Vascular Tissue

Vascular system throughout the body is biological pipeline for the blood transportation, and is also the main site for the material exchange between blood and tissue. The blood in the vessel carries a variety of nutrients and oxygen to all the tissues and organs of the body and discharges metabolic products. At the beginning of the twenty-first century, about 17 million people died of cardiovascular and cerebrovascular diseases [52, 53], and the number is continuing to rise, expecting to reach 25 million by 2020. With the rise in the incidence of cardiovascular disease (CVD), clinical treatment requires a variety of different diameter vascular substitutes to repair damaged vascular tissue. In recent years, vascular tissue engineering has been recognized as an effective way to provide a variety of methods to create vascular networks for the treatment of cardiovascular diseases [54, 55]. Vascular tissue engineering is the science of preparing, reconstructing, and regenerating vascular replacement materials by using normal cells in the vessel wall and biodegradable materials. With a large number of studies in the field going on, vascular scaffolds have been developed with no immunogenicity, high tissue and cell compatibility and certain mechanical properties, and show the potential to be widely used in clinical vascular replacement to solve the clinical treatment of ischemic diseases caused by vascular stenosis or occlusion [56-58].

Like most regenerative tissue cultures, regenerated vascular designs consist of three components, namely, vascular cells, scaffolds, and nurturing environment [59]. As an artificial ECM of vascular tissue, the scaffolds need to act as an effective temporary structure for supporting and promoting cell adhesion, differentiation and proliferation And then the scaffold should degrade over time when the cells precipitate to form a new regenerative ECM. Extensive materials from natural materials to synthetic materials have been studied as design candidates for tissue engineering scaffolds for vascular tissue engineering. Biomaterials include natural biomaterials, such as collagen, decellularized matrix and fibrin [60-64], synthetic polymeric degradable materials, such as polyglutamic acid, polylactic acid, polyurethanes, and polycaprolactone [16, 65, 66], and their composites or copolymers [67, 68]. Because of the complexity of the structure and function of natural blood vessels, a single material is difficult to meet the requirements of scaffolds. In recent years, fiber- or tube-reinforced scaffolds have been used for vascular tissue and have become the focus of extensive research. Such a reinforcing scaffold can be prepared by the use of two or more materials with different characteristics and mechanical properties through certain methods and manufacturing techniques.

Caves et al. [69] used ECM protein analogues to produce a multi-layered vascular graft consisting of a synthetic collagen microfibers-reinforced elastin-like protein matrix for the repair and regeneration of smaller diameter (<6 mm) grafts. For

Fig. 8.2 Multilamellar protein polymer microfiber composite vascular graft. The vessel wall has a multilamellar structure consisting of collagen microfiber (thickness 100 mm) embedded in protein polymer, and its orientation and density are established to achieve the mechanical design goals (Adapted with permission from Ref. [69]. Copyright 2010 Elsevier Ltd)

vein graft bypass transplantation, compliance under physiological stress is associated with intimal hyperplasia, which affects vascular patency. Early vascular tissue engineering has demonstrated the feasibility of the concept of ECM protein scaffolds from collagen gel. However, the strength of the scaffold is insufficient due to the deviation of the microstructures from the natural collagen fiber orientation, architecture, and packing density [60]. In order to create a densely packaged oriented collagen scaffold, continuous ultrafine fibers consisting of aligned, D-type periodic collagen fibrils were prepared by using a scalable spinning process [70]. In addition, a composite material has recently been described, consisting of a continuous, aligned, micro-crimped collagen fibers embedded into in an elastin-like protein matrix [71]. As Fig. 8.2 shown, the generation of cell-free arterial substitutes had a multilayer structure formulated from the recombinant elastin-like protein and integrated synthetic collagen microfibers. A simple description of manufacturing small diameter vascular grafts was shown in Fig. 8.3. The manufacturing scheme allowed a series of fiber orientation and volume fraction, which resulted in variable mechanical properties. The composites had a rupture strength of 239-2760 mm Hg, and their compliance and suture retention strength were 2.8-8.4%/100 mm Hg and 35-192 gf, respectively. According to the expected design criteria, the breaking strength, the compliance and the suture retention strength of the most reasonable material structure were 1483 ± 143 mm Hg, 5.1 ± 0.8%/100 mm Hg, and 173 ± 4 gf, respectively. Thus, recombinant elastin-based biomaterials can play an important role in improving mechanical properties of the scaffold by adding collagen microfibers.

In another study, Pooyan and colleagues [59] recognized that microstructural and mechanical properties of vascular stents were important parameters for further cellular activity and new tissue development, and designed a new cellulose nanowhiskers (CNWs) reinforced scaffold. As the most widely distributed and abundant polysaccharide in nature, cellulose is a macromolecular polysaccharide composed of glucose, and it is also the main component of plant cell wall. CNWs are usually extracted from cellulose by intense multistage chemical/mechanical separation methods, typically 1-100 nm in diameter and 0.5-2 pm in length [72, 73]. Figure 8.4

B. Pei et al.


Fabrication of fiber-reinforced small diameter vascular grafts

Fig. 8.3 Fabrication of fiber-reinforced small diameter vascular grafts. The graft consists of oriented synthetic collagen microfibers and an elastin protein polymer matrix. (a) Parallel arrays of fiber produced by winding about a frame. (b) Two arrays were transferred to a glass sheet at the desired angle. (c) Characterize the average fiber spacing and orientation with digital photographs. (d) Fiber orientation fast fourier transform analysis. (e) Fibers were surrounded with precision- thickness shims and a solution of elastin protein polymer was applied into a thin film. (f) The gelled film was rolled about a Teflon rod to produce a six-layered tube. (g) Schematic diagram of the average fiber spacing (d) and angle (ff) (Adapted with permission from Ref. [69]. Copyright 2010 Elsevier Ltd)

AFM morphology images of cellulose nanowhiskers (Adapted with permission from Ref. [59]. Copyright 2012 Elsevier Ltd)

Fig. 8.4 AFM morphology images of cellulose nanowhiskers (Adapted with permission from Ref. [59]. Copyright 2012 Elsevier Ltd)

showed the microstructure and general morphology of the CNW by AFM detection. For CNW has sufficient tensile strength and elastic modulus, the incorporation of CNW into fiber networks may help to get a variety of structures and functions. CNWs have a porous permeability, controllable biodegradability, stable mechanical strength, moldable properties and long-term storage capacity, whose excellent properties make them a revolutionary candidate for the use as a reinforcing material in

SEM images of the dispersion of CNWs within the CAP medium at 3% volume fraction (Adapted with permission from Ref. [59]. Copyright 2012 Elsevier Ltd)

Fig. 8.5 SEM images of the dispersion of CNWs within the CAP medium at 3% volume fraction (Adapted with permission from Ref. [59]. Copyright 2012 Elsevier Ltd)

vascular tissue engineering to provide a biocompatible environment for the cells growth and tissue development [74, 75]. In order to make full use of the properties of cellulose and its derivatives, a biological scaffold consisting of CNWs embedded in the cellulose acetate propionate matrix was designed by Parisa Pooyan et al. As Fig. 8.5 shown, when compared to carbon nanotubes or Kevlar, reinforced scaffolds with 0.2 wt.% CNWs exhibited significant strength and directional rigidity. When adding 3.0 wt.% CNWs, the value was almost double. In order to predict the enhancement effect of CNWs, the mechanical behavior of the nanocomposite was subjected to tensile tests in the non-linear range at a body temperature of 37 ° C. Based on these comparisons, the reason for the excellent mechanical stability of the composite structure at such a low CNWs filler content was that CNWs were connected to each other by strong hydrogen bonds to form a 3D rigid percolation network. Thus, the use of fibrous porous CNW microstructures to reinforce the scaffold under specific operating conditions provides a potential to improve mechanical properties for withstanding the physiological pressure, and simulates the structural features of natural ECMs in human blood vessels. The newly designed composite materials not only greatly expand the use of cellulosic-based materials in tissue engineering, but also provide potential candidates as reinforcements for vascular tissue, especially in small-diameter grafts.

Allen et al. [76] developed an absorbable arteriolar graft that didn’t require cell seeding or culture prior to implantation, so the substitute minimized the cost of production and eliminated the waiting time of patient. The composite vascular scaffold was made of microporous tubes of rapidly degradable elastomeric poly (glycerol sebacate) (PGS), whose outer surface was reinforced by PCL nanofibers to improve the long-term survival of acellular scaffold. The scaffold was inserted into the rat abdominal aorta. One year later, the graft regenerated a new “neoarteries” with a general appearance similar to that of the native rat aorta. At 30 days after implantation, the vast majority of composite scaffolds were completely absorbed, except for some PCL residues [77]. Rapid degradation and absorbability of the scaf-

Gross morphology and tissue structure of neoarteries similar to native arteries. (a) Gross morphology of neoarteries. Top left

Fig. 8.6 Gross morphology and tissue structure of neoarteries similar to native arteries. (a) Gross morphology of neoarteries. Top left: transformation of graft into neoartery in situ in 1 year. Top right: Transverse view of exploratory neoarteries. Bottom: Longitudinal view of exploratory neoarteries. Ah ruler ticks are 1 mm. (b) The H & E staining transverse sections of the middle of neoarteries show similar tissue architecture with native aortas. (c) Neoartery sections immunos- tained for von Willebrand factor (vwf, red) and а-smooth muscle actin (a-SMA, green). (d) En face view of the luminal surface of neoarteries shows complete coverage by CD31 positive cells. (e) Immunostaining for smooth muscle myosin heavy chain 11 (MHC-11, red). Scale bar 100 mm. L indicates vessel lumen. Nuclei stained with DAPI (blue) (Adapted with permission from Ref. [76]. Copyright 2013 Elsevier Ltd)

fold material accelerated the ability of host remodeling. The advantage of this scaffold was that it minimized the duration of host response to foreign body, which helped to reduce intimal hyperplasia [78], calcification [79, 80] and tissue degeneration [81] in the late-term. In fact, no signs of tissue degeneration (Fig. 8.6), neointi- mal hyperplasia (Fig. 8.7a), dilation (Fig. 8.7), or calcification (Fig. 8.7b) were seen at one year after implantation. Moreover, the regenerative arteries contained not only the nerves in perivascular tissues (Fig. 8.8) but also mature elastin in the same amount as native arteries. And mature elastin achieved complete regeneration in native tissue. Although the magnitude of neonatal arteries differed from the native aortas, neonatal arteries responded to vasomotor agents, which were primarily used to modulate vasotonia. There were some differences in matrix tissue, and the regenerative arteries in vivo showed a dynamic mechanical compliance similar to that of native arteries. This study shows that cell-free scaffolds with rapid resorbability and

Neoarteries resist common modes of late-term graft failure

Fig. 8.7 Neoarteries resist common modes of late-term graft failure. (a) Statistical diameter of neoartery and native aortas. (b) von Kossa staining of Neoartery and Native Aortas. Scale bar 500 mm. (c) Neoarteries with cells positive of macrophage marker CD68 in the outermost layer of the neoartery wall. Scale bar 100 mm. DAPI stained nuclei (blue) (Adapted with permission from Ref. [76]. Copyright 2013 Elsevier Ltd)

Regeneration of nerves in neoarteries

Fig. 8.8 Regeneration of nerves in neoarteries. (a) Schematic of en face imaging of the adventitial surface of neoarteries. (b) Nerves cover the adventitial surface of neoarteries with similar morphology to those in native aortas. Scale bar 100 mm (Adapted with permission from Ref. [76]. Copyright 2013 Elsevier Ltd) elasticity can be reconstructed at damaged tissue and result in regenerative arteries with long-term stability. Additionally, this design improves the performance of the cell-free scaffolds in repairing small arteries, which also can be applied to other soft tissue repair and regeneration.

Liu et al. [82] designed a silk-based scaffold as small diameter vessel prostheses, whose matrix and reinforcements were composed of the same material, silk fibroin. The reinforced scaffolds had a bilayer structure, that was an internal silk fiber reinforced silk fibroin tube embedded in the nanofibrous silk layers. The outer nanofibrous silk layers were prepared by lyophilization with a highly porous structure, in which the nanofibers similar to the ECM comprised the macropore walls. The function of the outer layers was to provide an appropriate growth environment for fibroblasts and smooth muscle adhesion and proliferation, while the inner silk fiber reinforcement utilized internal heparin to provide excellent blood compatibility for at least 1 month. Compared with previous silk scaffolds, the bilayer fiber-reinforced ones not only provided a better mechanical strength, burst pressure, and suture retention strength but also achieved a similar compliance to saphenous veins. The silk-based reinforced scaffold also had a good cytocompatibility and hemocompat- ibility in vitro. These results confirm that the silk reinforced bilayer constructs have the potential as small diameter vascular grafts, and the follow-on in vivo studies still need to be conducted to reduce the risk of graft failure, due to endometrial hyperplasia, poor suture retention and weak mechanical properties.

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