechanical strength of TEBVs.

In addition, the application of the developed technique might not be confined to producing blood vessel grafts. The rapid maturation of the compartmentalized endothelium and smooth muscles might be useful for in vitro tissue modeling or organ-chips investigations. Equipped with the flexibility of 3D cell printing, this technique may be employed as a novel manner of establishing arterial in vitro models with diverse geometries (e.g., straight, curved, or with a constricted lumen) that can be used to study the physiological associations between endothelial cells and smooth muscle cells, as well as to understand various cardiovascular diseases (e.g., atherosclerosis, stenosis, or aneurysm). Moreover, this digitally tunable technique, when adapted to other tissue-specific materials, might be broadened for tissue-engineering other circumferentially multilayered tissues and organs, such as the trachea, intestine, and ureter, for tissue regeneration and modeling.