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Keywords:

  • tissue engineering;
  • stem cell;
  • vasculature

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

Tissue engineering holds great promise to address complications and limitations encountered with the use of traditional prosthetic materials, such as thrombogenicity, infection, and future degeneration which represent the major morbidity and mortality after device implant surgery. The general concept of tissue engineering consists of three main components: a scaffold material, a cell type for seeding the scaffold, and biochemical, physio-chemical signaling and remodeling process. This remodeling process is guided by cell signals derived from both seeded cells and host inflammatory cells that infiltrate the scaffold and deposit extracellular matrix, forming the neotissue. Vascular tissue engineering is at the forefront in the translation of this technology to clinical practice, as tissue engineered vascular grafts (TEVGs) have now been successfully implanted in children with congenital heart disease. In this report, we review the history, advances, and state of the art in TEVGs. Anat Rec, 297:83–97. 2014. © 2013 Wiley Periodicals, Inc.

Abbreviations used
BMC

bone marrow cells

BMMNC

bone marrow mononuclear cell

CST

cell sheet technology

ECM

extracellular matrix

EB

embryonic bodies

ESC

embryonic stem cells

EC

endothelial cells

IVC

inferior vena cava

iPS cells

induced pluriopotent stem cells

PIPAAm

poly(N-isopropylacrylamide)

SMC

smooth muscle cell.

The clinical translation of therapies utilizing tissue engineering techniques has great potential for improving outcomes in patients with cardiovascular diseases. In particular, congenital cardiac anomalies affect nearly 1% of all newborns and represent the most common birth defect. Despite significant advances in surgical management, these anomalies remain a leading cause of death during the newborn period. Most reconstructive operations employ synthetic vascular grafts, valves, or patches to maintain vascular continuity. Associated complications, including thrombo-embolic events, infection, and poor durability related to the development of neointimal hyperplasia or ectopic calcification, remain the most common causes of morbidity and mortality following surgery. Limitations of currently used synthetic material include thrombogenicity, increased risk of infection, and lack of growth potential.

Tissue engineering has the potential to improve on all these complications with the ability to create biologically active graft materials for surgical repair. When constructed from autologous cells, these grafts lack immunogenicity, lower risk of infection, and have the ability to integrate, remodel, and grow with host tissue. So called tissue engineered vascular grafts (TEVGs) are particularly attractive for repairing congenital cardiac anomalies in the pediatric population where current materials such as Dacron and Gortex lack potential for growth and can require successive surgeries to up-size grafts.

We implemented the first clinical application of TEVGs for use in corrective surgery of congenital heart disease and confirmed their significant potential via an initial trial involving 25 children (Shin'oka et al., 2001; Shin'oka et al., 2001; Hibino et al., 2012a). The results of this study are promising and using a bench to bedside and back approach, we have elucidated some of the mechanisms behind this state of the art technology. Herein, we discuss common materials and methods to generate TEVGs, the mechanism behind this technology, and our own experience with our clinical investigations.

CONCEPT OF TISSUE ENGINEERING

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

In the mid-1980s the concept of tissue engineering was first proposed, seeking methods to generate materials to overcome a shortage of donor organs for transplantation. In 1993, Langer and Vacanti, pioneers in the nascent field, described tissue engineering as an interdisciplinary field with the goal of creating biological tissue to restore function of diseased tissue, or even entire organs (Langer and Vacanti, 2003). Biologic tissue is an assembly of interconnected cells that perform a similar function as host tissue which consists of the cells, the extracellular matrix (ECM), and the signaling systems to orchestrate organ function. The general idea of tissue engineering is to use three main components: scaffold material, cell type(s) for seeding the scaffold, and biochemical and physio-chemical signaling and remodeling processes (Fig. 1).

image

Figure 1. The basic concept of tissue engineering consists of three essential components, which include (1) Cells, (2) Scaffold, and (3) Signals. All three factors are interdependent and are indispensable in the formation of highly organized vascular tissue.

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MATERIALS USED IN TISSUE ENGINEERING

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

The incorporation of cells in reconstituted prosthetic tissue devices often can provide the signals needed for tissue building; however, the differentiation and resultant function of reconstructed tissue may be limited. There is little clinical data evaluating functional mimicry of endothelial cells (ECs) on the psuedointima of synthetic vascular grafts; however, one study suggested EC function of psuedointima of synthetic grafts was less than 10% that of native vessel (Walles et al., 2004). Conversely, using the tissue engineering approach, intima formed on a biodegradable scaffold showed EC function similar to that of native vein (Mastumura et al., 2006).

Biodegradable Scaffold

In the field of vascular tissue engineering, the role of the polymer scaffold is to provide a temporary, three-dimensional structure that facilitates cellular attachment and proliferation, ECM deposition, and finally, degradation into safe by-products. While there has been a vast array of biodegradable scaffolds employed in a variety of models, an ideal polymeric construction for vascular tissue engineering remains to be developed.

Polyglycolic Acid, Polylactic Acid, and Poly(ε-caprolactone)

Polyglycolic acid (PGA), polylactic acid (PLA), and poly(ε-caprolactone) (PCL), and their copolymers are commonly used synthetic degradable polymers for constructing TEVGs due to FDA approval for human implantation, a history of successful clinical applications, and a safe and well understood degradation process. The degradation of these polymers is controlled by hydrolytic cleavage of the ester linkage and can be tailored by adjusting the polymer's initial molecular weight, surface area to volume ratio, and crystallinity. In the final stage of degradation, polymer fragments of low molecular weight are phagocytosed by macrophages and broken down into naturally occurring metabolites.

PGA is a highly crystalline, hydrophilic polymer that degrades rapidly in vivo, typically losing tensile strength within 2–4 weeks. The PLA repeating unit has an extra methyl group when compared to PGA, which makes it less hydrophilic and more resistant to hydrolytic degradation once implanted, typically losing tensile strength over many months or even years. PCL has a more hydrophobic structure than either PGA or PLA, and therefore has a significantly slower degradation process making it most suitable for long-term implantation. PCL also has unique mechanical properties due to a low glass transition temperature of about −60°C, causing a rubbery state at room temperature (Pitt et al., 2009).

Copolymers such as polyglycolic-polylactic acid (PGLA) are attractive for TEVG engineering because the ratio of monomers can be selected thereby providing control over mechanical properties and degradation rate. However, due to immiscible components and reduced crystallinity, copolymers can sometimes degrade more rapidly compared with either PGA or PLA alone (Pachence and Kohn, 2008).

Polyhydoxyalkanoates and Polyhydroxybutyrates

Polyhydoxyalkanoates (PHA) and polyhydroxybutyrate (PHB) are aliphatic polyesters similar to PGA, PLA, and PCL in that they are thermoprocessable, biodegradable, and biocompatible. However, PHA and PHB are derived from microorganisms where they naturally accumulate in intracellular granules as a form of stored energy (Gogolewski et al., 1993). They are attractive biomaterials for applications in both conventional medical devices and tissue engineering because they are naturally polymerized leading to a material without any residual impurity, catalysts, or initiators.

Fabrication Method of Scaffold Material

There have been numerous methods proposed for polymer scaffold construction but the majority of techniques involve phase separation, electrospinning, and self-assembly.

Phase separation technique enables the creation of macro-porous scaffolds that facilitate cell seeding and infiltration (Stegemann et al., 2007). This method relies on dissolving a polymer into a solvent and then added some quantity of non-solvent to create an emulsion of polymer-rich and polymer-poor phases. The emulsion can be snap frozen in liquid nitrogen and lyophilized to remove both liquids, rendering an interconnected, porous polymer scaffold. A similar strategy termed thermally induced phase separation (TIPS) utilizes solvents with high melting temperatures and then cooling the mixture until liquid–liquid or solid–liquid phase separation occurs. Morphology of the scaffold can be controlled by varying the polymer concentration, solvent, and cooling rate, with low gelation temperatures typically creating open nanoscale fiber networks and high temperatures forming more platelet-like structures.

The electrospinning apparatus consists of a voltage generator, syringe pump, and grounded collection target. Briefly, a polymer solution is forced through an electrified capillary toward a grounded target via a syringe pump. As the charged polymer solution enters this large electric field (typically 10–30 kV), electrostatic repulsion exceeds the surface tension of the polymer solution creating a jet of entangled polymer chains. This unstable jet accelerates in the electric field creating a whipping effect and draws ultrafine polymer filaments toward the grounded target while simultaneously evaporating the solvent. The resulting electrospun fabric deposited on the grounded collector can range in fiber diameter from less than 50 nm to over 10 µm and can be controlled by adjusting polymer concentration, solvent type, voltage, tip to collector distance, and flow rate of the system (Rathore et al., 1981). The electrospun nanofiber material has unique topographies that not only have an immense surface area to volume ratio, but also resemble structural components of native ECM, thereby improving conditions for cellular attachment and proliferation (Greiner and Wendorff, 2007; Kumbar et al., 2005).

Self-assembly presents a unique method of scaffold production relying on peptide-based fibers that can self-assemble into nanofibers. The ECM proteins collagen and elastin determine the strength and elasticity of vascular tissue and so groups have focused on collagen-like and elastin-like structures to produce biomimetic scaffolds. Self-assembly of collagen-like structures have utilized peptide amphiphiles to create structures that are both biocompatible and stable (Koide et al., 2002; Paramonov et al., 2000). The self-assembly of elastin-like materials utilizes tropo-elastin, elastin-like polypeptides, and recombinant polypeptides to produce bioactive materials similar in mechanical properties to native elastin (Daamen et al., 2007). While this natural assembly of structural protein is exciting, the costs and complexity remain high, limiting its utility under current methods.

Decellularized Materials

Decellularized vessels contain intact and structurally organized ECM proteins and offer a natural material similar to autologous vessel for cardiovascular repair. A decellularized vessel can originate from allogeneic, xenogenic, or in vitro-engineered donor tissue and is subsequently processed to remove all cellular and antigenic components while retaining structural protein (Madden et al., 2008; Piterina et al., 2006; Dahl et al., 2011). The decellularization process uses any combination of detergents or surfactants, enzymatic digestion, and physical agitation to lyse cell membranes, solubilize, and remove cellular debris including proteins, lipids, and nucleotide remnants. For vascular tissues, the resulting decelled material is composed of approximately 90% collagen, fibronectin, growth factors, glycosaminoglycans, proteoglycans, and glycoproteins. However, in pursuit of complete decellularization, processing methods can also degrade or damage matrix proteins resulting in the loss of ECM integrity. Consequently, there is much ongoing research to optimize the most effective decell recipe that preserves ECM structure. Some early clinical studies using decelled xenogeneic tissue failed due to immunogenic response but strategies have shifted toward growing human tissue in bioreactors in vitro and then decellularizing the construct (Dahl et al., 2011). Simon et al. cultured cadaveric SMCs on a scaffold in a pulsatile bioreactor over 10 weeks, and after decelling, the vessel had a burst pressure over 3,300 mmHg (Simon et al., 2007). Some of the drawbacks limiting this approach are the costs and complexity in engineering consistent human tissue in vitro and effectively decellularizing the material before implantation.

Cell Sheet Technique

Cell sheet assembly into TEVGs relies on culturing cells in vitro on plates coated with thermoresponsive polymers such as poly(N-isopropylacrylamide) (PIPAAm) (Okano et al., 2010). At temperatures greater than 32°C, as in an incubator, the material is hydrophobic and therefore suitable for cellular attachment and culture. However, when the temperature of the material is reduced below 32°C, the surface alters its surface chemistry to become hydrophilic, causing the confluent sheet of cells to detach from the plate. Using this technique, entire cell sheets can be harvested without breaking cell-to-cell junctions thereby preserving the natural cellular matrix. This technique was applied in corneal surface reconstruction, esophageal ulceration, periodontry, and myocardiac tissue reconstruction (Elloumi-Hannachi et al., 2009).

STEM CELLS USED IN VASCULAR TISSUE ENGINEERING

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

Advances in stem cell science have enabled a new generation of vascular tissue engineering that utilize the intrinsic abilities of stem cells to indefinitely self-renew and differentiate into various mature cell types. Various stem cell types such as embryonic stem cells (ESC), mesenchyme stem cells, induced pluripotent stem cells, and multilineage differentiating stress enduring have been employed in TEVG strategies and enable a functional cell source for tissue regeneration (Naito et al., 2011).

Embryonic Stem Cells

ESC are pluripotent stem cells derived from the inner cell mass of the blastocyst and are capable of differentiating into cell types of all three germ layers. Human ESC were first reported by Thompson et al. in 1998, but ESC research has faced challenges due to restricted federal funding and ethical concerns (Thomson et al., 2004). When ESC are cultured in suspension in the absence of leukemia inhibitory factor they form embryonic bodies (EB) composed of cell types from all three primitive germ layers. In prolonged culture of EB, it was found that blood island-like structures composed of immature hematopoietic cells surrounded by EC-like structure could be derived. Analysis of primary EB cultures with EC-like structures suggested the presence of two characteristics of ECs: the cells labeled with fluorescent acetylated low density lipoprotein which is known to be actively taken up by ECs, and a small number of cells that stained positive for von Willebrand's factor (Wang et al., 2001). Yamashita et al. reported Flk1+ cells isolated from mouse ESC were vascular progenitor cells and have the ability to differentiate into both ECs and mural cells (smooth muscle cells and pericytes) depending on the cytokines administered to achieve differentiation (Yamashita et al., 2008). The effects of many growth factors have been shown to differentiate human ESC toward specific lineages, such as Activin-A and transforming growth factor (TGF)-beta driving mesodermal differentiation (Schuldiner et al., 2000). Certain ECMs have been shown to provide guidance cues for capillary morphogenesis (Davis and Senger, 2005). ESC differentiated into ECs in vitro have been shown to form vascular structures when implanted in collagen gel (McCloskey et al., 2003). This strategy of ESC manipulation represents a ground-up strategy for engineering limitless vascular tissue in vitro.

Several animal studies have been reported utilizing ESC to promote vascular regeneration. In one study, ECs derived from mouse ESC were seeded on the luminal surface of a smooth muscle cell and PGA scaffold construct and implanted subcutaneously in mice (Shen et al., 2003). Upon harvesting the vessel, it was observed that the PGA scaffold was completely degraded and histological and immunohistochemical analysis suggested the engineered vessel resembled typical blood vessel, with an intact EC lining from the ESC derived cells. Levenberg et al. showed that human ECs could be isolated from ESC by using platelet EC-adhesion molecule-1 antibodies and self assemble into vessel-like structures when cultured on matrigel (Levenberg et al., 1993). When implanted into severe combined immune deficiency (SCID) mice, the cells formed microvasculature that contained host blood cells. Although research in this field remains active, the use of ESCs in research and clinical investigation is hampered by the tumorigenic potential of undifferentiated ESC as well as the ethical considerations when isolating ESCs from a fertilized human embryo.

Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) originate from mesenchyme, a type of undifferentiated tissue that is derived from the mesoderm, and MSCs can differentiate into various cell types including: osteocytes, chondrocytes, adipocytes, and tissue of the circulatory system. MSCs have generated considerable interest in vascular tissue engineering because they can be differentiated and expanded into vascular tissue lineages including SMC (O'Cearbhaill et al., 2012) and EC (Lin et al., 2002), and are readily isolated from a variety of adult tissue sources such as bone marrow, fat (Bernacki et al., 2008), peripheral blood (Kuwana et al., 2010), umbilical cord blood (Wang et al., 1992), and adult blood vessel (Abedin et al., 2004). This presents a unique strategy within vascular engineering where cells can be isolated from a patient, expanded, and differentiated, and used to generate autologous tissue for transplant. This method eliminates the need for immunosuppressants and mitigates the risk of teratoma formation associated with ESCs and iPS cell types (Jiang et al., 2000). While this presents several advantages over ESCs, MSCs do not possess the capacity to generate an entire organ and it has been proposed to rename them to Multipotent Stromal Cells. In humans, a MSC phenotype is suggested by the presence of cell surface markers CD73, CD90, and CD105, coupled with a deficiency of markers such as CD11b, CD14, CD19, CD34, CD45, and HLA-DR surface markers.

There have been several animal studies utilizing MSC-seeded grafts that have demonstrated encouraging results suggesting this strategy may present a reasonable therapeutic option for vascular repair. Mirza et al. demonstrated rat bone marrow derived MSCs differentiated into vascular lineage in vivo following seeding on polyurethane vascular grafts and implantation into rat (Mirza et al., 2000). The MSCs were labeled with green fluorescence protein and 2 weeks after implantation the cells were found to express SMC markers, demonstrating successful induction of MSC to the vascular lineage. Zhang et al. reported novel MSC-seeded biodegradable scaffolds implanted in a canine abdominal aorta model (Zhang et al., 2008). After 24 weeks in vivo, the implanted grafts were fully patent, having no indications of occlusion or aneurysm and possessed excellent mechanical properties.

iPS Cells

Yamanaka and coworkers demonstrated the induction of adult fibroblasts into pluripotent stem cells using a retrovirus to transfect cells using four genes: Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi et al., 1996). These so called induced pluripotent stem cells, or iPS cells, have similar characteristics with other type of pluripotent stem cells, such as the expression of stem cell genes and proteins, embryoid body formation, teratoma formation, viable chimera formation, and capacity of self-renewal and differentiability. However, human iPS cells are autologous cells, harvested from a patient's own tissue, thereby eliminating the risk of rejection and circumventing the associated political and ethical concerns surrounding ESCs-based therapies. In the time since the discovery, much attention has focused on safer methods to generate iPS cells that avoid viral transfection which introduce risk of tumorigenicity and are not clinically viable. Okita et al. demonstrated a virus-free approach using a plasmid vector to deliver several transcription factors to produce safer iPS cells (Okita et al., 1995).

Hibino et al. reported the use of mouse iPS cells for seeding a TEVG implanted as an interposition graft in murine inferior vena cava (Hibino et al., 2010). All mice survived without complications and the grafts demonstrated endothelialization with von Willebrand factor and an inner layer with smooth muscle actin- and calponin-positive cells at 10 weeks. However, the number of seeded differentiated iPS cells decreased over time and none were found to co-localize with the von Willebrand factor or smooth muscle actin-positive cells. Based on these results, the authors concluded that the seeded iPS cells exerted a paracrine effect to drive neotissue formation and later succumbed to apoptosis (Hibino et al., 2010).

Muse Cells

Dezawa and coworkers reported a relatively new form of pluriopotent stem cell termed “multilineage-differentiating stress-enduring” (Muse) cells (Kuroda et al., 2008). The group isolated this cell population from cultured skin fibroblast, cultured bone marrow stromal cells, and native bone marrow aspirates showing capacity of both self-renewal and differentiation into three germ layers. Unlike ESCs and iPS cells, Muse cells appear to only rarely develop into teratomas, which is the most significant limitation preventing clinical adoption of stem cell therapies. As a result, Muse cells possess enormous potential in the field of regenerative medicine and cell-based therapies

MECHANISM OF TE REMODELING

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

The use of biodegradable polymeric scaffolds either in vitro or in vivo is currently the most common approach to constructing TEVGs. As such, there is a simultaneous process of scaffold degradation and vascular tissue formation. As the polymer of the scaffold degrades, inflammatory cells respond via a foreign body reaction; however, we have shown that inflammation is actually a critical component of the neotissue formation and remodeling process (Fig. 2) (Roh et al., 2010).

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Figure 2. Schematic and histology describing the remodeling of TEVG. Seeded bone marrow cells (BMC) and infiltrating monocytes release multiple angiogenic cytokines and growth factors (i.e., VEGF), which recruit host derived cells to the scaffold. Vascular cells potentially come from circulating progenitors and/or proliferation/migration of mature vascular cells in adjacent vessel segments. As the scaffold degrades, monocytes migrate away, leaving behind a completely autologous neovessel.

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The biologic response and success following TEVG implantation is a result of biochemical and biomechanical factors. This process begins when circulating proteins such as albumin, fibronectin, fibrinogen, and others, adsorb to the TEVG material surface. These proteins facilitate cell interactions and adhesion with host inflammatory cells and consequently drive the inflammation and wound healing responses (Jenney and Anderson, 2000; Brodbeck et al., 2003).

TEVG biomaterials are generally selected due to their biocompatibility and inertness and it is accepted that these materials are less immunologically active than autologous or allogenic materials (Hanker and Giammara, 1988). Nonetheless, there is an activation of the coagulation cascade and complement system once exposed to blood and a number of these products have been described to function as mediators for host inflammation (Jonatova, 2000).

The next step in this inflammatory process is marked by the migration and infiltration of monocytes and macrophages to the TEVG. It has been shown that these cells are guided in response to released cytokines and chemokines (Esche et al., 2005). Circulating monocytes that are attracted to the TEVG in this way may further differentiate (polarize) into either activated M-1 macrophages or the alternative M-2 phenotype (Mills et al., 1994). This process is highly dependent on the graft material properties (Tang et al., 1998; Janatova, 2011; Badylak and Gilbert, 2008).

When large foreign bodies such as TEVGs are present and exceed the phagocytotic capacity of a single macrophage (particles 10–100 μm in diameter), macrophages fuse together to form foreign body giant cells (FBGC) (Brodbeck and Anderson, 2009). Factors contributing to the adhesion and fusion of monocyte/macrophage include integrins, chemokine/cytokines (CCL-2, IL-4, IL-13, IFN-gamma), cell surface fusion mediators (Mannose receptor, CD13, E-Cadherin). In particular, IL-4 and IL-13 up-regulate mannose receptors on fusing macrophages which play a key role in endocytosis and phagocytosis, and localize at fusion sites between macrophages (DeFife et al., 1997; Apostolopoulos and McKenzie, 2001). While the role of FBGCs are not fully elucidated, they are known to release reactive oxygen intermediates, oxygen free radicals, degradative enzymes, and decrease local pH, thereby altering the environment between cell membrane and TEVG surface, and influencing the degradation process (Henson, 1971).

ECM Remodeling

Monocytes recruited to the TEVG infiltrate the porous structure, differentiate to macrophages and kick-off a cytokine cascade recruiting the migration of adjacent host SMCs and ECs into the scaffold. Macrophages, fibroblasts, and SMCs begin to deposit ECM while the scaffold begins to degrade and the ECM undergoes continuous remodeling throughout degradation. As macrophages take residence within the nascent ECM, monocytes are polarized into the M-2 anti-inflammatory phenotype, considered a “healing” macrophage, and characterized by enhanced secretion of regenerative trophic factors (Badylak and Gilbert, 2008). The M-2 macrophage is critical for ECM remodeling because it partially degrades ECM to facilitate new tissue in-growth and also secretes cytokines with chemotactic factors, both benefit early stage of wound healing and TEVG remodeling.

We have extensively analyzed ECM remodeling process in TEVG using C57BL6 mice model over the 4-weeks period of time (Naito et al., 2003). Masson trichrome staining highlighted collagen, which was associated with scaffold material over the 4-week study period. Abundant collagenous fibers formed between the polymer at early time points (Fig. 3a,b), but these fibers lost their volume by 4 weeks (Fig. 3c). Immunohistochemical staining revealed an initial increase in Type III collagen deposited between the scaffold fragments (Fig. 3i,j) followed by Type I collagen deposition. Type IV collagen was not detected at the luminal surface of neovessel until the 4-week time point. When viewed using polarized light (darkfield microscopy), picrosirius red staining revealed larger collagen fibers (possibly Type I collagen) as bright yellow or orange and thinner fibers (possibly Type III collagen) as green (Fig. 3m–o). At the 4-week time point, the predominant collagen was Type I. Gene expression of both Types I and III collagen peaked at 2-weeks time point, whereas Type IV collagen which primarily found in basement membrane, continued to increase over the 4-week period. Biochemical analysis showed total collagen production was greatest at 2 weeks which correspond with the result of gene expression (Fig. 4).

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Figure 3. Characterization of collagen subtypes by histology. Masson's Trichrome stain (a–d), immunohistochemistry of Type I (e–h), Type III (i–l), and Type IV (q–t) collagens, and Picrosirius red staining (m–p) demonstrated changes in the deposition of each collagen type over time. The layer between arrows indicates the medial layer of each neovessel (c, g, k).

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Figure 4. Collagen gene expression. Gene expression characterization of collagen subtypes (a–c) and biochemical quantification of total collagen (d). (*P < 0.05, N = 6 for gene expression, N = 3 for biochemical assay). Gene expression of collagen Type I (a) and III (b) and the total collagen trended toward peaks at the 2-week time point. The level of gene expression and amount of collagen approach those of normal vena cava at 4-week time point. The expression of Type IV collagen gene (c) was significantly lower at early time points compared to that of vena cava; however, it approached that of normal vena cava at 4-week time point. Total collagen quantified by a hydroxyproline assay indicated that the trend of total collagen in the TEVG peaked at 2 weeks. There was statistical significance between 2 week TEVG and normal vena cava, but collagen content was equivalent between the 4-week TEVG and normal vena cava.

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Histological characterization of elastin production demonstrated fibrillin-1 deposition at 1 week precedes with elastin production at 4 weeks (Fig. 5). Faint stain suggested elastic fibers near the luminal surface of the neovessels at 4 weeks. Fibrillin-1 (which presumably serves as a template for tropoelastin deposition) was up-regulated during early time periods (Fig. 5e). The gene expression of elastin increased over time throughout the entire study period (Fig. 5i); in contrast, that of fibrillin-1 was higher than elastin at early time points but then decreased at 4 weeks (Fig. 5j). Upregulation of fibrillin-1 gene expression at early time points is presumably in accordance with basic mechanisms of elastogenesis in early development, in which tropoelastin is deposited on a preformed template of fibrillin-rich microfibrils (Mecham and Davis, 1994]. Overall, elastin protein production increased over time in the neovessels (Fig. 5k).

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Figure 5. Characterization of elastin and its associated microfibril. Faint stain suggested elastic fibers formed on the luminal surface of neovessel at 4 weeks. Fibrillin-1 was up-regulated during early time periods (e–g). Our results showed that gene expression (i) and production (k) of elastin increased over time throughout the entire study period. The gene expression of fibrillin-1 (j) preceded that of elastin, and decreased over time.

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Histological characterization of matrix metalloproteinase (MMPs) revealed production of MMP2 is robust at each time point while MMP9 decreased over the 4-week period (Fig. 6). Our PCR results showed gene expression of MMP-9 peaked in the earliest phase of neovessel formation, but decreased thereafter; in contrast, expression of MMP-2 simply trend upward over time (Fig. 6i,j). These trends are similar to what is normally found in vascular injury models (Bendeck et al., 1994; Godin et al., 2000). The presumptive role of MMP-2 and −9 in TEVG neovessel remodeling includes induction of an early inflammatory response evoked by a foreign body reaction as well as ECM degradation to adapt to the mechanobiological/physiological milieu.

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Figure 6. Characterization of MMPs by histology. Histology showed robust production of MMP-2 at each time point (a–c), whereas MMP-9 decreased over time (e–g). Gene expression of MMP-2 significantly increased at 4 weeks (i), whereas MMP-9 significantly decreased at the 4 week time point (j).

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SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

The tissue engineering techniques described above were evaluated in a preclinical, large animal model to determine clinical feasibility (Shinoka et al., 2005). The pilot study utilized harvested ovine artery to isolate a mixed cell population of ECs and fibroblasts, which were expanded in culture and seeded onto synthetic biodegradable PGA tubular scaffolds. The TEVGs were cultured for 7 days to allow cells to reach confluence and then used to replace 2 cm segment of pulmonary artery in lambs; a control group received acellular polymer grafts coated with fibrin sealant. Animals were sacrificed between 11 and 24 weeks and all TEVGs were patent and demonstrated the capacity for normal diameter growth, without indication of aneurysmal change. Tissue analysis revealed that collagen content in TEVG group was comparable to that of native pulmonary arteries, and elastic fibers were identified in the medial layer of remodeled TEVGs. Additionally, endothelial specific factor VIII was present within the lumen in the TEVG group. Using a canine model, our group also investigated the use of this technique to replace inferior vena cava (IVC) (Watanabe et al., 2000). Vascular cells from femoral veins were isolated, expanded, and seeded onto PGA/PCLA scaffolds for implantation. Three to six months following implantation, explanted TEVGs demonstrated similar characteristics and architecture to native vessel with no evidence of dilation or stenosis.

Utilizing this technology, the first human pediatric patient successfully received a TEVG to repair an occluded pulmonary artery and was constructed with a PGA/PCLA scaffold and co-culture of isolated vascular cells (Shin'oka et al., 2001). Subsequently, several patients underwent tissue engineered vascular graft implantation using autologous venous cells for TEVG seeding. However, venous tissue harvest and culture with serum increases risk and complexity of this approach so other cell sources were investigated. Bone marrow mononuclear cells (BMMNCs) are one such cell source that can be aspirated in sufficient numbers from a patient's iliac bone on the same day of the TEVG surgery, thereby removing the need and risk of cell culture and xenoserums. To investigate the utility and feasibility of BMMNCs, we created TEVGs with BMMNCs and implanted them into the vena cava of dogs. Using comparable polymeric scaffolds and different types of cells (isolated venous cells; BMMNCs without culture; and acellular control grafts), we implanted the constructs for up to 8 weeks and the TEVG seeded with BMMNCs without culture showed comparable to TEVG seeded with cultured venous cells. In addition, BMMNCs were found to contribute to the creation of tissue engineered vascular autografts in vivo (Matsumura et al., 2004). Encouraged by these findings, the group pursued the first human clinical trial of TEVGs seeded with autologous BMMNCs and implanted TEVGs as conduits for extracardiac total cavopulmonary connection (EC-TCPC) in 25 patients with single ventricle anomalies (Fig. 7) (Naito et al., 2008). These patients were closely followed up for over 7 years, and over that time course, TEVGs functioned without evidence of graft related complications and there was no graft-related mortality (Hibino et al., 2012a). Post-implantation imaging revealed stenosis of graft, in 16% of patients, and was the only major complication (Fig. 8). These patients were generally asymptomatic and successfully treated with catheter-based angioplasty or stenting. Coumadin was administered postoperatively for 6 months to assuage potential blood clotting but after 6 months the therapeutic was successfully discontinued, demonstrating that TEVG remodeling was successful and a life time of anticoagulation therapy was not required.

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Figure 7. First clinical application of TEVG in extracardiac TCPC operation.

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Figure 8. TEVG stenosis successfully treated by balloon angioplasty. PTA, Percutaneous transluminal angioplasty.

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THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

The first concept of tissue engineering believed seeded cells created neotissue. However, there has been little discussion regarding the importance and contribution of cell seeding and only preliminary evidence that supports the contribution of seeded BMMNCs for histogenesis of neovessel (Matsumura, 2003). It was postulated that the seeded BMMNC population differentiated into mature ECs and SMCs, and so, we investigate their function using human BMMNC seeded scaffolds implanted into the IVC of severe combined immunodeficient/beige (SCID/bg) mice. Seeded human BMMNCs were tracked using CD68+ human monocyte and CD31+ human EC markers. To our surprise, only a minority of originally seeded human cell types could be found within the scaffold wall after 1 week, and no remaining human cells were detectable beyond that time point. Even though this study suggested the seeded cells did not directly develop into neovessel, when we tested seeded versus acellular interposition TEVGs, the overall cellularity was significantly greater for seeded scaffolds after 7 days in vivo. This increase in cellularity was primarily attributed to differences in the host inflammatory response, initially with more robust infiltration of monocytes into human BMMNC seeded scaffolds.

The effects of seeding BMMNCs onto TEVGs have been investigated using a mouse model and significantly improved patency rates were achieved using cell seeded scaffolds versus an acellular control group (Hibino et al., 2012b). Both seeded and unseeded TEVGs were implanted as IVC interposition grafts in mouse model and evaluated for rate of stenosis using ultrasound and quantitative histological morphometry. Results of both demonstrated that cell seeding decreased the formation of TEVG stenosis and increased luminal diameter, proving cell seeding is critical for proper neovessel formation (Figs. 9 and 10).

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Figure 9. Characterization of seeded or unseeded TEVGs. Patency rate was significantly higher in seeded grafts compared with unseeded grafts (a). Luminal diameter was significantly higher in seeded grafts compared with unseeded grafts (b). The number of F4/80 positive macrophages in the graft was significantly higher in unseeded grafts than in seeded grafts (c). Stenotic grafts included significantly higher numbers of F4/80 positive macrophages compared with patent grafts (d).

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Figure 10. Characterization of TEVG stenosis over time. Representative images of patent and stenosed TEVGs 2 weeks after implantation with histological morphometric analyses of the inner and outer diameters (a). The outer diameter remains constant in the stenosed grafts, suggesting wall thickening. Representative images of patent and stenosed (occluded) TEVGs 6 months after implantation with histological morphometric analysis of the inner and outer diameter (b). The outer diameter decreases in size in the stenosed graft, suggesting inward remodeling, whereas, patent graft showed appropriate remodeling mimicking native vessels.

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CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED

Currently, autologous blood vessels and synthetic graft materials such as Dacron and Goretex remain the gold standard materials for vascular reconstruction. However, autologous vessels are inherently limited in number and length and current synthetic graft materials have limited efficacy, increased risk of infection, and the need for chronic anticoagulation therapy. In the pediatric population, the utility of synthetic grafts is further limited because of the lack of growth potential, requiring additional procedures to upsize a graft. Vascular tissue engineering provides a novel technology to produce TEVGs, which develop into host blood vessel and potentially solve the problems associated with conventional vascular grafts. As we develop a better understanding of the neotissue formation and graft remodeling processes, we will be able to further improve patient outcomes using this technology.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. CONCEPT OF TISSUE ENGINEERING
  4. MATERIALS USED IN TISSUE ENGINEERING
  5. STEM CELLS USED IN VASCULAR TISSUE ENGINEERING
  6. MECHANISM OF TE REMODELING
  7. SUCCESSFUL IMPLANTATION OF TEVG INTO HUMAN PULMONARY CIRCULATION
  8. THE ROLE OF BONE MARROW DERIVED MONONUCLEAR CELLS IN CELL SEEDING
  9. CONCLUSION
  10. LITERATURE CITED
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