1Drug TargeT InsIghTs 2016:10(s1) Preliminary In Vivo Evaluation of a Hybrid Armored Vascular Graft Combining Electrospinning and Additive Manufacturing Techniques Supplementary Issue: Current Developments in Drug Eluting Devices Cristiano spadaccio1,*, Francesco nappi2,*, Federico De Marco3, Pietro sedati4, Fraser W.h. sutherland1, Massimo Chello5, Marcella Trombetta6 and alberto rainer6 1Department of Cardiothoracic Surgery, Golden Jubilee National Hospital, Clydebank, Dunbartonshire, UK. 2Cardiac Surgery, Centre Cardiologique du Nord de Saint-Denis, Paris, France. 3Laboratory of Virology, The Regina Elena National Cancer Institute, Rome, Italy. 4Unit of Imaging and Diagnostics, Università Campus Bio-Medico di Roma, Rome, Italy. 5Unit of Cardiac Surgery, Università Campus Bio-Medico di Roma, Rome, Italy. 6Tissue Engineering Laboratory, Università Campus Bio-Medico di Roma, Rome, Italy. *These authors contributed equally to this work. ABSTR ACT: In this study, we tested in vivo effectiveness of a previously developed poly-l-lactide/poly-e-caprolactone armored vascular graft releasing heparin. This bioprosthesis was designed in order to overcome the main drawbacks of tissue-engineered vascular grafts, mainly concerning poor mechanical properties, thrombogenicity, and endothelialization. The bioprosthesis was successfully implanted in an aortic vascular reconstruction model in rabbits. All grafts implanted were patent at four weeks postoperatively and have been adequately populated by endogenous cells without signs of thrombosis or structural failure and with no need of antiplatelet therapy. The results of this preliminary study might warrant for further larger controlled in vivo studies to further confirm these findings. KEY WORDS: vascular graft, computer-aided tissue engineering, electrospinning, additive manufacturing, heparin, drug release SUPPLEMENT: Current Developments in Drug eluting Devices CITATION: spadaccio et al. Preliminary In Vivo evaluation of a hybrid armored Vascular graft Combining electrospinning and additive Manufacturing Techniques. Drug Target Insights 2016:10(s1) 1–7 doi:10.4137/DTI.s35202. TYPE: Original research RECEIVED: september 25, 2015. RESUBMITTED: november 12, 2015. ACCEPTED FOR PUBLICATION: november 16, 2015. ACADEMIC EDITOR: anuj Chauhan, editor in Chief PEER REVIEW: Three peer reviewers contributed to the peer review report. reviewers’ reports totaled 507 words, excluding any confidential comments to the academic editor. FUNDING: authors disclose no external funding sources. COMPETING INTERESTS: Authors disclose no potential conflicts of interest. COPYRIGHT: © the authors, publisher and licensee Libertas academica Limited. This is an open-access article distributed under the terms of the Creative Commons CC-BY-nC 3.0 License. CORRESPONDENCE: cristiano.spadaccio@gjnh.scot.nhs.uk Paper subject to independent expert blind peer review. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti- plagiarism scanning. Prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. This journal is a member of the Committee on Publication ethics (COPe). Published by Libertas academica. Learn more about this journal. Introduction Cardiovascular diseases are nowadays emerging as an overwhelming clinical reality leading to high morbidity and requiring several surgical procedures such as arterial bypass and vascular replacing. Currently used prosthetic vascular graft materials such as polyethylene terephthalate and polytetrafluoroethylene (PTFE) are often inadequate because of limitations regarding thrombogenicity, graft fail- ure, and infection, especially when used for small-caliber ves- sel bypass grafting. On the other side, the use of autografts, such as internal thoracic artery or safenous vein, is harnessed by problems of poor quality, especially in the elderly, and inadequate size or length of the vascular conduit itself. Tis- sue engineering of vascular conduits is emerging as a corner- stone strategy to surmount the drawbacks experienced with surgical replacement with autologous vessels, allografts or xenografts, and prosthetic materials.1,2 Tissue-engineered vascular graft (TEVG) is emerging as a valid alternative to routinely used vascular prostheses. TEVG constructs are based on the use of biodegradable polymeric scaffolds that are used as frameworks for autologous vascular wall cells seed- ing and culturing.3 Animal studies and human trials demon- strated the effectiveness of TEVGs in vascular replacement for both large- (∅  .  6  mm)4 and small-diameter vessels (∅ , 4 mm).5 TEVGs seeded with autologous cells showed efficient endothelialization, stability over time, and freedom from infections. However, several issues concerning the clini- cal applications of TEVG still need to be answered. Reason- able and clinically suitable timing for scaffold preparation, mechanical properties, cell type, differentiation and growth inside the construct, donor scarcity, and thrombogenicity represent the major concerns. The minimum time from harvest of autologous cells to the generation of a mature and durable conduit is approxi- mately eight weeks.6,7 This limits the use of such conduits to elective procedures and requires local expertise and bioreactor facilities. Moreover issues concerning the patency rate of the graft should be con- sidered as the use of engineered scaffold or even small intes- tine submucosa in several animal models has been shown an Journal name: Drug Target Insights Journal type: Original Research Year: 2015 Volume: 9(S1) Running head verso: Spadaccio et al Running head recto: Hybrid armored vascular graft manufacturing techniques http://www.la-press.com/drug-target-insights-journal-j23 http://www.la-press.com http://dx.doi.org/10.4137/DTI.S35202 http://creativecommons.org/licenses/by-nc/3.0/ http://creativecommons.org/licenses/by-nc/3.0/ mailto:cristiano.spadaccio@gjnh.scot.nhs.uk http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 Spadaccio et al 2 Drug TargeT InsIghTs 2016:10(s1) overall patency rate of 75 per cent (48 weeks) nothwithstand- ing aspirin and warfarin administration.8 In this context, bioengineering experimental efforts have been oriented toward the fabrication of scaffolds behaving as biological equivalents with histoarchitecture similar to the native vessels with the aim to stimulate and support cell engraftment and proliferation. Different approaches to the fabrication of TEVGs have been described.9 In this context, electrospinning has been claimed among the most promising manufacturing techniques for the production of polymeric fibrillar meshes resembling the extracellular matrix (ECM) organization with fiber diameters ranging in the nanome- ter or micrometer scale.10 With this system, it is possible to manufacture interconnected porous structures displaying desirable morphologic characteristics in biological environ- ments as a high surface-to-volume ratio, as well as a high permeability.11 The manufacturing setup is amenable to pro- duce tubular shapes10 and associated to all the techniques of cell seeding.12 Additionally, fibers are amenable to be func- tionalized with several compounds and growth factors with- out impairing their biological function and activity. We have previously developed poly-l-lactide (PLLA) electrospun tubular scaffold functionalized with heparin with the final aim to both assist cell differentiation and realize a drug delivery device to prevent graft thrombosis with encourag- ing results.13 Moreover, electrospun scaffolds, simulating the arrangement of ECM fibrillar proteins, showed a permissive effect on vascular remodeling of both the cellular and extra- cellular components of a graft, and might therefore constitute a suitable candidate for TEVGs fabrication.14 However, the ability to simulate biomechanical characteristics and behavior of the natural vessel wall represents an obstacle.15–18 Despite a number of efforts experimental efforts lavished in this direction, the mechanical properties of electrospun fibrillar matrices are still inappropriate to sustain the pressure loads soon after implantation in the vascular tree and to ensure structural and functional integrity in the later stages during the remodeling phases.19 In order to overcome this drawback, we recently developed a hybrid technique associating electrospinning and bioprinting to fabricate a bioresorbable scaffold for vas- cular tissue engineering. With the aim to improve mechani- cal and functional properties of the TEVG, a single-layer helical poly-e-caprolactone (PCL) coil was bioprinted on the external surface as a reinforcement of an heparin-releasing PLLA tubular electrospun scaffold.20 This approach takes its inspiration from the so-called computer-aided tissue engineering (CATE), which has been reported to be an exciting resource to produce three-dimensional (3D) geom- etry constructs.21,22 Biofunctionalization with heparin per- mitted the creation of a drug delivery system that could overcome the thrombogenic issues of TEVG and at the same time provide a microenvironment suitable to stimulate endothelial differentiation. The PCL external reinforcement ameliorated the resistance to mechanical stress of the scaffold in comparison to nonarmored grafts, and more interestingly, when compared to autologous conduits, it showed better mechanical properties and stress–strain profile than a human saphenous vein while approximating to the ones of the internal thoracic artery. Additionally, it preserved the fibrillar ECM-like arrangement optimal for initial cell attachment and was able to stimulate the engraftment, proliferation, and endothelial differentiation of human bone marrow-derived mesenchymal stem cells. To our knowledge, no other approaches combining a heparin- releasing PLLA electrospun scaffold with a bioprinted PCL armor have been proposed in the literature. On the basis of these experimental results, we decided to perform a preliminary proof-of-principle study to test the effectiveness of the previously developed TEVG in an in vivo rabbit model of aortoaortic vascular bypass. Materials and Methods Fabrication of the graft. Armored vascular grafts were prepared as previously described.20 Briefly, a 13% w/w PLLA (Sigma-Aldrich) solution in dichloromethane was combined with unfractioned heparin (sodium salt, 5000  UI/mL; Mspharma) using methanol as a cosolvent. A final concentration of heparin (with respect to PLLA) of 830 mg/g was obtained, corresponding to the dosage routinely used in literature and clinical settings.23 A tubular scaffold, 5 mm in diameter and 6 cm in length, was manufactured by means of electrospinning according to previously described methods.13,20 Subsequently, an outer PCL (MW 80  kDa; Sigma- Aldrich) armor was constructed by fusion deposition model- ing techniques using a previously developed bioprinter.20 A detailed description of the morphological and mechan- ical characterization of the graft is reported elsewhere.20 In vivo experimental design and surgical procedure. A modified model of abdominal aorta vascular reconstruction was performed according to previously described protocols (Fig. 1).24 A total of 10 adult male New Zealand White rabbits weighting 1.8–2.1  kg were purchased (Charles River Labo- ratory) and housed under controlled conditions and normal diet for three weeks before experimentation. All animals were implanted with a PCL-armored heparin-releasing PLLA tubular scaffold according to the procedure further described. The study design did not include a control graft without hepa- rin loading, as previous preliminary experiments using PCL- armored PLLA grafts showed an excessively low patency rate due to acute graft thrombotic occlusion. Additionally, nonar- mored PLLA tubular grafts showed no adequate mechani- cal properties to support flow after implantation. Considering the preliminary nature of this study, designed as a proof- of-principle to test effectiveness of the newly developed armored heparin-releasing scaffold, we decided to not include control groups which would have been certainly inferior in http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 Hybrid armored vascular graft manufacturing techniques 3Drug TargeT InsIghTs 2016:10(s1) performance to the study group. All procedures, care, and handling of the animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Regina Elena Institute. An optimized anesthesia protocol was selected in order to guarantee stabilization of cardiovascular function dur- ing the open chest procedure.25 Anesthesia was inducted by intramuscular (i.m.) administration of ketamine hydrochlo- ride (25  mg/kg of body weight) and xylazine hydrochloride (15  mg/kg). After disappearance of the pedal reflex in the hindlimbs, rabbits were placed on a warming operative plat- form (37°C) in the supine position. A 23G vascular access was obtained through the marginal vein of the ear. The skin of the ventral abdomen was aseptically prepped with povidone– iodine solution. Briefly, a midline laparotomy incision was performed and the abdominal aorta exposed. Renal arteries were identified and following administration of 100  IU/kg of sodium heparin, proximal end-to-side anastomosis with the composite graft was constructed using a sidebiting microclamp  ~1  cm below the origin or renal arteries using monofilament 8–0 polypropylene suture. Aorta was bypassed for a tract of ~4 cm and the distal end graft was then end-to- side anastomosed to aorta before the origin of iliac arteries using monofilament 8–0 polypropylene sutures. The infrare- nal aorta between the two anastomoses was ligated so that all the blood flow to the inferior limbs was dependent on the graft (Fig. 1). Muscle layer and skin were closed with 3–0 polygla- ctin absorbable suture (Vycril; Ethicon). After closure of the abdomen, the animals were allowed to recover on a warming pad. The surgical procedure was completed within 30 minutes after the initiation of anesthesia. When responsive to stimuli and able to maintain an upright posture, rabbits were returned to the home cage and analgesia initiated with buprenorphine (0.5 mg/kg) and paracetamol (1 mg/kg). The first three days after surgery, buprenorphine (0.5 mg/kg b.i.d.) and cefurox- ime (100  mg/kg b.i.d.) were administered subcutaneously. The  animals received no antiaggregation therapy. Daily controls using handheld Doppler ultrasound system were performed to assess patency of the grafts and animals were examined for signs of inferior limbs weakness or paralysis. Four weeks after implantation, animals were humanely sacri- ficed and grafts were explanted for evaluation. Contrast-enhanced vascular imaging. Before sacrifice, animals underwent CT scanning with intravenous contrast agent. Briefly, rabbits were anesthetized with midazolam at a dose of 2 mg/kg i.m. Iodinated contrast medium was injected in the marginal ear vein, and cardiac CT scans were obtained and reformatted in 3D using maximum intensity projection (MIP) and volume rendering algorithms. Histology. Samples obtained were fixed in 4% parafor- maldehyde and embedded in paraffin. Serial sections (6 mm thick) were cut and used for hematoxylin–eosin staining. Results At the moment of surgery, no evidence of transgraft leakage of blood was demonstrated. During clinical follow-up, no neurological events, infection, or other surgical complica- tions have been noted. Use of sidebiting clamp and the end- to-side fashion of the anastomosis used prevented the known tendency to paraplegia resulting from cross-clamping of infra- renal aorta. All the grafts and iliac axes were patent at four weeks postimplant and perfusion preserved in the inferior limbs. Kidneys were adequately perfused (Fig. 2). At explant the graft preserved their integrity with no signs of significant intraluminal thrombosis or exuberant foreign-body inflammatory reaction. At histology scaffolds appeared uniformly cellularized with a variety elements colo- nizing the scaffold fibrillar framework. Polymer fibers could be seen with cells engrafted within scaffold meshes with deposition of ECM. Interestingly, the inner surface was populated by elongated cells with rare cytoplasm, a high Figure 1. (A) schematic diagram of the experimental procedure. (B) Intraoperative photograph showing the PLLa armored scaffold implanted and the ligature of the segment of infrarenal aorta between the two anastomoses. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 Spadaccio et al 4 Drug TargeT InsIghTs 2016:10(s1) nucleus/cytoplasm ratio, and nuclei protruding in the lumen. This endothelial-like morphology was not encountered within the wall of the scaffold, in which cellular elements looked more irregular and immersed in an eosinophil connectival atmosphere. The outer side of the tubular graft was populated by spindle-shaped cells with characters resembling proliferat- ing fibroblasts (Fig. 3). Taken together, these findings might reliably suggest the idea of self-colonization of the scaffold by endogenous cells that progressively acquired different pheno- types within the scaffold. Figure 2. Computed tomographic angiography study. Left: contrast-enhanced imaging. note patency of the TegV. right: 3D reconstruction with MIP and volume rendering algorithms. Figure 3. histological analysis. hematoxylin–eosin staining. The scaffold appeared to be densely colonized by different cellular elements that progressively acquired different phenotypic characteristics according to the region of the TeVg in which they engrafted. (A) 5× magnification. (B) 10× magnification. (C) 40× magnification of the inner side of the TEVG. Note flat elongated cells with nucleus protruding in the lumen (arrow) organized in an endothelial-like fashion. (D) 40× magnification of the outer side of the TEVG. Note spindle-shaped cells reliably representing fibroblasts (arrow). F indicates fibers of polymer in both cross and long axis section. http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 Hybrid armored vascular graft manufacturing techniques 5Drug TargeT InsIghTs 2016:10(s1) Discussion The two major concerns in TEVGs rely on the prevention of thrombosis and mechanical resistance of the construct when implanted in vivo. Acute graft failure for thrombotic occlu- sion or structural deterioration with aneurysmal degenera- tion or rupture are considered the main drawbacks of TEVG harnessing their actual clinical application.26–28 In this proof- of-principle study, a PCL-armored heparin-releasing PLLA graft showed good patency rate and structural integrity in a previously described model of aortoaortic bypass.24 We avoided an aortic interposition graft model for a number of reasons. First, to avoid the well-known tendency to paraplegia after infrarenal aortic cross-clamp in rabbits.29,30 Second, to simu- late the clinical scenario normally characterized by end-to-side anastomoses rather than end-to-end sutured grafts. Third, to test the antithrombogenic properties of the TEVG in a condi- tion characterized by a higher risk of turbulence and hemo- dynamic irregularity, as for a 90° anastomosis, rather than a full-channel straight anastomosis between conduits of similar diameters. The TEVG remained patent throughout the dura- tion of the study and no signs of embolic or neurological events were noted. The latter acquires a significant value considering that, for the characteristics of the model used, the entire limbs perfusion was dependent on the graft and no antiplatelet agents were given as thrombosis prophylaxis. The scaffold appeared to be densely colonized by different cellular elements that pro- gressively acquired different phenotypic characteristics accord- ing to the region of the TEVG in which they engrafted. The inner side was populated by elongated flat cells organized in an endothelial-like fashion. Cells within the scaffold wall and in the outer side showed characters of both quiescent and active fibroblast, indicating the contemporaneous presence of differ- ent degrees of cell activity in terms of neo-ECM deposition. The outer layers of the scaffolds were colonized by spindle- shaped cells, which might either represent fibroblasts or smooth muscle cells. As shown in our previous work, heparin func- tionalization exerted a significant influence on cell differentia- tion toward vascular endothelium.13 Besides the well-known action on thrombosis prevention,31,32 heparin is essential for endothelial cell adhesion and homeostasis, ameliorates cell engraftment into the scaffold, and provides signals for cells survival and differentiation.33,34 Additionally, heparin binds a number of angiogenic growth factors, such as Vascular endo- thelial growth factor (VEGF) and Basic Fibroblast Growth Factor (bFGF).33 Therefore, scaffold functionalization with heparin would permit to attract and concentrate in the scaf- fold the soluble angiogenic growth factors released in the blood stream during a vascular injury. This might reliably explain the flourishing cell colonization within the scaffold after four weeks and their differentiation toward an endothelial-like phe- notype. However, specific immunophenotype analysis of both resident and blood stream cells should be performed to support this hypothesis. Conversely, the fibroblastic/smooth muscle cell phenotype observed in the outer side of the scaffold might represent either a further evolution of the same population of cells colonizing the scaffold from the blood stream or the results of a foreign body reaction from the tissue surrounding the graft. In the first case, we might reliably speculate that both biologi- cal and mechanical factors (ie, shear stress at luminal side) have influenced the differentiation of the cells. However, in the in vivo hemodynamic conditions, several other factors might be involved in these phenomena and exert specific effects on the system. In the second case, despite PLLA is known to be highly biocompatible and to not elicit strong inflammatory reactions, the connective tissue surrounding the TEVG might have constituted a fibroblastic layer around the prosthesis. Clearly, further studies are required to elucidate the mecha- nism underlying these findings. Different from other approaches and in light of a trans- lational inspiration, in this study, we did not preseed scaffolds with autologous cells. Indeed, in order to overcome many of the biological, economical, logistic, and ethical concerns, which are currently known to hurdle the clinical application of cellular-based therapy, we decided to explore a strategy that avoids the use of cells but mostly relies on the biomimetic design of the scaffold and its paracrine effect as a drug-eluting device and might, therefore, be more rapidly translatable to the clinical practice. The rationale underlying this study concerned the possibility to exploit the endogenous repara- tive capabilities of the body and to guide these regenerative resources toward tissue restoration by means of a tailored absorbable material. Circulating endothelial progenitor cells or bone marrow-derived cells recruited from the blood stream might therefore colonize an artificial matrix that, for its inter- nal structure, closely simulates the native ECM atmosphere and actively emanates biological signals to support their survival and differentiation. Recruited cells would in fact dif- ferentiate within a 3D environment that closely mimics the organization of the vascular ECM and guarantees progressive cell growth and neotissue formation. The concept of fabricating a scaffold recapitulating native ECM and delivering molecules like heparin, which are able to stimulate stem cells differentiation and also to produce crucial systemic effects, has been previously explored and validated by our group also in nonvascular fields.35–38 In this study, we aimed at developing a device able to boost the endogenous process of reendothelialization and, contemporaneously, to simplify and improve the pharmacological handling and the outcomes of the graft once in the clinical scenario. The idea of combining biomaterials with different resorption timings to progressively accompany vascular reconstitution over time is also novel. Additionally, the proposed manufacturing tech- nique is amenable to be used in the context of GMP facilities in view of a potential translational application. In conclusion, this preliminary study assessed the fea- sibility and the in vivo effectiveness of a heparin-releasing armored tubular scaffold. Scaffold has been adequately populated by endogenous cells and did not show signs of http://www.la-press.com http://www.la-press.com/drug-target-insights-journal-j23 Spadaccio et al 6 Drug TargeT InsIghTs 2016:10(s1) thrombosis or structural failure with no need of antiplate- let therapy. If confirmed by larger studies, we might reliably speculate that this construct could constitute an attractive alternative in the panorama of tissue engineering of vascu- lar grafts overcoming the majority of the limits currently known for TEVG and allowing for a potentially easier clini- cal management. Limitations Among the limitations of this study, the authors acknowledge the lack of an immunohistological analysis of the cells colo- nizing the scaffolds to demonstrate the phenomena of endo- thelialization and smooth muscular differentiation speculated in the text. The scarcity of a specific antibody to detect rab- bit markers limited this study. Second, the lack of a quan- titative and qualitative analysis of the neo-matrix deposited within the biopolymer in order to support the findings on the vascular remodeling of the TEVG. Third, the lack of a control group implanted with a nonfunctionalized PLLA graft or with current state-of-the-art graft routinely used in clinical practice. As described earlier, our preliminary in vivo study demonstrated an excessive acute failure of the graft for thrombosis in nonheparin-functionalized scaffolds, which led us to abandon this approach. Conversely, a number of rea- sons prevented to add a polyethylene terephthalate or PTFE graft control group in the study. First, the technical diffi- culty in performing the vascular reconstruction model used considering the discrepancy in caliber of the native aorta and the smallest clinically available prosthetic graft, as no other experiences with these type of grafts are reported in the lit- erature for a rabbit model. Second, the discrepancy in caliber would have added an additional bias considering the possi- bility of thrombosis because of hemodynamic irregularity or turbulence. Third, this group would have implied to admin- ister antiplatelet agents, as normally required in clinical set- tings to avoid thrombosis, weakening the main aims of this study, ie, to test the actual ability of a TEVG to constitute a valid alternative to autologous conduits guaranteeing patency and mechanical resistance. In this context, a longer term follow-up would have been useful to evaluate vascular recon- stitution and potential side effects. Finally, this study was only preliminary and speculative in its nature and was performed as a proof-of-principle to test the in vivo effectiveness of our previously developed armored prosthesis in terms of patency and resistance. The results of this preliminary study might warrant for further larger controlled in vivo studies to further confirm these findings. Author Contributions Conceived and designed the experiments: CS. Analyzed the data: FN, FdM, AR. Wrote the first draft of the manuscript: CS, PS, FS. Contributed to the writing of the manuscript: FN, AR, MC, MT. Agree with manuscript results and con- clusions: AR, FS, MC, MT. Jointly developed the structure and arguments for the paper: CS, AR, FN, FS, PS. Made critical revisions and approved final version: FS, MC, MT, AR, FN. All authors reviewed and approved of the final manuscript. REFERENCES 1. Xu ZC, Zhang WJ, Li H, et al. Engineering of an elastic large muscular vessel wall with pulsatile stimulation in bioreactor. Biomaterials. 2008;29(10): 1464–1472. 2. Yang F, Murugan R, Wang S, et al. Electrospinning of nano/micro scale poly(L- lactic acid) aligned fibers and their potential in neural tissue engineering. Bioma- terials. 2005;26(15):2603–2610. 3. Shinoka T, Shum-Tim D, Ma PX, et al. Creation of viable pulmonary artery autografts through tissue engineering. 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