Trends in Tissue Engineering for Blood Vessels

Over the years, cardiovascular diseases continue to increase and affect not only human health but also the economic stability worldwide. The advancement in tissue engineering is contributing a lot in dealing with this immediate need of alleviating human health. Blood vessel diseases are considered as major cardiovascular health problems. Although blood vessel transplantation is the most convenient treatment, it has been delimited due to scarcity of donors and the patient's conditions. However, tissue-engineered blood vessels are promising alternatives as mode of treatment for blood vessel defects. The purpose of this paper is to show the importance of the advancement on biofabrication technology for treatment of soft tissue defects particularly for vascular tissues. This will also provide an overview and update on the current status of tissue reconstruction especially from autologous stem cells, scaffolds, and scaffold-free cellular transplantable constructs. The discussion of this paper will be focused on the historical view of cardiovascular tissue engineering and stem cell biology. The representative studies featured in this paper are limited within the last decade in order to trace the trend and evolution of techniques for blood vessel tissue engineering.


Introduction
Many recent studies have focused on cell therapies, scaffold based and cell based, for cardiovascular diseases primarily because these pathologies still rank among the top ten leading causes of mortality and morbidity worldwide. Annually, thousands of people die due to myocardial infarction, congestive heart failure, stroke, valvular heart diseases, and vascular diseases. According to the recent report of the American Heart Association, in 2008, an average of one death occurred every 39 seconds accounting to more than 2200 American deaths due to cardiovascular diseases (CVD) each day, with greater rate among black than white Americans [1]. Bearing in mind that the administration of various drugs caused a number of chemical reactions in the body and even led to autoimmune complications instead of treating the primary disease, it is therefore noteworthy that advancements on regenerative medicine and tissue engineering are highly beneficial. Tissue engineering and regenerative medicine do not only offer fast recovery but also lessen the medical and economic burden among the patients suffering from cardiovascular diseases.
At present, a vast of information and techniques on biofabrication and stem cell biology have been established; however, the effectiveness based on the clinical applications of these techniques remains to be elucidated. This paper will highlight the updates of regenerative medicine and tissue engineering techniques which addressed cardiovascular diseases. In particular, this will include the evolution of scaffold and scaffold-free cell therapies for the blood vessel defects as well as the various stem cells used for engineering blood vessels. The updates on tissue engineering for heart valve and the myocardium will be reviewed subsequent to this paper.

History of Tissue Engineering for Cardiovascular Tissues
It is globally known that in the developed countries, cardiovascular diseases are the primary causes of mortality and morbidity. Based on the review done by Zaragosa et al., both genetic and environmental factors are associated with the cardiac and vascular complications. Therefore, these complex multifactorial pathologies are very difficult to prevent [2]. Although prior papers emphasized that new drugs and innovative devices have improved quality of life for patients inflicted with cardiovascular diseases, they have not necessarily reduced the mortality and morbidity rate [3]. Then, organ transplantation provided a new therapeutic path. Transplantation offers an immediate "cure" by replacing the damaged tissues or organs with normally functional substitutes [4].
Since it is the preferred treatment for organ failure, there is a growing need for transplantable human organs, either autologous or from certain donors [5]. However, there are two major lifelong obstacles that have restricted organ transplantation, namely, the critical paucity of donors and the high risk of graft rejection [6]. In fact, successful treatment to cardiovascular diseases has been limited due to lack of suitable autologous tissue to restore injured cardiac muscles or to serve as vascular conduits to replace or bypass diseased or occluded vessels. On the other hand, the immunosuppressive agents bridged the mortality and morbidity of the organ failure to the risk of infection, cancer, and cardiovascular diseases [3,4]. Hence, tissue engineering has been projected as an alternative treatment to these problems by replacing the damaged tissue or organ function with constructs which are biofabricated based on the required tissue or organ features [3]. In particular, cardiovascular tissue engineering is more valuable and relevant compared to other fields of tissue engineering mainly because it increases life expectancy, preserve, the extremities, and caters a vast number of patients [7]. Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of functional substitutes for damaged tissues. It is anchored on the fundamental concept of utilizing the body's natural biological response to tissue damage in conjunction with engineering principles [8,9]. In addition, tissue engineering is designed to produce biomimetic constructs, which resemble normal tissues, to replace the damage tissues. Moreover, the main objective of tissue engineering is the restoration of function through the delivery of living elements which become integrated in the patient [10].
Tissue engineering strategies have three basic components: firstly, the cells or source which must express the appropriate genes and maintain the appropriate phenotype in order to preserve the specific function of the tissue [11], secondly, the bioreactive agents or signals that induce cells to function, and thirdly, the scaffolds that house the cells and act as substitute for the damaged tissue [12]. The source may either be embryonic stem cells (ESC) or adult stem cells (ASC) in origin, the scaffolds may be categorized as synthetic, biological, or composite, and the signals may include growth factors/cytokines, adhesion factors, and bioreactors [13]. In many studies, the tissue engineering components have been considered and explored focusing on single component only (S component only-source, scaffold, or signal only), or in combination (S-S components-Scaffold/Signal, Scaffold/Source, and Signal/Source), or altogether (S-S-S-Source, Scaffold, and Signal) ( Figure 1).
Currently, the evolution of bottom-up and top-down approaches ( Figure 2) in tissue engineering has been continuously investigated by many groups as the most promising tissue engineering approaches. The bottom-up approach usually employs implantation of precultured cells and synthetic scaffold complexes into the defect area. The cells or source, generally isolated from host target tissues, are expanded in vitro and preseeded into the scaffold to provide a porous three-dimensional structure that accommodate the seeded cells and form extracellular matrix. [9,[14][15][16][17]. Subsequently multiple methods such as cell aggregation, microfabrication, cell sheeting, and cell printing are utilized in generating modular tissues. They are then assembled through random assembly, stacking of cell sheets, or directed assembly into engineered tissues with specific microarchitectural features. Thereafter, the engineered tissue is transplanted into the defected area. Therefore, this approach allow, scientists to finely transform the nanostructure of materials by balancing polymer degradation rates with the extracellular matrix (ECM) production and cellular infiltration which caused the increased cell binding sequences, enzymatic cleavage sites, and tethering of chemoattractant molecules [17][18][19][20]. Conversely, in the top-down approach, there are two ways to manufacture the engineered tissue: (1) cells and biomaterial scaffolds are combined and cultured until the cells fill the support structure to create an engineered tissue [17] or (2) the acellular scaffolds, incorporated with biomolecules, are delivered immediately after injury. The biomolecules are released from scaffolds in a controlled manner, and they may recruit the progenitor cells in injured area and promote their proliferation and differentiation and eventually repair the injured tissues [9,[14][15][16].

Blood Vessel Structure, Functions, and Cell Sources
Blood vessels extend throughout the body and mediate gas exchange, nutrient and waste transport, and immune defense. The blood vessels consist of endothelial cells that are in contact with the blood, vascular smooth muscle cells that cover the endothelial cells as well as form the middle layer, and the fibroblasts and matrix that form the vessels' outer layer. Based on the earlier evaluation, cells of the blood vessels perform different functions in relation to cardiovascular physiology ( Figure 3) [21][22][23][24][25][26][27][28][29]. Altogether, these layers of cells play a role in repairing, remodeling, and maintaining the blood vessels following an injury. All these cell types have been involved not only in cardiovascular pathology but also in therapy. In a recent review, Chen Figure 1: Tissue engineering triad of cells or source, signals (provided chemically by growth factors/cytokines or physically by a bioreactor), and the scaffold which acts as a template for tissue formation allowing the cells to migrate, adhere, and produce tissue. These components make up the Triple S (Source, Scaffold, and Signal) of tissue engineering. Any combination of these triad components has been considered in various studies in cell therapy which accounts for SS (Scaffold/Signal, Scaffold/Source, and Signal/Source). Finally, these three components (Source, Scaffold, and Signal or SSS) have also been considered altogether in some research projects.
"Bottom-up" approach "Top-down" approach cardiac repair particularly in acute myocardial infarction; the pericytes, on the other hand, contribute to the regeneration of dystrophic skeletal muscles, while the adventitial cell participates in vascular remodeling and regeneration of the surrounding tissues [28,30,31].
Blood vessel diseases such as atherosclerosis and arteritis [30,32], Chronic Venous Insufficiency (CVI) [33], and thrombosis remain as major vascular problems globally. Hence, the increasing morbidity of cardiovascular diseases in the modern society has made it crucial to develop blood vessel substitutes especially for those small diameter vessels (less than 6 mm) for the replacement of diseased coronary and below the knee vessels. Current therapies for such diseases include surgical replacement with autologous vessels or synthetic materials [34,35]. Although autologous vessels remain the standard for small grafts, many patients have a limited vessel suitable for use because of vascular disease, amputation, or previous harvest [36]. Despite a clear clinical need for a functional arterial graft, success has been limited to arterial replacements of large-caliber vessels such as the thoracic and abdominal aorta, arch vessels, iliac, and common femoral arteries; however, small-caliber arterial substitutes, which account for a majority of the demand, have generally proved inadequate largely because of acute thrombogenicity of the graft, anastomotic intimal hyperplasia, aneurysm formation, infection, and progression of artherosclerotic disease [37]. From this time, tissue engineering is the promising approach to address the shortcomings of such treatment [36,37].
Although advances in vascular tissue engineering have had limited clinical application because of the cell's limited replicative capacity, loss of telomerase activity of adult somatic cells, and the long-term production of the biological construct [38,39], many investigators have further explored techniques with the objective of fabricating biomimetic tissue-engineered blood vessels (TEBV). Creating an engineered blood vessel substitute requires the incorporation of both smooth muscles and endothelial cells into the tubular scaffold so as to establish an architecture like that of a native blood vessel [40]. The first production of completely biological tissue-engineered blood vessels, composed of intima, media, and an adventitia, using cultured mature smooth muscle cells and endothelial cells in bovine collagen gels was pioneered by Weinberg and Bell [41] and this rapidly expanded the number of studies akin to blood vessel diseases. It is now well known that the smooth muscle layer of the blood vessel plays an important role in maintaining homeostasis of blood vessels [42]. Additionally, vascular smooth muscle cells do not only play important roles in the physiological function of the blood vessels but also in their remodeling under pathological conditions [31]. Thus, generating functional smooth muscle layer is a prerequisite for successful blood vessel construction through tissue engineering approach. Smooth muscle cells and endothelial cells were the early sources broadly utilized to construct vascular implants [42,43]. Nevertheless, due to the limited proliferation ability and loss of contractile phenotype of mature-differentiated smooth muscle cells (SMCs), various alternative sources of cells have been explored for the production of blood vessel replacements. Their corresponding characteristics, some of these were previously described, [42] are summarized in Table 1.

Scaffolds for Blood Vessels
To date, despite numerous scaffolds that have been manufactured through varied forms of tissue engineering techniques, the construction of an entirely biomimetic blood vessels is still underway. To achieve a successful clinical application of tissue-engineered blood vessels, the biofabrication of vascular grafts necessitates a vigorous yet time-efficient biotechnological process [39]. Several tissue engineering (i) readily accessible autologous cell source (ii) BMC aspiration is less invasive and associated with much lower morbidity at the donor sites (iii) have the potential to regenerate vascular tissues (iv) improve patency in tissue-engineered small-diameter vascular grafts (i) may induce calcification and thrombus formation [63,64] Human artery-derived fibroblast (HAFs) (i) promotes enhanced ECM formation and maturation [39] Human umbilical cord vein endothelial cells (HUVEC) (i) important in endothelialization after transplantation (ii) prevent platelet adhesion (iii) largely and routinely cultured from a readily available supply of discarded tissue (iv) have reproducible and enhanced angiogenesis capacity (in vitro) (i) time-consuming isolation (ii) cell culture includes risk of infection and requires exogenous growth factor (iii) low proliferative capacity [39,65,66] strategies have emerged to address biological flaws at the blood-material interface of the synthetic scaffolds, hence, paving the way to vascular cell seeding and design of bioactive polymers for in situ regeneration. Moreover, advances in biomaterial design have been directed towards the generation of suitable materials that does not only mimic the native vascular tissue's mechanical properties but also promote cell growth, inhibit thrombogenicity, and facilitate extracellular matrix production [18]. In addition, an important characteristic of artificial scaffolds in advanced biomaterial vessel substitutes is not just the tolerance of the cells but the capacity to mimic the natural ECM in order to regulate extent and strength of cell adhesion, growth activity, cell differentiation, and maturation to the desired phenotype [84][85][86]. The extracellular matrix proteins such as collagen, elastin, fibronectin, vitronectin, and laminins which mediate cell-material adhesion have been thoroughly assessed in an earlier review [31].
Materials for vascular replacements should be biomimetic in such a way that they should be resistant not only to thrombosis, but also to inflammation, and neointimal proliferation, and for all intents and purposes, they should resemble the native vessels [3,31]. For these reasons, it is necessary to investigate the physical, chemical, and 6 Journal of Biomedicine and Biotechnology  (1) shear stress application maintained the intact monolayer of EC in the vessel's lumen (2) endothelial cells (ECs) are aligned in long axis parallel to the blood flow (3) shear stress also increased PECAM-1 and E-cadherin and integrin αγβ3 expressions (4) amniotic fluid tube reduced the TEBV fabrication through sheet-based engineering in vitro [74] 2012 Tubular hydrogels of circumferentially aligned peptide amphiphile nanofibers SMCs by applying low shear stress and ionic crosslinking (1) arterial cell scaffolds encapsulates and orient vascular cells (2) direct cell organization without external stimulation or gel compaction in vitro [75] biological properties and modifications of materials to further understand the molecular mechanism of the cellmaterial interaction [31]. The lack of endothelial cells on the luminal surface of the artificial grafts contributes to synthetic graft thrombogenicity and promotes intimal proliferation within the graft. Endothelial cell (EC) seeding on the synthetic grafts has been attempted to mitigate these problems. Herring et al. [87] were among the first to perform endothelial cell (EC) isolation and their subsequent transplantation into vascular graft. Current researches indicate the significance of such process in vascular tissue engineering. The polymer surfaces which have been formerly investigated for endothelial attachment, proliferation, and function had been listed in an earlier review [3]. On the other hand, the synthetic polymers for reconstructing blood vessels for clinical practice which are based on polyethylene 8 Journal of Biomedicine and Biotechnology  (i) fibroblasts seeding on fibrin gel (ii) direct injection of cell/fibrinogen suspension into glass mandrel tubular molds (iii) two weeks static culture system (iv) nine weeks multigraft pulsed flow-stretch culture system in a bioreactor (v) noninvasive strength monitoring (1) cells cultured in pulsed-flow bioreactor produced more collagen and with higher burst pressures (2) the tissue suture retention force was suitable for implantation in rat model and in ovine model using poly(lactic acid) sewing rings in vitro in vivo in rat and ovine model in vitro in vivo in rabbit model [83] terephthalate (PET) or polytetrafluoroethylene (PTFE) had been previously reviewed [31]. Furthermore, blood vessel stem cells have been studied in combination with recent and alternative types of scaffolds/polymers. Parallel to this, in scaffold-based blood vessel engineering, bioreactors and pulsatile flow systems, designed by many scientists, have been found to progress the mechanical property of the engineered blood vessels by augmenting the deposition and remodeling of extracellular matrix as well as the maturation and differentiation of self-assembled microtissues [38,39,67,68,76]. Bioreactors, which were originally designed for industrial use, have high degree of reproducibility, control, and automation for specific experimental bioprocesses and these have been the reasons for their transfer to largescale applications including vascular tissue engineering. The bioreactors allow scientists to manipulate the environment and the parameters such as pH, temperature, pressure, nutrient supply, and waste removal in order mimic the in vivo physiological condition and allow biological or biochemical processes to occur and subsequently develop the desired tissue [88]. Taken together, the formation of a microvasculature within a tissue-engineered organ or tissue will depend on multiple factors: the biochemical environment, EC type, the micro-architecture presented by the scaffold material, and mechanical signals [89,90]. Due to the goal of developing biomimetic blood vessel scaffolds, many groups have designed such biomaterials. The representative studies on the different blood vessel scaffolds are depicted in Table 2. The polymers used in scaffold fabrication for tissue engineered blood vessels started from polyglycolic (PGA) to varied types such as polyglycolic acid-poly-L-lactic acid (PGA-PLLA), Collagen/Elastin, chitosan, Poly (glycerol sebacate) (PGS), and very recently polyglycolide knitted fiber, and an L-lactide and ε-caprolactone copolymer sponge crosslinked to amniotic fluid. Furthermore, amniotic membranes have been used as scaffolds which signify that scaffoldbased tissue-engineered blood vessels can be fabricated from autologous cells at a reduced manufacturing period.

Scaffold-Free Techniques for Blood Vessels
The chronic inflammation, thrombosis, rejection, and poor mechanical properties of allogeneic or xenogeneic and synthetic vessels have impaired their clinical applications [79]. In addition, due to the failure cell to cell interaction and the assembly and alignment of ECM components, and the complex host response to scaffolds, the scaffoldfree techniques had recently emerged [80]. In scaffold-free tissue engineering approach, the fabrication of the tissue construct is anchored in the crucial capability of the cells to manufacture their own extracellular matrix [39]. In 1998, the first scaffold-free tissue-engineered human blood vessel was established by L' Heureux and has been replicated for further preclinical evaluation using rat and mice models in 2006 [76,79]. Years later, groups of scientists reported a fully biological self-assembly approaches by implementing rapid prototyping bioprinting method and stimulation via bioreactors for scaffold-free small diameter vascular reconstruction [39,80].
Similar to scaffold-based technique, in tissue engineering for scaffold-free blood vessels, the bioreactors were also used to provide specific biochemical and physical signals to regulate cell differentiation, ECM production, and tissue assembly by using chemical, mechanical, or electromagnetic stimulation techniques to produce de novo tissue with properties comparable to the damaged or desired tissues [91,92]. There are many types of launched bioreactors however, in engineering the vascular tissues, designs of various bioreactors have been based on the expansion and recoil properties of blood vessels, and so the combinations of stress, strain, and perfusion stimulation in biomimetic bioreactors have successfully developed vascular tissues [92].
In case of cell senescence problem, lifespan extension via telomerase expression in vascular cells (smooth muscle cells and endothelial cells) from elderly patients has been found as an effective strategy for engineering autologous blood vessels and eventually provides bypass conduit for atherosclerotic diseases [38]. Human telomerase, composed of an RNA component and a reverse transcriptase (hTERT), maintains the telomere length at the ends of the chromosomes [93]. Absence of hTERT expression in mature somatic cells induces lack of telomerase activity thus its ectopic expression has been shown to restore telomerase activity, arrest telomere shortening and senescence in some cells [94]. While high cell population is essential in cell-based vessel biofabrication and the expansion process is lengthy, cell-based therapies are more promising in terms of efficacy despite the fact that they are more complex and costly than scaffold-based techniques [7]. Therefore, many researches have focused on this approach and the representative studies are presented in Table 3. Among the well-studied scaffold-free techniques are the coculture system, sheet-based engineering, decellularization, direct cell injection, bioprinting, and biofabrication in a bioreactor system.

Conclusion and Future Directions
At present, the previously established treatments for cardiovascular disorders, such as organ transplantation, surgical reconstruction, usage of mechanical and synthetic devices, or administration of metabolic products, although promising are still yet constraints and complication-free [3]. Hence, the developments in vitro and in vivo generation of biomimetic constructs for specific target organ or tissue are more suitable for regeneration of damaged cardiovascular organs [95]. Albeit the points of view of Demirbag et al. [12] on the benefits and drawbacks of the applications scaffold and scaffold-free approaches over each other are indeed noteworthy. Due to the uniqueness of cells, tissues, and nature of cardiovascular defects, these approaches should be considered complementary instead of being competing components of vascular tissue engineering [12]. The treatment for blood vessel defects (Figure 4)   vessels may be directly utilized for treatment. Otherwise, especially in the cases congenital defects, tissue-engineered blood vessels manufactured, either scaffold based or cell based, may be considered as the alternative treatment. The current blood vessel constructs still have their corresponding limitations, therefore, further reassessment and modification have to be conducted. However, once a biomimetic blood vessel is produced, then transplantation can be the convenient treatment for blood vessel defects most especially in the infants whose transplantable large blood vessels are not yet ready for harvest.
In the future, further combination of techniques may be considered in the production and design of cardiovascularengineered tissues for the blood vessels. For instance, the High-Density Suspension System (HDSS) initiated by Lee et al. [96] for constructing spherical spheroids composed of cartilage and synovium cells was found to be an effective and efficient culture system, then the spheroids were successfully implanted and able to heal the bone defects of the knee in rabbit model. Such technique may be applied for expanding stem cells for the cardiovascular diseases especially if the construct has to be composed of a mixture of cells such as myofibroblast, smooth muscles, EPCs, and many others for blood vessel defects. Hence, this strategy may enhance the existing coculture system applied in the construction of engineered blood vessels. However, it would be better if researchers will be able to design a completely scaffold-free product since it is more beneficial in a sense that complications can be prevented and might promote greater life expectancy of the individuals affected with vascular diseases. It will remain a challenge to devise blood vessel engineered tissue until completely biomimetic construct is produced. Furthermore, since angiogenesis is essential in almost all types of organ remodeling and tissue engineering, it is indeed a crucial step for the complex organ reconstruction [77]. Hence, it is also interesting to explore the importance and the interrelationships of the vascularization signaling pathways in designing biomaterials. Perhaps, along the process of biofabrication, certain growth factors and other signaling molecules may be incorporated while regulating their release into the scaffold or scaffold-free construct in order to enhance blood vessel reconstruction on the injured site. The designs of the bioreactors may be further improved to hasten vascular tissue construction through their efficient and effective stimulation and by combining them with automated cell tissue culture systems to reduce time-consuming expansion period which primarily delimit the clinical application of the current vascular grafts especially in cases of emergencies. This may be the future mechanism in vascular tissue engineering.