Due to the lack of efficacy of synthetic vascular substitutes in the replacement of small-caliber arteries, vascular tissue engineering (VTE) has emerged as a promising solution to produce viable small-caliber tissue-engineered vascular grafts (TEVG). Previous studies have shown the importance of a cellular intimal layer at the luminal surface of TEVG to prevent thrombotic events. However, the cellularization of a TEVG seems to be a critical approach to consider in the development of a TEVG. To date, no standard cellularization method or cell type has been established to create the ideal TEVG by promoting its long-term patency and function. In this review, advances in VTE are described and discussed with a particular focus on the construction approaches of cellularized small-caliber TEVGs, the cell types used, as well as their preclinical and clinical applications. These diseases are often associated with a dysfunctional vasculature caused by inflammatory, metabolic and proliferative alterations leading to the narrowing or complete obstruction of blood vessels. Thus, tissue damage is induced by diseased blood vessels that are unable to convey oxygen and essential nutrients to cells. Common presentations of CVD are coronary heart disease, peripheral arterial disease, deep vein thrombosis, and cerebrovascular disease4.Cardiovascular diseases (CVD) are the major cause of death worldwide6. For the case of myocardial revascularization, coronary artery bypass grafting (CABG) remains the most common cardiac surgery procedure in the world, with about 200,000 CABGs performed annually in the US, while in Western European countries an average incidence of 62 per 100,000 people is reported7. It is worth mentioning that vascular conduits are also required to create arterial–venous fistulas for frequent hemodialysis access.Consequently, CVD have resulted in an increasing need for vascular grafts in order to reconstruct or bypass vascular stenosis. Vascular bypass grafting remains one of the most effective treatment approaches for patients who need long-term revascularization4.Although autologous arteries and veins are the preferable conduits for vascular grafting, their use may be impossible due to their limited availability, their variable quality , and the complications associated with their removal . As an alternative to autografts, synthetic vascular grafts can be used as they have shown a long-term efficacy in the replacement of large—(>8 mm) and medium—(6–8 mm) diameter arteries. However, they present poor patency rates when used for bypassing small-diameter arteries (<6 mm)9.Globally, small vascular graft failures are commonly due to intimal hyperplasia, thrombosis, atherosclerosis, or even through infections. Indeed, one of their major limitations is the absence of their luminal cellularization, leading to thrombosis via the adherence of blood proteins and the activation of clotting mechanisms10. The production of a tissue-engineered vascular graft (TEVG) for the replacement of small-diameter vessels has become the most heavily investigated and challenging areas of vascular tissue engineering (VTE). A TEVG with long-term patency, capable of being remodeled by the host organism, and showing self-repair capacities in vivo would be of great benefit11. Such a TEVG may represent a major advance in the field of cardiovascular surgery by providing an allogeneic “off-the-self” solution for clinical use allowing a new therapeutic option in various pathological states such as myocardial and lower limbs revascularization, pediatric vascular disease, useful also as arteriovenous bridges for hemodialysis. Furthermore, an “off-the-shelf” TEVG will be perfectly adapted for mini-invasive surgeries performed by robots. In fact, surgical robotic technology has emerged to perform remote operations in limited spaces and activities and is considered as the surgically invasive version of coronary bypass, avoiding a complete thoracotomy or sternotomy, thus preserving the thoracic integrity and suffering of the patient, and significantly reducing hospitalization period and costs.The limitations of small-diameter vascular grafts are a real obstacle to the vascular bypass substitutes, making this a real public health issue® mesh, and collagen to create a tubular scaffold. On this scaffold, ECs, SMCs, and bovine adventitial fibroblasts were co-seeded on the luminal surface, the internal wall and on the external side, respectively12.The first TEVG developed was reported by Weinberg and Bell in 1986. It was produced by mimicking the blood vessel architecture using bovine aortic endothelial cells (ECs), smooth muscle cells (SMCs), thin DacronSince this first TEVG, several small-diameter cellularized vessel substitutes have been elaborated and tested with different production approaches which are presented in Fig. A variety of scaffolds from natural sources can be used, such as decellularized vessels or in vitro developed scaffolds, e.g., synthetic or biosynthetic grafts. Associations of different cell sources and different combinations of microenvironmental conditions to lead cell differentiation or maintain cell integrity have also been described. Despite these considerable advances in VTE, only few grafts have reached clinical studies and the perfect TEVG has not been yet developed.13.A key factor to consider in the development of a TEVG is the cellular component. In fact, a very recent meta-analysis of preclinical studies using small-diameter TEVG has found that recellularized grafts were more patent than acellular grafts (85.1% vs. 64.4% respectively)15. Thus, in order to ensure optimal hemostasis, a continuous cell layer on the luminal surface of the vascular substitute is required. For this purpose, different cellularization approaches are described in the literature, using variable amounts of cells according to the seeding technique and the maturation period of the graft. The cell adhesion and retention can be highly improved by a prior coating deposit on the luminal surface of the graft. The addition of a luminal coating in the TEVG manufacture protocol is highly recommended as it can modify the microenvironment and guide cell behavior by promoting cell adhesion, proliferation and/or differentiation16. Several types of coatings have been used, composed of one or a combination of proteins which are in most cases of animal origin. Our team has recently developed an innovative approach to coating surfaces using a natural extracellular matrix (ECM) of human origin, extracted from the umbilical cord Wharton’s jelly (WJ) and is totally natural, biocompatible, and perfectly controllable17.The cell adhesion, proliferation, as well as differentiation and several other contributions to hemostasis is crucial to create a hemocompatible small-diameter vascular graft. In physiological conditions, the blood vessel hemocompatibility is ensured by a monolayer of ECs localized at its luminal surface (termed the “endothelium”). This blood contact surface maintains hemostasis through several antithrombogenic mechanisms, such as the synthesis of plasminogen activator (tPA); inhibition of platelet aggregation via secretion of nitric oxide (NO) and prostacyclin; and by the presence of glycosaminoglycans on the endothelial cellular surface preventing blood coagulationThis review will focus on the cellularized vascular grafts described in the literature, the different manufacturing protocols, the various quality control tests, the preclinical in vivo evaluation in several animal models and different implantation sites, as well as the few human clinical trials. The results will be presented and discussed critically, with an emphasis on the weak points for each TEVG and the proposition of solutions for their improvement.Several approaches have been described for the cellularization of TEVG using different types of cells in order to recreate the intimal and medial layers. Cells used in VTE can be from autologous or heterologous origins, as discussed in the following paragraphs of this review.18, which may be incompatible with the critical situation of the patient.The advantage of using autologous cells is the avoidance of any host immunological reaction which induces the graft rejection. Two cellularization approaches have already been reported in the literature: (1) an in vitro method involving the harvesting of autologous animal cells before the preparation of the substitute and (2) an in vivo method which uses the animal model as an “incubator” by placing an acellular tube in the animal before vascular grafting. These studies are part of a proof-of-concept approach; they propose TEVG manufactured using autologous animal cells that are not directly transferrable to human implantations. Moreover, the use of this cell type requires fairly long preparation times of up to several months19. Cells were harvested from the left external jugular vein. Cells were cultured until the 4th passage and then seeded by rotational precipitation into a decellularized aortae of fetal pigs, clamped on both sides (arteries are rotated 120° every 10 min for 30 min). After the EC deposit the graft was placed in a bioreactor for 7 days, allowing a step of dynamic culture where the perfusion rate was increased from 10 to 60 mL/min, followed by 3 days of static culture. The autologous graft was then implanted as a carotid graft replacement in seven dogs for a period of up to 6 months. Results showed an absence of thrombi and stenosis and all the grafts remained patent over the period of the study. After 6 months, the intimal layer of the graft was composed by EPCs and ECs whose origin has not been precisely determined. Moreover, the graft was remodeled by host cells, notably muscular fibroblasts which settled in a “media-like” layer. Although the graft has shown satisfactory results, some issues could be addressed further, especially in regards to the animal model used in the study. Thrombogenesis mechanisms and vascular viscoelastic properties in dogs are very different from the human physiological parameters20. Thus, these results cannot necessarily be transferrable to humans.Ma et al. have reported the use of mature autologous canine ECs in the manufacture of their graftECs can also be obtained from EPCs that are collected non-invasively from the host’s peripheral blood. These cells can be used directly to cellularize a scaffold or can be subjected to a prior differentiation step before being seeded.21. Seeding was accomplished under dynamic rotating conditions for 6 h. The graft was then matured in a laminar flow bioreactor for 6 days with a gradually increasing shear stress and implanted in an end-to-end carotid artery replacement model. Two groups of animals were compared under the same grafting conditions: seven animals receiving seeded grafts vs. four animals with unseeded grafts. The grafts patency was monitored by ultrasound Doppler every day, for a period ranging from 15 to 130 days. In the first 5 days of implantation, three of the four unseeded grafts were 100% clogged. The 4th graft was 50% clogged 15 days after the implantation. Concurrently, all seeded grafts remained patent over the entire study period . At 15 days, a layer of ECs derived from originally seeded EPCs was present, however the media of the substitute remained acellular. At 130 days, the results showed a thickening of the media that was infiltrated by host cells void of smooth muscle alpha-actin (α-SMA) expression, as well as an intimal layer colonized by α-SMA+ cells. Only 10% of the ECs identified at the luminal surface at 130 days were derived from the originally seeded EPCs. After 130 days, collected grafts showed a vasoconstriction capacity following exposure to serotonin and norepinephrine, reflecting the functionality of SMCs found in the intimal layer. On the other hand, these same grafts showed a relaxing capacity probably mediated by the release of NO, similar to that found in the native carotid artery.In 2001, Kaushal et al. evaluated the in vivo efficiency of a TEVG built from a pig decellularized iliac artery in a sheep model. The artery was seeded with autologous EPCs harvested from the internal jugular vein of 1 to 2 week(s) old sheep22. In their study, the substitute was developed by seeding differentiated autologous circulating EPCs on a decellularized porcine carotid artery segment. The seeding was carried out in a bioreactor by injecting the cells into the lumen of the vessels for 2 h. The graft was then subjected to a gradually increasing nonpulsatile flow for 5 days. Graft maturation was then completed after 9 days under pulsatile flow. This substitute was evaluated for arteriovenous flow and needle punctures in the ovine model. “Short-term” and “long-term” groups were monitored. The “short-term” group, consisting of a study period of 2 months, included seven grafted animals. Grafts of this group were punctured 3 times a week during the second month. In the “long-term” group, eight animals were implanted and followed for 6 months. From the 5th month, grafts were punctured 3 times a week. During this experiment, of the total number of grafts, four were stunted in the first month of transplantation  and occlusions appeared from 3.3 months. This type of graft using decellularized blood vessels as scaffolds have shown resistance deficiencies to repeated punctures. Other scaffolds, such as those built by mesh construction could be more effective in needle resistance.Otherwise, EPCs can be subjected to a prior differentiation step before seeding, as achieved by Tillman et al. in 201223. Results were compared with those obtained from TEVG seeded only with ECs. The manufacture of this graft consisted of the decellularization of a pig carotid artery, followed by an incubation with a SMC suspension under rotation into an outer chamber. Then, the graft was placed in a bioreactor where the lumen was perfused with a suspension of ECs for 10 to 14 days. Results have shown that the addition of SMCs to the graft wall allowed a better cellularization of the graft after implantation. This method of combined cellularization also reinforced the TEVG contractility without a negative impact on its permeability. However, this contraction capacity was much weaker than that of a native carotid artery (15–20%).Previous studies have described a co-seeding method with a combined cellularization, using autologous ECs and SMCs to improve the TEVG resistance. This approach was evaluated by Neff et al. in a carotid artery interposition grafting model in pigs for 4 months24 and Ju et al.25 have applied the co-seeding technique to a fibrin scaffold supported by a poly(L/D)lactide 96/4 [P(L/D)LA 96/4)] mesh and an electrospun bilayered vascular scaffold, respectively. The same cellularization protocol as previously described was utilized by Ju et al. Koch et al. produced their scaffold by a SMC/fibroblasts/fibrinogen suspension that was left to polymerize inside a mold. After polymerization, the luminal surface was cellularized with an ECs suspension into a rotating chamber26. Both TEVG were evaluated in a sheep carotid arterial interposition model. After an implantation period of 6 months, promising results have shown that the TEVG remained patent and well-integrated into the host tissue. Although these grafts appear to be suitable for clinical applications, some issues must be addressed, such as the long processing time. In fact, the scaffold preparation requires 7 days, followed by several weeks of autologous cell expansion until the 3rd–5th passage. An evaluation of this graft in a CABG or peripheric vascular bypass model would be beneficial to validate its efficiency in different representative clinical situations.Koch et al.27. A first implantation attempt showed a high thrombogenicity due to a weak production of NO by ELCs. Therefore, in order to increase the NO production by ELCs, a transfection of ASC was achieved with an adenoviral vector containing the endothelial nitric oxide synthase (eNOS)28. The TEVG with modified cells was tested in five rabbits in an abdominal aortic interposition graft model. The control group consisted of five rabbits implanted with unseeded grafts. After 2 months of implantation, all grafts, seeded and unseeded, had remained patent. Histological analyses have demonstrated that unseeded grafts exhibited a thrombus formation and/or a thrombin and fibrin staining at their luminal surface. Seeded grafts on the other hand, presented a smooth luminal surface without any traces of thrombin or fibrin. Although transfected cells have allowed the improvement of the graft characteristics via the overexpression of eNOS, the long-term patency and efficiency need to be evaluated in larger animal models which have a more similar physiology to humans. Moreover, the originality of this graft seems to be a major default since gene editing for clinical applications is at the center of ethical debates29.Another approach to obtain suitable efficient TEVGs is to modify seeded cells to confer them an additional functionality or to enhance a specific function. McIlhenny et al. have proposed a TEVG consisting of a decellularized human saphenous vein seeded with rabbit autologous adipose derived stem cells (ASCs). Cells were obtained from dorsal fat pads and seeded by a rotating perfusion of the graft’s luminal surface. The cellularized graft was then matured for 1 week in a bioreactor with a linearly increasing shear stress rate, thereby inducing the ASCs differentiation toward endothelial-like cells (ELCs)30, poly(methyl methacrylate) (PMMA) tube31, poly(vinyl chloride) (PVS)31, etc. Watanabe et al.32 developed a biotube by subcutaneously implanting a silicone rod of 3 mm diameter into rabbits for 2 months. In their previous study, the resultant biotube was described as an autologous connective tissue of 76 ± 37 µm of thickness, composed of collagen and fibroblasts33. This biotube was then evaluated in a carotid implantation model in the same rabbit, for 26 months. Histomorphological analyses of explanted grafts showed the absence of neointimal thickening. The luminal surface was entirely covered with PECAM-1 positive ECs, under which α-SMA positive cells and elastic fibers were found. Also, these α-SMA positive cells and collagen were remarkably oriented. Although these results seem promising, this study focused more on the manufacturing protocol than on the arterial implantation, since the latter was achieved in only one rabbit. Results need to be confirmed in a larger study including more animals with a control group. In 2016, the same fabrication approach was performed in vivo by Rothuizen et al. in pigs34. Rods were subcutaneously implanted for 4 weeks and harvested with the surrounding tissue. The biotubes produced were principally composed of collagen (type I and III) and glycosaminoglycans. Despite an important graft wall colonization mostly by fibroblasts and leukocytes, no endothelium-like layer was observed. 8 TEVGs have been studied in 4 female Landrace pigs  for 4 weeks. After 4 weeks of vascular integration, 7/8 grafts were still patent. The described occlusion was presumably due to a pre-anastomotic intimal hyperplasia which was absent in the contralateral graft. Histological analyses of the integrated graft have shown an increase in the wall thickness and an acquisition of a contractile phenotype by fibroblasts. Interestingly, a confluent layer of lectin positive cells on the graft luminal surface was present, which may suggest a graft endothelialization had occured during vascular integration. However, the major inconvenience of this “biotube” technique remains the long in vivo maturation period and the complicated transposition for clinical application. To overcome these limits, very recently, Yamanami et al.35 used the same principle by testing biotube cellularization in Beagle dogs for 4 weeks. After explantation, biotubes were decellularized and stored at −20 °C for 1 week. Before implantation, decellularized grafts were treated with a direct thrombin inhibitor—argatroban, to confer antithrombogenic properties to the graft. Grafts were then implanted at the infrarenal abdominal aorta in three adult male Wistar rats. At 1-month post implantation, grafts were still patent, and seemed to be infiltrated by host cells. However, the tested grafts were only 1 cm long. To meet clinical needs, longer grafts should be tested in larger animal models and in different implantation sites to evaluate their patency over long periods.Grafts developed according to this method are called “biotubes” and are derived from the “in-body tissue architecture technology”. The preparation consists of a natural or synthetic rod implanted subcutaneously into the host tissue and left to be colonized/encapsulated by host cells. After the in vivo incubation period, the encapsulated rod is harvested. The rod is then removed, and the host tissue surrounding the rod is used as a TEVG. Different kinds of tubes were used in this context, such as a PGA tube18. Production methods described in this section will be divided into conventional and innovative production techniques.In order to address the issue of small-diameter vascular substitutes, numerous studies have included allogeneic cells in the fabrication protocol of TEVG. The main advantage of these cells is the possibility of obtaining a “ready-to-use” cellularized graft. However, two limitations arise: (1) the host immunological reaction towards foreign cells and (2) the storage conditions of cellularized grafts before their use. Different solutions are proposed to overcome these obstacles and to produce readily available “universal” small vascular grafts. Various models are described in the literature, such as grafts manufactured directly using modified or unmodified allogeneic cells, or using several cell types of mixed allogeneic and autologous sources. Moreover, some authors are trying to provide an “off the shelf” graft by promoting allogeneic cells to produce their own ECM that will constitute the graft’s scaffold. The latter will be subjected to a decellularization step and can be recellularized with patient autologous cells just before the implantation. This approach provides a “personalized” cellularized graft for the patient36. Modified cells were also used in order to enhance the proliferation capacity of allogeneic cells (which decline with the donor age) or to confer antithrombogenic properties to the used cells.Conventional TEVGs production approaches using allogeneic cells can be produced directly by a single “simple cellularization”. Several cell types have been evaluated, such as bone-marrow mononuclear cells (BMNCs), EPCs, SMCs, ECs, and induced Pluripotent Stem Cells (iPSCs)37. To create their own small-diameter vascular graft, Roh et al.38 used commercial human BMMCs (hBMMCs) that were suspended into a fibrin gel solution and seeded onto a biodegradable Polyglycolic Acid–Poly-L-Lactide and -ε-Caprolactone (PGA-P(CL/LA)) scaffold 39. The in vivo study consisted of an inferior vena cava interposition into 3–4-month-old female SCID/bg mice for up to 24 weeks. At the end of the study, 9 of 10 seeded grafts were still patent. Histological analysis has shown an endothelial cell layer at the luminal surface and two layers of smooth vascular cells. However, cells composing the graft were exclusively from murine origin. Authors have suggested that the graft colonization by host cells was mediated by MCP-1 (monocyte chemoattractant protein-1) released by hBMMCs. The MCP-1 release led to monocyte infiltration and to inflammatory mechanisms involved in blood vessel formation and remodeling. Regarding the immunodeficient small animal model used, results do not allow the evaluation of the grafts immunological state. Indeed, several extensive studies are required to assess the host immune response triggered towards the graft. Furthermore, hBMMCs have not shown expected results concerning their differentiation potential into vascular cells in order to remodel the graft. This may be due to the inappropriate animal model used, lacking immunomodulatory cells that may be involved in blood vessel remodeling.The BMMCs population is known to have a differentiation potential in both endothelial and SMCs, meaning they are widely used in VTE40 tested a PGA-PLCL TEVG (inner diameter of 1 mm) in an inferior vena cava interposition model in C57BL/6 mice for 6 months. Seeded grafts were studied in comparison with unseeded grafts to determine the role of BMMCs. Grafts were seeded with allogeneic BMMCs and harvested from donor mice, followed by a perfusion of the cell suspension into the scaffold lumen for 10 min. Seeded grafts were then incubated overnight at 37 °C prior to the implantation. At the end of the study, 9 of 10 seeded grafts remained patent. Moreover, this study demonstrated that the BMMCs seeding prevented the graft narrowing and stenosis that were observed in unseeded grafts. This phenomenon seems to be mediated by a macrophage modulation and a prevention of platelet activation, induced by seeded BMMCs. This study complements other studies that have reached clinical trials for arteriovenous grafts with an inner diameter of 10 mm and could provide a new cellularization approach for next generation grafts.In an another study, Fukunishi et al.42, the generation of a regulatory T-cell population and the modulation of Natural Killer cells (NK) and macrophage responses43. In order to demonstrate the antithrombogenic potential of MSCs, Hashi et al.44 produced a cellularized small TEVG by seeding human bone-marrow MSCs on an electrospun nanofibrous membrane for 1 day. This cellularized membrane was then rolled and sutured around a 0.7 mm diameter mandrel. After removal of the mandrel, the graft was kept in culture for 2 days and then implanted for up to 60 days as a common carotid artery replacement in 6–8 week athymic rats. Results suggested that MSCs can modulate the host inflammatory response by preventing platelet adhesion and aggregation. These cells also showed antithrombogenic capacities by decreasing platelet aggregation (similar to ECs capacity) compared to unseeded graft controls. This was supported by a low and stable intimal thickening in seeded grafts 1-month post implantation, whereas a significantly higher thickening of the vascular wall was observed in unseeded implanted grafts. Both unseeded and seeded grafts showed an efficient recruitment of host ECs and SMCs which led to an organized layered vascular wall similar to a native artery. The data seems to demonstrate a short-term engraftment activity and efficacy of MSCs which are rarely found 2 months post implantation. MSCs appear to have beneficial effects, making them a promising cell source to consider in VTE. MSCs seeded grafts should be further evaluated under more challenging conditions, with immunocompetent larger animal models, longer implantation periods and at different implantation sites. Moreover, a more in-depth analysis regarding the fate of MSCs in the graft would also be interesting in order to understand their specific effects on the vascular wall remodeling, the recruitment of host cells, the modulation of the inflammation induced by the graft and their in vivo differentiation potential.In addition to their differentiation potential into vascular phenotypes, MSCs exhibit important immunomodulatory capacities, such as T cell45. The substitute wall was composed of BM-MSC cellularized gelatin hydrogels and PCL fibers which were automatedly arranged to reproduce the J-shaped mechanical response and compliance of human coronary arteries. Preliminary in vivo tests were conducted in six rabbits, as a carotid replacement model, divided into three groups: animals receiving cellularized grafts, animals receiving acellularized grafts and a control group consisting of an incision-anastomosis. Histological studies were performed at 14 days and 30 days post implantation to particularly observe the graft remodeling. Although a thrombus development was observed in both grafts (cellularized and acellularized), complete vascular wall remodeling was achieved at only 30 days in cellularized grafts. This study encourages the consideration of MSCs for TEVG cellularization as they prevent inflammation and promote graft remodeling after implantation.Inspired by the human coronary artery structure, a study has described a TEVG produced by an innovative approach, involving the deposition of layers of cell-laden hydrogel, by combining a dip-spinning technology with a solution blow spinning device (dip-spinning-SBS technology) within 30 min46. These cells are preferentially harvested from bone marrow, but they are also found abundantly in many other tissues such as dental pulp, adipose tissues, or perinatal tissues. For VTE, MSCs derived from perinatal sources, such as the WJ of the umbilical cord, may be an appropriate cell type for VTE applications. Wharton’s jelly derived MSCs (WJ-MSCs) have all the characteristics of MSCs, possess a more primitive phenotype than other mature MSCs and their use is totally safe as they do not induce teratoma formation47. They were shown to differentiate toward vascular phenotypes50. Moreover, for VTE purposes, our team has already shown that WJ-MSCs subjected to an endothelial differentiation, conserved their immumodulatory properties after differentiation and remained immunosuppressive by inhibiting the proliferation of T cells, reducing the toxicity of NK cells and generating a population of regulatory T cells51.MSCs seem to be a promising cell source for VTE52. Nieponice et al.53 compared three different TEVGs: (1) unseeded Poly(ester urethane)urea biodegradable tubular scaffolds produced by thermally induced phase separation (unseeded TIPS-PEUU), (2) TIPSS PEUU seeded with MDSCs (TIPS-PEUU), and (3) a MDSC seeded graft manufactured by combining TIPS with an electrospinning technique (ES-TIPS-PEUU). Autologous MDSCs were isolated, labeled by transfection with the LacZ reporter gene  and amplified, before being seeded on grafts of 15 mm length and 1.5 mm inner diameter, with a rotational vacuum seeding device54. Grafts were then cultured for 48 h under agitation. After 8 weeks of implantation in a rat aorta interposition model in Lewis rats, results reported that 73% of seeded TIPS-PEUU grafts showed signs of stenosis or aneurysmal dilatations and 65% of ES-TIPS-PEUU were patent without aneurysms, whereas only 10% of unseeded TIPS-PEUU grafts remained patent. Grafts were colonized by host cells, with von Willebrand factor positive cells at the luminal surface and cells with a contractile phenotype in the graft tissue. Moreover, a few transfected cells were still found in the grafts. Thus, the ES-TIPS-PEUU construct produced by double processing techniques could provide a promising solution for VTE. However, further analyses are required in larger animal models with an adaptation of the construction protocol to meet the clinical requirements.MDSCs have also been integrated in the cellularization protocol of some TEVGs. MDSCs are known for their high proliferation rates, a low expression of major histocompatibility complex class I and their in vivo and in vitro multilineage differentiation potentials toward muscle, neural, and ECs55 have used commercial male mouse iPSCs that were differentiated into sheets of ECs and SMCs, before being seeded onto PGA-P(CL/LA) with a diameter of 0.8 mm. This graft was implanted as an infrarenal inferior vena cava in female SCID/bg mice. After 10 weeks of implantation, all grafts were still patent with no signs of aneurysms or calcification. Immunohistological analysis of collected grafts revealed the gradual loss of the originally seeded cells and their replacement by host ECs at their luminal surface and SMCs at the inner layer. Wang et al.56 have also described an in vivo study where the TEVG were cellularized with iPSCs derived from primary human aortic fibroblasts which differentiated into proliferative SMCs. The cell seeding was carried out by an incubation of 24 h on PLLA scaffold. The vascular graft was placed in the subcutaneous pocket into nude mice for up to 2 weeks. This study has not evaluated the graft capacities and function but has explored an original approach, allowing the obtention of a personalized TEVG using the patient autologous cells.iPSCs are among cells that have been investigated by several teams for the cellularization of small-caliber TEVG. Hibino et al.48. In addition, even after differentiation, a residual presence of undifferentiated cells represents a high risk of tumorigenicity that may lead to the development of teratomas and malignant tumors, which affects their safety for human medical applications57.Although iPSCs seem to be a very promising source for new therapeutic strategies and regenerative medicine, due to the major ethical concerns around the use of embryonic stem cells, their use is still debated. Human iPSCs are produced with poor efficiency, moreover, the reprogramming protocols involve viral vectors that lead to safety issues for clinical applications58. Itoh et al. have used this technology to produce a TEVG in 4 days by assembling 500 multicell spheroids as a tube of 1.5 mm inner diameter and 7 mm in length59. These MultiCS (size of 615 ± 51.3 µm) were randomly composed of commercial human umbilical vein ECs (40%), human aortic SMCs (10%), and human dermal fibroblasts (50%). The tubular construct was matured in a bioreactor with a triple medium mix for 4 days under an increasing perfusion rate. It was then integrated in an abdominal aorta replacement model in nude rats for a period of 2 to 5 days. At the end of the study, ECs that were originally integrated into MultiCS were found at the luminal surface of the graft, forming an intima-like layer. Interestingly, an increase of the graft inner diameter was also observed. Although the immunodeficient model is essential to avoid immune rejection when using mature adult cells, it did not allow investigations into TEVG safety and the interactions between MultiCS and the host immune cells, in addition to the potential modulation of the host inflammatory response. Moreover, the graft length was only 7 mm which is incompatible with clinical applications . As already mentioned for other studies, this graft should be adapted for clinical use regarding its length and the origin of the integrated cells. A deeper characterization should be conducted around its safety, its patency and efficiency.Bio-printing is a biomedical application using the principles of 3D printing to artificially produce living biological tissue. Bio-printing is a fast-growing field today because its potential applications are vast and promising in the medical field as well as in research60. Moreover, in allogeneic implantation models, they may induce a strong host immune response against the foreign graft. In order to allow advanced studies in the context of allogeneic implantations in immunocompetent animals, undifferentiated stem cells are more appropriate. BMNCs seem to be a potential suitable candidate for TEVG cellularization because of their differentiation potential into both types of vascular cells. However, BMNCs are a heterogenous population composed of a large mix of cells, among them EPCs, ECs, MSCs, hematopoietic stem cells, T cells, and B cells, which induce complex interactions with host cells that are difficult to fully decipher and understand. This excessive heterogeneity could be a real obstacle for potential clinical applications where total control of the graft production is required, as well as a complete understanding of graft–host interactions. Several homogeneous stem cells populations possess the same differentiation potential as MDSCs or MSCs. Only a few studies on MDSCs are found in the literature compared to MSCs, which seem to be more appropriate for TEVG production. Indeed, their use could be more relevant since they have a homogeneous population, having adhesion capacity and differentiation potentials into both SMCs and ECs. Moreover, these cells are easily harvested from different tissues and are known to have important immunomodulatory properties which can be relevant to modulate the host immune system and avoid the graft rejection in the case of allogeneic cells61.In the VTE field, allogeneic cells are widely studied. In fact, the cell type is an essential and determining criterion in the construction of TEVG with regard to the required functionality. TEVG with mature ECs and SMCs are still in the preliminary study phases, in which nude animals are used to allow the observation of the graft development once integrated into the blood circulation. However, several problems inherent to the use of mature cells are to be considered. First of all, mature cells cultured in vitro over long periods present the risk of dedifferentiationVTE combines cells, biomaterials and a suitable microenvironment for the production of small-caliber TEVG and aims to optimize the graft cellularization together with the biomechanical signals and scaffold structure. As described above, many studies have proposed the cellularization of a scaffold which will then be matured and implanted. Besides, other teams have developed innovative approaches to address several problems related to the TEVG production time and storage. The major challenge with cellularized TEVG is to find a suitable system and the combination of appropriate conditions for their storage without affecting the cell viability or the scaffold structure. Innovative cell-mediated production approaches concern the “Off-the-Shelf” and “Precast” grafts.62. They evaluated this original approach in several in vivo bypass models as a femoral bypass in sheep for 24 weeks63 and more recently as an arteriovenous bypass in baboons for up to 6 months64. In the last study, the graft was manufactured by seeding commercial human dermal fibroblasts into a bovine fibrinogen solution. The cell suspension was then injected into a tubular mold carrying a glass mandrel. Grafts were then cultured for 2 weeks and matured in a bioreactor for 3 supplementary weeks62. After maturation, grafts were decellularized and then stored in PBS at 4 °C. Grafts implanted as arteriovenous fistulas in baboons were evaluated by ultrasound assessments at 1, 2, 3, and 6 months, then scanned by angiography at the end of the study, just prior to collection. Graft patency rates were estimated at 83% at 3 months and 60% at 6 months. Moreover, an increase of the graft diameter was observed. This was probably induced by a remodeling of the graft tissue by vimentin and/or α-SMA positive cells and the colonization of the luminal surface by CD31 positive cells. However, some early thrombosis problems have been detected, probably due to the implantation site which has been changed to address these events in eight baboons. This led to a considerable reduction of the cohort. Results are promising and are similar to actual clinical solutions for arteriovenous fistula based on an ePTFE substitute. This model, although encouraging, is not representative of the small-diameter vessel bypass clinical situations, where grafts are subjected to challenging conditions like the blood pressure, the resistance to sutures and mechanical strains.Several teams are trying to produce a “ready-to-use” and storable graft that can be implanted in any patient, i.e., a “universal” or “off-the-shelf” graft. In order to propose such a graft, Tranquillo’s team has developed a decellularized graft originally composed by synthetic ECM18 have proposed a storable decellularized graft which can be recellularized with autologous cells. Here, the graft was tested in a porcine carotid replacement model for a period of 30 days. A 4 mm diameter PGA mesh scaffold was seeded with a suspension of porcine aortic SMCs for 10 weeks under pulsatile conditions. The graft was then decellularized. Two cells sources were studied for the recellularization, either autologous ECs or EPCs, which were seeded onto the graft under pulsatile and rotational conditions for 2 h, followed by 2 h of static culture. The graft was then preconditioned in a bioreactor under a progressively increasing shear stress for 4–5 days. Three EPC-seeded grafts and two EC-seeded grafts were implanted and compared with a control group of three unseeded grafts and another control group consisting of 8 internal jugular veins. All the seeded grafts were still patent at 30 days post implantation compared to three control grafts . Unseeded grafts implantations have confirmed that the recellularized “precast graft” did not induce substantial inflammation. Moreover, a slow host cell colonization of the seeded graft matrix was highlighted by the presence of α-actin positive cells into the graft wall. In addition, tissue remodeling was also observed on the luminal surface where seeded cells were partially replaced by host ECs. Although the “precast graft” innovative approach seems to reduce the time required for the TEVG development, there is still a considerable amount of time for the patient autologous cell culture, plus 6 days of seeding and preconditioning. Moreover, the authors did not set up and define the storage conditions of their TEVG, which is one of the main criteria of this promising approach.The use of autologous cells is limited by the lengthy manufacturing time, which may be incompatible with the clinical situation of the patient. In order to reduce the production delay, Quint et al.65 by seeding a PGA scaffold with allogeneic canine SMCs  for 7 to 10 days, followed by a decellularization step. The decellularized matrix was then washed and stored at 4 °C. The endothelialization step was performed by a fibronectin coating of the luminal surface of the TEVG followed by an autologous EC seeding under rotational conditions. The graft was then matured before implantation, by an increased shear stress preconditioning in a bioreactor for 35 h. The potential alterations of the grafts due to the storage conditions have been studied by mechanical characterizations, before and after a period of 12 months storage in PBS at 4 °C. After this storage period, no significant change was observed for burst pressure, compliance or suture strength. Eight grafts have been tested in vivo in dog models as follows: five in a carotid bypass model for up to 12 months and 3 grafts in a coronary bypass model for up to 1 month. During the study, two animals died with a patent graft and only one graft occluded at 1 week. Despite the histological analysis showing an incomplete EC coverage of the grafts luminal surface, 5 out of 6 grafts remained patent until the end of the study. Patent grafts showed a considerable remodeling, notably by α-SMA cells presumably derived from adjacent native vasculature.A relatively similar approach is reported by Dahl et al.66, and the graft of 18 mm of diameter produced by Shin’oka et al. by autologous mononuclear BMCs seeded on a polymer tube67. Nevertheless, due to their specific clinical contexts, different from the problematic of this review, they will be excluded from this part of the review.This part of the review focuses on TEVGs with an inner diameter smaller than 6 mm that have progressed beyond the preclinical studies to phase I clinical trials and discusses the reported results as well as the encountered obstacles. Several medium (6–8 mm) and large (>8 mm)-diameter TEVGs have reached clinical trials such as the one of 8 mm of diameter developed by Olausson et al. consisting on a decellularized iliac artery seeded with autologous ECs and SMCs68. However, the mechanical behavior, i.e., the blood pressure applied to TEVG in an arteriovenous shunt model, is much less compared to that found in other potential applications of the graft in blood vessel bypass grafting models, which makes it difficult to evaluate its effectiveness and performance under more challenging conditions.The most commonly used model in clinical trials is the arteriovenous fistula model, which allows the graft to be evaluated under nonlethal conditions. Moreover, this is a clinical situation in which TEVG would be of a great interest since the substitutes currently used are expanded polytetrafluoroethylene (ePTFE). In fact, ePTFE have a limited lifespan due to the damage of the graft wall by numerous punctures, thrombosis, infections and intimal hyperplasia in the long term69 and nonhuman primates70, the first clinical trial performed with a TEVG was between 1989 and 199171. The study evaluated an endothelialized ePTFE graft in a femoropopliteal bypass model in 49 patients, in whom saphenous vein bypass was not applicable71. ECs were harvested from the right external jugular vein. An ePTFE graft with a diameter of 6 mm and a length of 60 cm was precoated with fibrin glue and seeded with autologous ECs, by filling the lumen with the cell suspension under rotation for 3 h. Then, the graft was implanted after 11.6 ± 3.0 days of a maturation period. Graft patency was determined by duplex sonography and Doppler ultrasonography after 9 days, 3 months, 6 months and 1 year of implantation. After 32 months, patency rates were estimated at 84.7% for endothelialized grafts vs. 55.4% for control non-endothelialized grafts. This endothelialized ePTFE TEVG was then included in a more generalized study involving 310 patients over a period of 15 years. Patients were divided into two groups, one group having a TEVG with a diameter of 6 mm, the other receiving a 7 mm diameter graft. The 6 mm diameter graft group has shown patency rates of 62% at 5 years and 55% at 10 years, vs. 78% and 71%, respectively, for the 7 mm diameter group72. Histological analysis of occluded grafts suggested that occlusions were not induced by an endothelialization defect but by large inflammatory foci located under the endothelium, causing a hyperplastic narrowing. Overall, the study demonstrates the interest of the integration of a VTE procedure as a routine therapy and confirms the benefit of autologous endothelialization of ePTFE grafts in clinical cases of infrainguinal bypass where no autologous vein is available. Major limitations of this graft include the use of an ePTFE synthetic scaffold that forms a static structure, preventing any possibility of growth and thereby excluding the graft use in a pediatric context. Moreover, the cellularization by autologous ECs implies several inconveniencies, such as the invasive collection of cells and the long-term in vitro culture that may increase the risk of contamination and loss of EC phenotype73.Following in vivo preclinical studies on dogs65.Before performing clinical trials with the “Humacyte graft”, which is a complete “off the shelf” small-caliber graft, Niklason and her team tested a preliminary version of their TEVG using harvested human aorta SMCs. This graft has been evaluated in a baboon model of arteriovenous bypass for 1 to 6 months and has presented a patency rate of 88%This Humacyte graft, a “human acellular vessel” (HAV), was produced by seeding human SMCs onto a biodegradable polymer scaffold (PGA) of 32 to 45 cm in length, for 8 weeks. The construction was matured in a bioreactor, applying pulsatile cyclic distension to allow the expansion of cells and the synthesis of their own ECM. The graft was then decellularized in order to obtain a tube of secreted ECM composed of different matrix proteins, such as collagens (I and III), fibronectin and vitronectin.74. The patency rates were checked by intraoperative ultrasound or Doppler and were valued at 63% and 28% at 6 and 12 months, respectively, (mostly due to thrombosis). The graft diameter increased during the implantation and reached 8.63 mm. During the clinical trial, some grafts at 16 to 55 weeks were collected after surgical revision procedures. Histological analysis revealed that at 16 weeks, the region near the vein anastomosis was colonized by myocytic cells expressing CD68 and α-SMA conjointly or separately, including myofibroblasts from surrounding tissue. The endothelialization of the graft was demonstrated by the detection of cells expressing CD31, VE-Cadherin, and eNOS at the luminal surface. At 46 weeks, the presence of these cells in the middle of the collected grafts seems to confirm that this phenomenon occurs from circulating blood and not from native adjacent vasculature. These two clinical studies have been extended to include 240 patients with a follow-up until 200 weeks75. Further histopathological analyses were conducted on collected grafts at different implantation times during the study to evaluate the HAV in vivo remodeling. These studies have revealed a tissue remodeling similar to that observed during embryonic vasculogenesis, which was characterized by the presence of Nestin+/CD34+ cells in the vascular wall at early stages. The number of these cells had decreased over time during implantation. In conclusion, the permeability results obtained with the “Humacyte” graft are substantially similar to those obtained with endothelialized ePTFE grafts. Despite its mechanical characteristics, similar or even superior to those of mammary arteries or saphenous veins, it suffers from the same shortcomings as synthetic grafts regarding repeated punctures. Indeed, several pseudoaneurysms were mentioned during these studies which are well-known complications of dialysis access with fistula or grafts.In 2014, enrollment of phase II trials (USA and Poland) was completed and 60 patients with end-stage renal disease (not candidates for fistula) had been grafted with the HAV “Humacyte graft” as an arteriovenous bypass for a period of 16 or 24 months77. This graft was clinically tested in an arteriovenous shunt model in 10 end-stage renal disease patients. Patency rates were 78% and 60% at 1 and 6 months, respectively. The principal inconvenience of this autologous graft is the production time which reaches up to 26 weeks. Considering this limitation, the authors have recently proposed a SBTE graft with a shorter production time, manufactured with only allogeneic fibroblasts without ECs at the luminal surface78. This allogeneic graft was tested in the same model of hemodialysis access in three patients. Despite the nuclear debris that remained in the graft’s outer layer after decellularization, this study has shown that the Lifeline allogeneic graft did not induce deleterious immune responses. Grafts were implanted for up to 11 months, during which no aneurysms were detected, however surgical interventions were required to ensure secondary patency (2/3) and only one graft was patent for 7 months. These disappointing results could be due to an incomplete endothelialization of the graft which could have more severe consequences in coronary or peripheric bypass models. Currently, Magnan et al. are working on a new TEVG manufactured with a “textile approach”, also based on an ECM fibroblast sheet which is transformed into ribbons79. These ribbons are actually threads used to produce a TEVG of a diameter of 4.2 mm which is resistant to burst pressure. The first study on its immunogenicity and efficacy was carried out in a sheep carotid bypass model for 1 day and did not induce any complications.L’Heureux and his team have developed an allogeneic small vascular graft called the “Lifeline graft” through “Sheet-Based Tissue Engineering” (SBTE). The TEVG was developed as follows: autologous fibroblasts were cultured and expanded, then the sheet of fibroblasts was decellularized (by dehydratation) and rolled up around a tube for 10 weeks, in order to obtain a homogenous cylinder. Another sheet of autologous fibroblasts was then used to cover the cylinder and the graft was kept in culture for a further 10 weeks. Autologous ECs were seeded into the graft several days before implantation (3–7 days), during which the graft was submitted to an increasing pulsatile flow in a bioreactorCurrently, vascular replacement techniques using synthetic substitutes are successfully used for the replacement of large and medium diameter arteries. However, these substitutes are not suitable for the replacement of small-diameter arteries (<6 mm), principally due to a compliance mismatch and a lack of endothelialization of their luminal surface, leading to thrombotic events. Thus, one of the greatest challenges in VTE is to develop and propose to cardiovascular surgeons an effective endothelialized vascular prosthesis able to replace small-diameter arteries.Since the first TEVG produced in 1986, the field of VTE seems devoted to a strong development and has never stopped to evolve, in order to create the “gold standard”, “readily available”, and “off-the-shelf” TEVG.13. As previously discussed, cells are widely used in the development of a small-caliber TEVG, in order to confer them with essential capacities that determine their success, such as hemocompatibility, patency, contractility, remodeling, growth or robustness. However, it should be noted that the cell types used in the TEVG manufacturing protocols are highly variable and can be from xenogeneic, allogeneic or autologous sources (summarized in Table Recellularization of TEVG is one of the major factors that contributes to a better patency in a clinically relevant contextWhen using autologous cells, the cellularization of TEVG involves several issues, such as the very long production time, high manufacturing costs, and the storage conditions for allogeneic grafts. The storage factor can be addressed by a decellularization step after ECM production. Grafts tested in the last two described clinical trials were developed according to this method and appear to be efficient as arteriovenous shunts. However, the use of these TEVGs in other arterial bypass contexts, such as coronary or femoral arteries bypasses, have still not been evaluated, even though these are among the most challenging situations for demonstrating the performance of a vascular graft.81. In the long term, minimally-invasive robotic surgery should be accessible and performed for bypass surgeries on a daily basis. Thus, the development of a “gold standard” TEVG could supply a bank of ready-to-use grafts that would be available to cardiovascular surgeons.In addition, grafting technology is in constant progress. In fact, robotic surgery is potentially the most advanced medical technology and has been well documented in recent yearsIn conclusion, our review shows the large heterogeneity of data produced in the study of cellularized small-diameter TEVG. This reflects the important need of small-diameter grafts and the considerable progress made in the VTE field in order to address this issue. However, this huge amount of data complicates the TEVG efficiency evaluation and the choice of the best development approach. There is an urgent need for standard guidelines for developing and testing TEVG in large animal models, at least regarding the cell type used for the recellularization. This would allow a proper comparison of the different strategies for the production of vascular grafts. The journey for finding the “gold standard” TEVG is on track to succeed, but there is still a long way to go for both research and clinical applications in the VTE field.


Acinetobacter baumanniitriggers autophagy, affects the degradation of autophagy, and causes severe inflammatory injury. LncRNA growth arrest-specific transcript 5 (LncRNA-GAS5) and Yin and Yang 1 (YY1) are known to play an important role in the regulation of autophagy, however, the precise role of LncRNA-GAS5 and YY1 in the damage to autophagy caused by Acinetobacter baumanniiremains unclear. The aim of this study was to investigate the role of LncRNA-GAS5 and YY1 in the regulation of autophagy induced by Acinetobacter baumannii. We found that LncRNA-GAS5 was up-regulated following infection with Acinetobacter baumannii, thus resulting in the degradation of STX17, autophagy disorders, and the aggravated replication of Acinetobacter baumannii. We also analyzed the mechanism of interaction between LncRNA-GAS5 and YY1 and found that YY1 regulated its expression in a negative manner by binding to the promoter of LncRNA-GAS5. LncRNA-GAS5 and YY1 had opposite effects on the expression of STX17, this process maintained the stable expression of STX17. Following Acinetobacter baumannii infection, YY1 was down regulated and then separated from the binding region of LncRNA-GAS5, thus resulting in the activation of LncRNA-GAS5 transcription and reduction in STX17 protein expression. Finally, we infected LncRNA-GAS5 knockdown mice with Acinetobacter baumannii, the expression levels of IFN-β in the lungs increased significantly, this alleviated lung injury. In conclusion, our work demonstrated the mechanism by which Acinetobacter baumannii infection can cause the degradation of STX17. We also demonstrated that LncRNA-GAS5 may be a potential therapeutic target for the treatment of lung injury induced by Acinetobacter baumannii. Acinetobacter baumannii is an opportunistic nosocomial pathogen that causes hospital-acquired pneumonia, septicemia, and urinary tract infection, especially in critically ill patients ). Significant differences were assigned to p values<0.05, <0.01 .Statistical analyses were performed using a two-tailed Student’s Acinetobacter baumannii on LncRNA-GAS5 expression, we used real-time PCR to detect the expression of GAS5 in HeLa cells infected with Acinetobacter baumannii at different times and concentrations. We found that the expression levels of GAS5 increased with the increased Acinetobacter baumannii infection time, reaching a peak at 24 hours (in situ hybridization (FISH) analysis to detect the subcellular localization of LncRNA-GAS5 in HeLa cells. We found that GAS5 was mainly localized in the nucleus of HeLa cells , to search for potential binding sites for YY1 within the promoter region of GAS5  into the lungs of mice to generate GAS5 knockdown mice and then infected the mice with Acinetobacter baumannii for 48 hours. Firstly, we examined the knockdown efficiency of AAV-shGAS5 in different tissues from the experimental mice. We found that the expression levels of GAS5 in the lung, liver, and spleen, decreased significantly three weeks after the injection of AAV-shGAS5  staining. The results showed that compared with the non-infected group, the lung tissue of mice infected with Acinetobacter baumannii showed inflammatory cell infiltration, the alveolar septum had widened and thickened, the size of the alveoli sac had decreased, and there was also evidence of bronchial hemorrhage. However, the degree of lung injury in mice infected with Acinetobacter baumannii but pretreated with AAV-shGAS5 was better than that in the Acinetobacter baumannii infection group  to regulate the expression of various genes at the proximal or distal end . By usinte STX17 .Figure 8Acinetobacter baumannii activated macrophages in the spleen of mice and induced higher levels of IFN-β release than in the Acinetobacter baumannii infection group. Furthermore, the number of macrophages in the spleen, and the release of IFN-β in the serum of AAV-shGAS5 mice infected with Acinetobacter baumannii, also increased significantly. These effects further weaken the ability of Acinetobacter baumannii to survive and exert protective effects on tissue damage. However, the mechanism responsible for how LncRNA-GAS5 regulates the release of IFN-β mediated by Acinetobacter baumannii remains unclear and needs further investigation.It is well known that type I IFN not only has an antiviral effect, but also plays an active role in a host’s resistance to bacterial infection. However, some bacteria can use their own virulence factors to control the release of type I IFN and disturb the immune inflammatory response ,35. In oAcinetobacter baumannii and promoted the invasion of host cells [Acinetobacter baumannii leads to the production of autophagosome and lysosomal markers in large quantities. This behavior seems to be more beneficial for the host to eliminate infection, but is not conducive to the survival of the bacteria. We believe that the real reason for the enhanced invasiveness of Acinetobacter baumannii after the activation of TFEB does not lie in changes in the amount of autophagosome and lysosome markers, but in terms of whether it interferes with the degradation function of autolysosomes. In the future, we should include TFEB to further analyze the destructive effect of Acinetobacter baumannii on autophagy degradation.It is worth noting that Parra-Millán R et al. found that the transcription factor TFEB was activated by st cells . TFEB plAcinetobacter baumannii infection. Our findings provide a potential therapeutic target for the treatment of inflammatory injury caused by Acinetobacter baumannii infection.In summary, we investigated the mechanism by which LncRNA-GAS5 interacts with the YY1 transcription factor to regulate STX17 in the autophagy degradation disorder induced by Click here for additional data file.


Resistant starch (RS) is a part of insoluble dietary fiber, and it could be recognized as a functional food ingredient in some types of confectionery products that lack dietary fiber. Unlike dark and milk chocolate, white chocolate does not contain fat-free cocoa solids rich in dietary fiber. In the present study, 5%, 10%, and 15% of white chocolate were substituted with RS in order to improve the nutritional value of enriched white chocolate. The influence of RS on rheological, textural, and thermal properties of the chocolate fat phase was firstly investigated, and then further influence on physical properties, dietary fiber content, and sensory characteristics of enriched white chocolates were investigated. The obtained results showed that enriched chocolates had increased content of total dietary fiber and reduced total fats and protein content in accordance with the added amount of RS. At the same time, RS increased viscosity and reduced the hardness and volume mean diameter in enriched chocolates in accordance with the added amount. RS improved the nutritional composition of white chocolate by increasing the content of dietary fiber. At the same time, RS did not impair the color and sensory characteristics of enriched white chocolates. Modern concepts of nutrition include the great importance of functional food because functional food products, apart from their basic nutritional functions, provide many physiological benefits and reduce the risk of chronic diseases. These food products either contain or add a component with a positive health effect or eliminate a component with a negative one . DietaryResistant starch refers to a part of starch present in the diet that escapes digestion and absorption in the small intestine and is fermented in the large intestine of humans, with the production of short–chain fatty acids (SCFA) ,2,3. TheResistant starch may not be digested for several reasons, such as a compact molecular structure that limits the accessibility of digestive enzymes or the starch granules are structured in a way that prevents the digestive enzymes from breaking them down. Additionally, thermal and mechanical treatment lead to the formation of starch crystals that are resistant to enzymes digestion or selected starches that have been chemically modified by etherification, esterification, or cross–bonding, cannot be broken down by digestive enzymes ,2.The application of RS in food products provides many benefits, such as desirable physicochemical properties. Its functional characteristics, e.g., swelling, viscosity increase, gel formation, and water–binding capacity, ensure good handling, improves texture in the final product, as well as crispiness and expansion . ResistaVarious bioactive compounds can be used in the development of functional foods, such as soluble and insoluble fibers, prebiotics, vitamins and minerals, herbal extracts, and other phytochemicals as the main ingredients which can be used as enrichment agents for chocolate ,8.White chocolate is a confectionery product made of sugar, milk solids, cocoa butter, lecithin, and vanillin where the particles of sugar and milk solids are covered by a continuous fatty phase (mainly cocoa butter). Milk solids commonly consist of whole milk powder, and in some formulations skimmed milk powder, whey powder, and lactose can be used. To be named “white chocolate”, white chocolate must include whole milk powder and cocoa butter in its composition, therefore it does not contain cocoa products other than cocoa butter . TherefoTaking into account all the features of resistant starch there is a very good possibility to apply RS for the development of functional white chocolate enriched with dietary fibers. Investigation of Ares et al.  showed tThe material used in this work included cocoa butter , white chocolate consisted of powdered sugar, cocoa butter, milk powder, whey powder, sunflower lecithin, and vanilla aroma , and resistant starch ActiStarTM RT 75330  which is a white neutral powder with moisture content 12%, pH 5,5, according to the product specification. In the first stage of the experiment, the model system (MS) was created with cocoa butter, which is also contained in white chocolate mass. The resistant starch (RS) was added to cocoa butter (MS0) in following amounts: 5% (MS5), 10% (MS10), and 15% (MS15). The rheological, textural, and thermal properties of the model system were examined.The pre-crystallization of cocoa butter and the mixture of cocoa butter and resistant starch was carried out in a modified Brabender farinograph where the original kneader is connected with two thermostats  with two-way taps. 100 g of chopped cocoa butter was placed in a farinograph kneader to melt at 50 °C for 45 min. Then the stirrer of the farinograph was switched on and the cocoa butter was stirred at 50 °C for 15 min, followed by another 10 min at 25 °C and another 15 min at 32 °C. The pre-crystallized cocoa butter was then poured into 10 × 10 g plastic molds and cooled in a refrigerator at 5 °C for 60 min. The same procedure was carried out in order to produce the sample MS0 and model system samples , where a certain amount of resistant starch was added to melted cocoa butter after 45 min at 50 °C. In the second stage of the experiment, the influence of RS on the quality of white chocolate was observed. The examined samples were: white chocolate without RS (WC0) and enriched white chocolate with 5% (WC5), 10% (WC10), and 15% (WC15) of RS.The pre-crystallization of white chocolate mass was also carried out in a modified Brabender farinograph where 100 g of chopped chocolate was placed in a farinograph kneader to melt at 40 °C for 45 min. Then the stirrer of the farinograph was switched on and the chocolate mass was stirred at 40 °C for 15 min, followed by another 10 min at 26 °C and another 15 min at 29 °C. The pre-crystallized chocolate mass was then poured into 10 × 10 g plastic molds and cooled in a refrigerator at 5 °C for 60 min. The same procedure was carried out in order to pre-crystallize the control sample of chocolate (WC0) and enriched white chocolate , where certain concentrations of resistant starch were added to melted cocoa chocolate mass after 45 min at 40 °C.Moisture, protein, fat, and ash contents of chocolate samples were determined after one day of storage, according to the methods described in the AOAC  . The tot−1 to 60 s−1 during a period of 180 s and then was kept constant for 60 s at max. speed of 60 s−1 and after that was reduced from 60 s−1 to 0 s−1, within 180 s. The rheological properties of samples were determined by a rotational rheometer Rheo Stress 600  at the temperature 40 ± 1 °C . The floThe determination of textural properties of the model system and chocolate was performed using Texture Analyser  following the 3-Point Bending Rig HDP/3PB method. Differential scanning calorimetry DSC 910, Thermal analyzer 990, and Dynamic mechanical analyzer  were used to determine the thermal profile of the model system and chocolate according to Lončarević et al. . Particle size distribution of resistant starch and chocolate samples were determined using the Mastersizer 2000 laser diffraction particle size analyzer . The Scirocco dispersion unit was used for dispersing resistant starch in the air, while the Hydro 2000 μP dispersion unit was used for dispersing chocolate in sunflower oil. The samples were added at ambient temperature until an adequate obscuration was obtained (5–10% for dry samples and 10–20% for liquid samples). The results were quantified as the volume-based particle size distribution by means of the Mastersizer 2000 software.0*, a0*, and b0* are values for the control white chocolate sample  decrease with increasing the amount of RS. However, there is no significant difference between samples WC0 and WC5 and samples WC10 and WC15. The volume mean diameter D decreases from 21.32 µm in WC0 to 18.82 µm in WC15 which is in accordance with the fact that particles in chocolate are desirable to be in the interval 15–30 μm [The addition of all amounts of RS to white chocolate does not significantly affect the d(0.1) parameter in enriched chocolates and significantly  highest L* value and significantly (p < 0.05) lowest a* and b* value indicating almost white surface color with a small share of a red and yellow tone. However, the addition of all amounts of RS to white chocolate did not significantly affect the lightness of the enriched chocolate surface. On the other hand, all enriched chocolate samples have significantly (p < 0.05) lower values of a* compared to the control sample of white chocolate. Furthermore, the addition of 5–15% of RS generally caused the decrease in b* values of enriched chocolates, where WC10 and WC15 have significantly (p < 0.05) lower values compared to WC0. Thus, the addition of RS in the white chocolate structure reduced the amount of red and yellow tone in observed white chocolate samples compared to the control one, according to the instrumental color determination.RS has the significantly  were obtained among all samples compared to the control sample, observing all chemical characteristics. Samples with starch addition had significantly higher moisture content compared to the control sample. In relation to the fat and protein parameters all samples were statistically different (p < 0.05) among them. As starch does not contain high amounts of fat and protein, it was expected that the amount of these parameters would decrease evenly with increasing starch content in the chocolate samples.Mean values for the chemical composition of chocolate samples are presented in As white chocolate contains a negligible proportion of fiber, the primary goal of our research was to increase fiber content in this product. Resistant starch contains 86.45% of fiber (the result is not presented in p > 0.05) among the chocolate samples  did not differ significantly ( samples . All samp < 0.05) from the other chocolate samples. With the increase in the proportion of resistant starch in the chocolate samples, there was a decrease in hardness. Sample WC15 had the lowest value for hardness, significantly lower than other samples, while samples WC5 and WC10 did not differ significantly in hardness, according to the panelists. These results are in accordance with the instrumental hardness measurement presented in The control sample (WC0) presented the highest mean value for hardness and differed significantly , while it did not have a significant influence on the chocolate melting in the mouth.The pleasant flavor and taste are the most important sensory attributes of chocolate because they determine the acceptance by consumers . As RS hAres et al.  mentioneObserving the results of sensory analysis, it can be said that the addition of resistant starch to white chocolate up to 15% did not affect the deterioration of sensory characteristics. The differences in the sensory attributes between samples, noticed by trained panelists are very small so that consumers probably would not even be able to notice them.The effects of resistant starch introduction to the formulation of white chocolate samples were investigated in terms of physical properties, nutritional value, and sensory characteristics. Resistant starch addition increased the water and dietary fiber content of white chocolate samples. The rheological properties of white chocolate samples showed strong dependence on the resistant starch addition to the formulation. Viscosity values of tested samples significantly increased with the increasing amount of resistant starch in the sample formulation. However, resistant starch supplemented chocolates had similar sensory characteristics as the control samples.


Case Report. This article describes a 9-month follow-up of the use of a flexible denture above a bone graft taken from the anterior iliac crest for adolescent patients with resected mandible due to ameloblastoma. Taking into account prosthetic considerations, radiography, and clinical observation, no complications were seen with the graft.  The use of bone grafts is a common procedure after excision and reconstruction of the mandible, although it is rare in children and adolescents due to incomplete growth, which means a long transition period until reaching an appropriate age for implants or more predictable outcomes.  It is safe to use a flexible denture as a prosthetic over an iliac bone graft block during the healing period. Ameloblastoma is a rare tumor, but it affects all ages, especially between the second and fifth decades, and equally, in both sexes, its incidence in the lower jaw is 80% compared to the upper jaw. Although it is a benign tumor, it has a pandemic effect because it grows without symptoms and tends to be recurrent in 90% of conservative approach  –4. ThereA 12-year-old male patient reported to the Department of Oral and Maxillofacial Surgery at the National Hospital of Hama with visible swelling; intraorally, there was a noticeable expansion of the mandible body in the posterior section. After the radiographic examination, the presence of multicystic ameloblastoma was suspected , which wA titanium reconstruction plate was adapted and contoured to the mandible body to reinforce and help stabilize a block graft, but we waited 8 months before applying the bone graft to ensure that there was no recurrence of excision, and due to the lack of technology required to operate a microsurgical reconstruction beside, the patient was unable to travel to another place where these experiences are available, so a nonvascularized bone graft was taken from the anterior iliac crest, and because the patient's bone is spongy and fragile, we waited a whole year, and after that, we confirm the success of the graft and the stability of its dimensions. During this period, a transpalatal arch was applied to prevent rotation of upper molars and maintain the arch width Figures  and 4. TIntraoral examination showed an obvious volume of soft tissue in the iliac graft region which had healed well , and allAs usual, the procedures began with making a primary impression using irreversible hydrocolloid material. The custom tray was made by autopolymerizing acrylic resin  and checked in the patient mouth. To fill the missed area of the resected mandible in the tray, impression compound sticks  were used to support the final impression material and to make the border molding; then, the functional impression was made with irreversible hydrocolloid material  and poured with the pink gypsum type IV  .The occlusal relationship was registered using a register plate made with autopolymerizing acrylic resin and modeling wax  to make the rim that holds the medium , taking into account the situation of the mandible and its deviation, and because this stage is considered critical, the relationship was manually directed until reaching the most stable position, taking advantage of the remaining teeth on the natural side, and then was confirmed during the clinical trial.The denture was processed with flexible acrylic resin  and delivered to the patient after adjustment procedures and assessment of both speech and mastication Figures  and 8; aA digital panoramic radiograph was made with a silicone bite on the biting block of the radiographic device to ensure that the patient bites the same situation after the end of the observation period which was 9 months .Its flexibility allows obtaining additional stability and retention by increasing the flange extension under hard or soft tissue undercuts, in addition to extending the clasp of more teeth , 29Softer surface compared with acrylic resin means that patients feel better when wearing it and there appears to be no problem with the fit  , 29Thermoplastic resins have hygienic advantages due to their low water absorption and solubility Aesthetically, it is better than acrylic resin The initial plan chosen for this patient was a flexible partial denture that will be used for a considerable period. All options for the type of the removable partial denture have been taken into account, starting with the cast metal one. But it is not considered a logical option due to the incomplete growth of young patient besides its high price, especially with the possibility of making a new denture after a while, in addition to the difficulty of controlling the lever effect around the rest with the presence of flabby tissue which may cause damage to the bone graft or natural teeth, as leaving the patient without a denture in this age is also illogical because of the need to restore the mastication and cosmesis in addition to the possibility of food trapping and possible infections of the surgical area due to the difficulty cleaning or impaired neuromuscular function . The secTaking into account some of its disadvantages, such as difficulty to repair or poor resistance to scratching , the patTo reduce the functional efforts transferred to the graft by reducing the total occlusal load applied in the supporting tissue, the lingualized articulation at the premolars was adopted but anatomic one at the first molar. For more effective chewing but with the deletion of the second molar , 24, panWithin the limits of this case and the duration of observation of a bone graft taken from the anterior iliac crest under a flexible partial denture, adhering to prosthetic considerations, it can be said that it is safe to use a flexible denture as a prosthetic over iliac bone graft block during the healing period .


Pseudomonas aeruginosa PAO1 in the presence (or absence) of one of three clinical isolates of the same species (Recipient) over ten serial transfers. We find that inhibition decreases in the absence of a recipient. In the presence of a recipient, antagonism evolved to be different depending on the recipient used. Our study shows that the evolution of interference competition by toxins can decrease or increase, experimentally demonstrating the importance of this type of interaction for the evolution of species interactions.The importance of interference competition, where individuals compete through antagonistic traits such as the production of toxins, has long been recognized by ecologists, yet understanding how these types of interactions evolve remains limited. Toxin production is thought to be beneficial when competing with a competitor. Here, we explore if antagonism can evolve by long-term selection of the toxin (pyocin) producing strain  Ecologists often distinguish between two types of competitive interaction. The first, exploitative competition, occurs through the depletion of a common resource: interactions among individuals are indirect, mediated solely through resource availability. The second is interference competition, where negative interactions occur directly through antagonistic traits such as the production of toxic chemicals, overgrowth, or aggressive behavior . While tThe evolution of interference competition between a focal strain that causes antagonism (the producer) and a competitor strain that is harmed (the recipient) involves a trade-off between the cost of producing the antagonistic trait and the benefits derived from eliminating competitors. Clearly interference competition should evolve only when the benefits outweigh the costs, however identifying the conditions under which this happens has proven challenging. Work on the production of antagonistic traits in bacteria like bacteriocins, proteinaceous toxins that specifically inhibit closely related  competitors, has demonstrated a preferential benefit to the producer when growing in structured environments . Under wThe relative costs of production could further be modulated by the strength of resource competition. Where resource competition is absent because no competitors are present, there is no advantage to producing an antagonistic trait: a genotype that avoids producing the antagonistic trait should enjoy a fitness benefit and so antagonism should steadily degrade over time . In the Pseudomonas aeruginosa (PAO1) in a spatially structured environment for ten serial passages in the absence or presence of one of three non-evolving clinical “recipient” strains whose provenance is described in Here, we performed an exploratory study on the evolution of antagonism. We evolved a laboratory strain of the pathogenic bacterium Pseudomonas aeruginosa PAO1 was used as the evolving strain , strains carrying the lacZ gene give rise to blue instead of white colonies. Using competition experiments with the original PAO1 strain and the PAO1 strain with lacZ inserted, we verified that this insertion had no effect on fitness. The recipient differ in their genetic similarity to PAO1  and the nalidixic acid resistance marker was introduced by selecting a spontaneous resistant mutant from the original strain. Using competition experiments with the original recipient strains and the recipient strain with the antibiotic resistance marker, we verified that the presence of this marker had no effect on fitness. Unless otherwise specified, strains were grown in liquid lysogeny broth (LB) at 37°C for 24 h.Standard laboratory strain g strain . Three cpatients , were usorescens . On agarPAO1 see  . To initiate a new round of selection, 5 μL of each M9 suspension  was transferred to a fresh plate with semi-solid overlay. Periodically, samples of transferred cultures were checked for absence of recipient using the selectable markers for PAO1  and the recipient , confirming that there was no (observed) carry-over of recipient strains during serial transfer. After seven transfers, one lineage from the Recipient 3 treatment was lost. The number of mitotic generations per serial transfer can be calculated assuming exponential population growth in a fully mixed culture (resulting in around 8 generations per growth cycle) or by using the doubling time of the cells and assuming linear colony expansion on a solid surface (resulting in around 50 generations per growth cycle). The actual number of mitotic generations most likely lies between these estimates.The ancestral evolving strain was cultured on semi-solid agar plates with or without a recipient for ten serial transfers, each with a 3-day growth cycle. Recipient, when present, were not propagated over multiple cycles. Rather, recipient strains were grown up from frozen stocks for every transfer and used to make semi-solid (0.7% agar) LB + X-gal overlays on solid (1.3% agar) LB + X-gal plates (100 μL recipient culture/5 mL overlay). For the Yield of the ancestral strain and evolved populations was determined in triplicate under the same conditions as the selection experiment as a proxy for fitness. After incubation, colonies were cut out of the plate and vortexed in 15 mL M9 for at least 30 s. The number of colony forming units (CFU) was then estimated using serial dilutions and plate counts on LB + tetracycline. For each environment, relative fitness was calculated using the yield of the ancestral strain as a reference. Inhibition (killing efficiency) was estimated in triplicate for the ancestral strain and evolved populations using the dilution method . BrieflyNo recipient, Recipient 1, Recipient 2, and Recipient 3), assay environment , and the interaction between these terms on fitness and killing, we used linear mixed effects models with plate and lineage within plate as random effects. Local adaptation to each social environment  was assessed using models in which the ‘assay environment’ term was replaced with these newly defined factors. A linear mixed effects model  was also used to evaluate the effect of genetic similarity  on killing. To assess whether the observed changes in inhibition could explain the observed changes in fitness, we used correlation tests for each environment separately, fitting the model on the mean inhibition and fitness for each lineage in each recipient environment instead of on the raw data (to account for non-independence). In cases of multiple comparisons, we used sequential Bonferroni tests. All statistical analyses were performed in R version 3.4.1 , and (2) the three populations from the Recipient 1 treatment with the highest relative inhibition score . Genomic DNA was extracted from 1 mL overnight culture in rich medium according to the ‘DNA purification from gram-negative bacteria using the Gentra Puregene Yeast/Bact Kit’ protocol of QIAGEN®. The DNA concentration was determined with QuBit. Library (PCR-free short-insert library) construction and whole genome shotgun sequencing was performed at BGI Tech Solutions in Hong Kong using an Illumina HiSeq4000 platform and 150 bp short-read sequencing technology (PE150) with 100x coverage.To determine which genomic changes that had occurred during our selection experiment, we isolated genomic DNA from: (1) the ancestral evolving strain, (2) the three populations from the Q < 30), and discarded if very short (<50 bp). Mapping to the reference P. aeruginosa PAO1 genome was performed using standard settings. Variant detection for the evolved populations was performed using the low frequency variant detector, again using standard settings, except that we only considered variants with a minimum frequency of 10%, and used a relative read direction filter of 5%. Variants were filtered against control reads, i.e., the mapping of the ancestral strain, and kept if fewer than three control reads supported the variant. Annotation of variants was performed using the P. aeruginosa PAO1 CDS (consensus coding sequence) track.We used the CLC Genomics Workbench 9.5.3 for all sequencing analyses. Reads were trimmed to remove adapter sequences and low-quality regions  of one of three non-evolving recipient strains over multiple rounds of propagation. Evolved populations were assayed for fitness and the capacity to inhibit the growth of recipient strains for all evolved populations relative to the ancestral PAO1 strain that founded the evolution experiment.F9,361 = 5.03, P < 0.0001; fitness higher than 1 in all four cases; P ≤ 0.0015). Some degree of local adaptation also evolved. Specifically, strains evolved in the presence of Recipient 1 and Recipient 2 performed better in the presence of their own vs. any other recipient. Conversely, strains evolved in the presence of Recipient 3 performed worse in the presence of their own recipient , suggesting there has been adaptation to the conditions of culture and a general adaptation to the presence of a competitor (recipient).Populations at the end of the experiment had increased yield, a proxy for fitness, relative to the ancestor in the environment in which they evolved . Strains evolved in the presence of Recipient 1 and Recipient 2 had a higher inhibition in the presence of their own vs. other recipient, while strains evolved in the presence of Recipient 3 had a lower inhibition in the presence of their own vs. other Recipient .The results for inhibition (killing efficiency) show that levels of inhibition have changed during our selection experiment, suggesting that our experimental protocol succeeded in selecting on antagonism. Populations from the Recipient 1 environment, inhibition and fitness were positively correlated, whereas in the Recipient 2 and Recipient 3 environment, these measures were negatively correlated. However, this correlation was significant only for the Recipient 3 environment .To assess whether the observed changes in inhibition could explain the observed changes in fitness, we determined whether these two measures correlated in each of the three recipient environments. In the No recipient treatment with the lowest inhibition , and the three populations from the Recipient 1 treatment with the highest inhibition . Our results revealed that on average 4.17 SNPs or small indels were segregating in each population at frequencies > 10%, and the mean number of such mutations per individual  was 1.83 . The large majority of all mutations (22/25) conferred a non-synonymous change, and evolution was highly parallel at the gene and functional level. Three genes were hit independently multiple times , and most mutations were related—either directly or indirectly—to quorum sensing  or motility . With the exception of mutations in pilA, found exclusively in the No recipient populations, populations from different treatments did not differ substantially in terms of mutation identity.We performed whole genome sequence analysis of the three populations from the was 1.83 . These nP. aeruginosa Liverpool Epidemic Strain (mvfR in population NO_R.4), and in a second case, the prophage integrated into the same gene in two independent populations . LESB58 Φ4 also integrated into two uncharacterized genes in populations E3 and E5 . While fitness (yield) increased in all environments, we found inhibition (killing) decreased in the absence of recipient. In the presence of recipient, inhibition showed a range of responses: it could decrease or increase, and be more or less specific to a particular recipient, depending on which recipient was used. Whole-genome sequencing of evolved populations revealed a range of possible mechanistic explanations for these results.We evolved a laboratory strain of the opportunistic pathogen At the phenotypic level, we found that the direct response to selection, that is, the change in fitness among strains selected and assayed in their selection environment, was positive for all strains. This shows that selection was effective at causing adaptation over ten serial transfers, both in the presence and absence of competitor strains during selection. Interestingly, all strains, even those that evolved in the absence of Recipient, increased their performance when assayed in the presence of Recipient, likely due to generic adaptation to the laboratory selection environment. While selection in the absence of competitors did not compromise performance when assayed in the presence of competitors, the reverse was not true. Adaptation in the presence of competitors does not improve fitness to the same extent when competitors are absent, suggesting that adaptation in the presence of a recipient involves recipient-specific genetic effects. This interpretation is even more strongly reinforced when we consider evolutionary changes to the level of inhibition: prolonged evolution in the absence of recipient leads to a highest loss of antagonism. These results thus suggest that the social environment can impact the outcome of adaptive evolution and, in particular, that prolonged selection in the absence of competitors can lead to dramatic phenotypic changes involving the loss of antagonistic traits.Recipient 1 and 2: inhibition of the ‘own’ strain was generally higher than for ‘other’ strains. There was no evidence for local adaptation in inhibition in Recipient 3, however. It is difficult to draw general conclusions from these results beyond the broad statement that the response to selection in the presence of un-evolving competitors can be highly variable and that the degree of inhibition is a trait that can evolve both upwards and downwards. The factors governing the extent to which the suite of antagonistic traits have general or specific effects on recipient remains a topic for further exploration. It may be that prolonged coevolution could lead to more specific adaptation, as has been observed previously in phage-bacteria experiments  both inhibition and fitness increased in the producer strain. This result supports the notion that interference competition by means of anti-competitor toxins, while costly to the producer, can be associated with an increase in fitness. Moreover, it provides a direct demonstration that antagonistic traits could be linked to positive selection. A caveat to this interpretation is that we do not know precisely which of the range of toxins available to producers are under selection; chemical and other analysis that the inhibition we observed is due to these toxins is required , it may interact with the existing arsenal of other prophages and bacteriocins in the evolving strain to increase their efficacy  the study. SS and CT-M carried out the laboratory selection experiments. CT-M performed the DNA extractions for sequencing. FG and SS performed the data analysis. FG made the figures. FG, RK, and SS drafted the manuscript with input by CT-M. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.