Scaffolds for vascular constructs synthesized using hydrolyzed elastin.Abbreviations: SMC: smooth muscle cell, EC: endothelial cell, EPC: endothelial progenitor cells, Fb: fibroblast, TCPS: tissue culture polystyrene, PLGA: poly(D,L-lactide-co-glycolide), PLLA: poly(L-lactide), PCL: polycaprolactone, PLCL: poly(L-lactide-co-ε-caprolactone), PDO: polydioxanone, PDS: polydioxanone, PGC: poliglecaprone
Elastin is a key structural protein found in the extracellular matrix (ECM) of all mammals. As the dominant part of the elastic fiber, elastin confers the mechanical properties of resilience and elasticity essential to the function of elastic tissues. Elastin interacts with cells through specific biochemical mechanisms. This chapter considers the (1) mechanical and biochemical roles of elastin in elastic tissues and the subsequent disease phenotypes that result from the degradation and loss of elastin, (2) development and success of current elastin based biomaterials including sources of elastin for tissue engineering and their application, and (3) vascular constructs that our laboratory has developed from recombinant human tropoelastin. These constructs mimic the physical and biochemical properties of native elastin.
2. Elastin formation in vivo
Elastin is formed in the process of elastogenesis through the assembly and cross-linking of the protein tropoelastin (Figure 1). The tropoelastin monomer is produced from expression of the elastin gene during perinatal development by elastogenic cells such as smooth muscle cells (SMCs), endothelial cells, fibroblasts and chondroblasts (Uitto, Christiano et al. 1991). The tropoelastin transcript undergoes extensive alternative splicing leading to the removal of entire domains from the protein. In humans, this splicing results in several tropoelastin isoforms, the most common of which lacks exon 26A (Indik, Yeh et al. 1987). Mature, intracellular tropoelastin associates with the elastin binding protein (EBP) and this complex is secreted to the cell surface (Hinek 1995). Competition from galactosides results in the dissociation of EBP from tropoelastin and the return of EBP to the cell (Mecham 1991). Released tropoelastin on the cell surface subsequently aggregates by coacervation. During this process, the hydrophobic domains of tropoelastin associate and tropoelastin molecules become concentrated and increasingly aligned allowing for subsequent formation of cross-links (Vrhovski, Jensen et al. 1997).
Coacervated tropoelastin is deposited onto microfibrils which probably serve as a scaffold to direct tropoelastin cross-linking and consequential elastic fiber formation. Cross-linking is facilitated by the enzyme lysyl oxidase, which deaminates lysine side chains in tropoelastin to form allysine sidechains that can subsequently react with adjacent allysine or lysine side chains to form cross-links (Kagan and Sullivan 1982). These cross-links can then further react to form desmosine and isodesmosine cross-links between tropoelastin molecules (Umeda, Nakamura et al. 2001). Multiple cross-links result in the mature insoluble elastic fiber.
3. The role of elastin in vivo
Elastin plays a key structural role in elastic tissues including arteries, skin, ligament, cartilage and tendons (Sandberg, Soskel et al. 1981). As the dominant part of the elastic fiber, elastin confers resilience and elasticity essential to the function of these tissues. The arrangement of elastin in the ECM varies between different tissues to yield a wide range of structures with tailored elastic properties. For example, elastin in the form of thin lamina in the arterial wall is mostly responsible for the strength and elasticity necessary for vessel expansion and regulation of blood flow (Glagov, Vito et al. 1992). In the lung, elastin is arranged as a latticework that helps to support the opening and closing of the alveoli (Starcher 2001). In skin, elastin fibers are enriched in the dermis where they impart skin flexibility and extensibility (Roten, Bhat et al. 1996; Pasquali-Ronchetti and Baccarani-Contri 1997).
3.1. Mechanical properties of elastin
Elastin is extremely durable protein with a mean residence time of 74 years (Shapiro, Endicott et al. 1991). It comprises almost 90% of the elastic fiber where it dominates its elastic, mechanical properties. The Young’s modulus for elastic fibers typically ranges from 300 - 600 kPa although it can measure as low as 100 kPa for arterial elastin, highlighting the versatile nature of these structures within the ECM (Mithieux and Weiss 2005; Zou and Zhang 2009). Although the mechanism for elasticity has not been fully elucidated, elastic recoil likely to be entropically driven whereby extension of the protein results in a more ordered structure and thus recoil occurs so the protein can return to a disorder state (reviewed by (Rosenbloom, Abrams et al. 1993; Vrhovski and Weiss 1998). This elasticity is due to the inherent elastic properties of the monomer (Holst, Watson et al. 2010; Baldock, Oberhauser et al. 2011).
3.2. Biological properties of elastin
Elastin plays key biological roles in the regulation of cells native to elastic tissues. Studies of elastin knockout mice reveal a crucial role for elastin in arterial morphogenesis through regulation of SMC proliferation and phenotype (Li, Brooke et al. 1998). This model is supported by in vitro studies showing that elastin can inhibit SMC proliferation in a dose dependent manner (Ito, Ishimaru et al. 1998). Elastin can also mediate the attachment and proliferation of endothelial cells from several vascular origins (Ito, Ishimaru et al. 1998; Williamson, Shuttleworth et al. 2007; Wilson, Gibson et al. 2010). Similar effects have been observed for dermal fibroblasts (Bax, Rodgers et al. 2009; Rnjak, Wise et al. 2011). Additionally elastin is a chemoattractant for SMCs, endothelial cells and monocytes (Senior, Griffin et al. 1980; Wilson, Gibson et al. 2010).
Several cell receptors have been identified for elastin (Figure 2). The most well documented of these receptors is EBP, which binds to multiple sites including the VGVAPG sequence on exon 24 of tropoelastin (Rodgers and Weiss 2005). Upon binding elastin, this receptor activates intracellular signaling pathways involved in cell proliferation, chemotaxis, migration and cell morphology for a range of cell types including SMCs, endothelial cells, fibroblasts, monocytes, leukocytes and mesenchymal cells (Senior, Griffin et al. 1980; Indik, Abrams et al. 1990; Faury, Ristori et al. 1994; Faury, Ristori et al. 1995; Kamisato, Uemura et al. 1997; Jung, Rutka et al. 1998; Mochizuki, Brassart et al. 2002). Other cell receptors, including a less documented glycoprotein termed elastonectin and G protein-coupled receptor can bind elastin through the VGVAPG sequence (Hornebeck, Tixier et al. 1986). Interactions of vascular cells with elastin via these receptors have been shown to dictate focal adhesion formation, cell proliferation and migration (Hornebeck, Tixier et al. 1986; Karnik, Brooke et al. 2003; Karnik, Wythe et al. 2003). Glycosaminoglycans on the SMC and chondrocyte cell surface dominate binding to the C-terminus of bovine tropoelastin (Broekelmann, Kozel et al. 2005; Akhtar, Broekelmann et al. 2011). Cell interactions with human tropoelastin C-terminus specifically occur through the integrin αvβ3 (Rodgers and Weiss 2004; Bax, Rodgers et al. 2009). Elastin binding for some cell types is likely to occur through multiple receptors (Bax, Rodgers et al. 2009; Wilson, Gibson et al. 2010; Akhtar, Broekelmann et al. 2011).
4. Elastin and disease
Disease phenotypes manifest due to the degradation and loss of elastin through injury, genetic mutation or age. For example, autosomal dominant and recessive forms of cutis laxa mutations can arise from genetic modifications to the elastin gene and impaired vesicular trafficking, and have been reviewed elsewhere (e.g. (Hucthagowder, Morava et al. 2009; Callewaert, Renard et al. 2011). In skin, the loss of elastin in the dermal layers in severe burns leads significant physical injuries including scarring, wound contraction and loss of skin extensibility (Rnjak, Wise et al. 2011). In the vasculature, genetic mutations in the elastin gene or genes associated with elastic fiber formation result in severe, debilitating diseases (reviewed by (Kielty 2006)). Supravalvular aortic stenosis can arise from point mutations, deletions or translocations within the elastin gene that typically lead to haploinsufficiency and an altered organization of elastic lamellae in the artery, SMC hyperproliferation, increased media thickness and obstruction of the aorta (Urban, Zhang et al. 2001). Elastin is also associated with several vascular pathologies. Damage and fragmentation of elastin in the artery have been linked with deregulation of SMC phenotype, SMC hyperproliferation and invasion which cause vessel occlusion and cardiovascular complications (Brooke, Bayes-Genis et al. 2003). The failure of inelastic materials as arterial replacements further indicate the essential need for intact elastin in functional arteries (Abbott, Megerman et al. 1987).
5. Elastin biomaterials
Common to all elastin diseases is the catalogued in vivo inability to adequately regenerate and repair dysfunctional elastic fibers leading to subsequent failure of tissue function. This deficiency is mostly attributed to exclusive expression of elastin during early development (Mecham 1991), which results in poor renewal of elastin in adult tissues. Materials that can serve as elastin replacements in adult tissues are in demand. This demand is most apparent in vascular tissue engineering as cardiovascular disease is the major contributor to adult mortality worldwide (Lloyd-Jones, Adams et al. 2010). Current synthetic vascular biomaterials, particularly expanded polyterafluoroethylene (ePTFE) and polyethylene terephthalate (Dacron) are poorly mismatched to native arteries in terms of mechanical properties, endothelial cell and SMC interactions and thrombogenecity which lead to a high failure rate in patients (Chlupac, Filova et al. 2009). Elastin can restore properties that are deficient in current grafts, including compliance and strength to match native vessels and regulation of endothelial and smooth muscle cells.
6. Decellularized tissues as elastin biomaterials
Decellularized tissues, generated by the removal of the cellular components of tissue explant are useful as biomaterials as they a priori possess much of the complex architecture of the native ECM. Elastic tissues are particular amenable to this method as the stability and insolubility of the elastin protein means it is resistant to many treatments used during decellularization processes.
Decellularizing elastin-rich tissues have been proposed as a path towards the potential replacement of artery, heart valves, bladder skin and lung (Daamen, Veerkamp et al. 2007; Petersen, Calle et al. 2010; Price, England et al. 2010). Enriched elastin vascular grafts generated by decellularization and removal of collagen with proteases from porcine carotid arteries can support fibroblasts in vitro (Chuang, Stabler et al. 2009). Cell infiltration has also been observed for other decellularized vascular constructs in vitro and in vivo (Schmidt and Baier 2000; Conklin, Richter et al. 2002; Dahl, Koh et al. 2003; Uchimura, Sawa et al. 2003). Skin replacements formed from decellularized porcine dermis containing 30% elastin show vascularization and support of cultured keratinocytes when examined in a rat excision model. Degradation of the collagen component of the material also occurs (Hafemann, Ensslen et al. 1999). Transplant of a repopulated decellularized human trachea demonstrates the feasibility of acquiring functionality and improved mechanical capabilities in a patient after 4 months (Macchiarini, Jungebluth et al. 2008).
Despite these advantages, decellularized tissue sources are generally animal derived and are therefore restricted in shape, size and supply. Additionally, decellularization methods involve chemical, physical or enzymatic treatments that can individually or collectively compromise mechanical and biological properties (Gilbert, Sellaro et al. 2006). The common use of detergents can limit the degree of cell repopulation. Decellularization methods are highly specific to a particular tissue thus their broader application to different tissues yields viable results in terms of remaining ECM structure and degree of decellularization (Gilbert, Sellaro et al. 2006). Lack of uniformity and versatility can limit the use of decellularized materials as commercial tissue replacements.
7. Tissue derived elastin constructs
7.1. Insoluble elastin materials
Elastin used for in vitro work is generally obtained by purifying the protein directly from elastin-rich tissues. Tissues are treated with chemicals such as NaOH or guanidine-HCl and/or high heat to remove other proteins and cellular material and leave insoluble elastic fibers. However extensive cross-linking and the consequential insolubility of elastin makes it difficult to manipulate in vitro (Daamen, Veerkamp et al. 2007).
Freeze-dried scaffolds of insoluble elastin fibers and purified collagen fibers present mechanical properties consistent with those of elastic tissues (Buttafoco, Engbers-Buijtenhuijs et al. 2006). Furthermore, these scaffolds appear to be compatible with SMCs (Buijtenhuijs, Buttafoco et al. 2004; Engbers-Buijtenhuijs, Buttafoco et al. 2005; Buttafoco, Engbers-Buijtenhuijs et al. 2006), endothelial cells (Wissink, van Luyn et al. 2000) and platelets (Koens, Faraj et al. 2010) pointing to potential vascular applications. Also, insoluble elastin/collagen scaffolds have been explored as possible dermal replacements as these materials can support fibroblasts (Daamen, van Moerkerk et al. 2003) and keratinocytes (Lammers, Tjabringa et al. 2009). Other insoluble elastin composites such as elastin/fibrin biomaterials have been generated but characterization of these materials is limited to mechanical capacity (Barbie, Angibaud et al. 1989).
7.2. Hydrolyzed elastin materials
The solubility of tissue-derived elastin can be improved by partial hydrolysis. A fragmented elastin preparation termed α-elastin is obtained by hydrolysis with oxalic acid and is often used in in vitro studies of elastin (Partridge, Davis et al. 1955). Hydrolysis can be performed with potassium hydroxide to yield κ-elastin or through mild digestion with proteinases (Partridge, Davis et al. 1955; Jacob and Hornebeck 1985). Hydrolyzed preparations of elastin display various properties that are similar to the native protein including temperature-induced aggregation (coacervation) and regulation of SMC and fibroblast phenotype (De Vries, Zeegelaar et al. 1995; Ito, Ishimaru et al. 1998). Fragmentation of elastin is associated with reduced protein structural integrity and altered cellular signaling properties (Daamen, Veerkamp et al. 2007; Bax, Rodgers et al. 2009).
Multiple vascular materials have been synthesized from hydrolyzed elastin preparations (Table 1). Hydrogels, cross-linked films and electrospun fibers containing hydrolyzed α-elastin all show preferable vascular material properties including regulation of SMC phenotype and increased mechanical elasticity. Electrospun materials are of particular interest as architecturally, these materials closely mimic the dimensions of elastic fibers in vivo (Li and Xia 2004).
Hydrolyzed elastin materials have also been proposed for use in the repair of elastic cartilage. In porous PCL scaffolds, infusion of α-elastin demonstrates enhanced scaffold elasticity and attachment and proliferation of articular cartilage chondrocytes in vitro (Annabi, Fathi et al. 2011). Replication of auricular-like cartilage has also been explored using alginate, collagen type I and κ-elastin containing hydrogels with auricular cartilage chondrocytes (de Chalain, Phillips et al. 1999). When these materials were implanted in mice and harvested after 12 weeks, matrix components including collagen and elastic fibers were present.
Dermal replacements containing hydrolyzed elastin demonstrate improved properties over elastin-free materials in regards to wound contraction and tissue regeneration (Rnjak, Wise et al. 2011). For example, MatriDerm, a collagen based scaffold with α-elastin shows improved skin elasticity (Ryssel, Gazyakan et al. 2008). Hydrogels formed exclusively from α-elastin (Figure 3) favorably support attachment and proliferation of dermal fibroblasts in vitro (Annabi, Mithieux et al. 2009; Annabi, Mithieux et al. 2009).
|α-elastin film||-Low elastic modulus|
-attachment & proliferation of SMCs
|-reduced SMC proliferation compared to TCPS||(Leach, Wolinsky et al. 2005)|
|elastin/gelatin gel||-Young’s modulus matched to native artery|
-proliferation & infiltration of SMCs
|-reduced SMC growth compared to TCPS||(Lamprou, Zhdan et al. 2010)|
|Collagen type I gels containing α-elastin||-SMC proliferation inhibited||- EC proliferation inhibited at high α-elastin concentrations||(Ito, Ishimaru et al. 1997)|
|α-elastin & collagen electrospun blended conduit||-attachment & proliferation of SMCs||-no mechanical testing||(Buttafoco, Kolkman et al. 2006)|
|α-elastin electrospun sheet||-SMC proliferation inhibited|
-α-SMA expression observed
|-no mechanical testing||(Miyamoto, Atarashi et al. 2009)|
|α-elastin electrospun fibers||-attachment & proliferation of embryonic mesenchymal cells||-complete 3D constructs not created||(Li, Mondrinos et al. 2005)|
|α-elastin, PLGA & gelatin electrospun sheet||-mechanical properties tuned to artery through polymer content|
-proliferation of ECs on scaffold surface & infiltration of SMCs.
-expression of functional EC molecules
|-mechanical properties tested on electrospun sheets, not tubes||(Han, Lazarovici et al. 2011)|
|α-elastin, collagen type I & PLGA electrospun conduit||-matched compliance to bovine iliac artery|
-proliferation of ECs on inner & SMCs on outer surface of conduit
-no immune reaction when implanted in mice
|-scaffold contraction in vitro||(Stitzel, Liu et al. 2006; Lee, Yoo et al. 2007)|
|α-elastin, collagen type I & PLLA, PCL or PLCL blended electrospun conduit||-growth of bovine ECs|
-infiltration and α-SMA expression of SMCs
|-scaffold contraction of PLCL blends in vitro||(Lee, Yoo et al. 2007)|
|α-elastin & PDO blended electrospun conduit||-mechanical properties matched to femoral artery with increased elastin content|
-increased cell infiltration with increased elastin content
-increased graft burst pressure with suture reinforcement
|-suture reinforcement lowers compliance||(Sell, McClure et al. 2006; Smith, McClure et al. 2008)|
|Elastin, collagen type I & collagen type III tri-layered electrospun conduit||-growth of EC, SMC and Fb in separate layers||-delamination of layers|
-no mechanical testing
|(Boland, Matthews et al. 2004)|
|α-elastin, gelatin & PDS blended electrospun conduit||-matched tensile properties & elastic modulus to femoral artery||-loss of tensile properties due to in vitro degradation|
-no cell studies performed
|(Thomas, Zhang et al. 2009)|
|α-elastin, gelatin & Maxon multi-layered electrospun conduit||-comparable mechanical properties to femoral artery||-no cell studies performed||(Thomas, Zhang et al. 2007)|
|bovine elastin, PGC, PCL & gelatin bi-layered electrospun conduit||-tensile strength matched to native artery|
-attachment & proliferation of EC & EPCs
|-no SMC characterization||(Zhang, Thomas et al. 2010; Zhang, Thomas et al. 2010; Zhang, Xu et al. 2011)|
|α-elastin, collagen, PCL tri-layered electrospun conduit||-mechanical properties matched to native artery by modulation of elastin & PCL content||-no cell studies performed||(McClure, Sell et al. 2010)|
8. Elastin-sequence based materials
8.1. Synthetic elastin-based peptides
Synthetic peptides based on key elastin sequences present elastin-like properties including self-assembly, cross-linking and cell interactions (Long, King et al. 1989; Faury, Garnier et al. 1998; Bellingham, Woodhouse et al. 2001; Karnik, Brooke et al. 2003; Karnik, Wythe et al. 2003). Coating of materials with elastin peptides can improve biocompatibility by providing protein sequences required for cell binding (reviewed by (Almine, Bax et al. 2010)). Some three dimensional materials formed from elastin-based peptides demonstrate elastin-like properties, including hydrogels that support cell growth and possess high degrees of elasticity (Keeley, Bellingham et al. 2002; Trabbic-Carlson, Setton et al. 2003). However as with hydrolyzed elastin preparations, synthetic peptides can lack the full repertoire of properties of the fully intact protein and are associated with inflammation (Faury, Ristori et al. 1995).
8.2. Recombinant human tropoelastin
Recombinant human tropoelastin (rhTE) is expressed and purified can be made as a recombinant protein in Escherichia coli. rhTE exhibits many properties of native tropoelastin including the ability to coacervate under physiological conditions and be cross-linked in vitro to form insoluble elastin fibers (Vrhovski, Jensen et al. 1997; Muiznieks, Jensen et al. 2003). rhTE promotes endothelial cell and fibroblast attachment, spreading and proliferation when used as a surface coating (Bax, Rodgers et al. 2009; Rnjak, Li et al. 2009; Wise, Byrom et al. 2011) and improves the biocompatibility of implanted devices (Yin, Wise et al. 2009; Wilson, Gibson et al. 2010).
Three dimensional biomaterials are produced by cross-linking rhTE to form synthetic human elastin. Synthetic elastin has advantages over decellularized tissue and hydrolyzed elastin preparations as it utilizes human protein avoiding potential problems arising from species differences while benefiting from homogeneity to improve reproducibility and uniformity.
These types of synthetic elastin hydrogels can be made by chemical cross-linking (Mithieux, Rasko et al. 2004), enzyme treatment (Mithieux, Wise et al. 2005) or raising the pH (Mithieux, Tu et al. 2009) of rhTE solutions (Figure 4). The hydrogels demonstrate mechanical properties that are consistent with native elastin including low elastic moduli, support of attachment and proliferation of dermal fibroblasts (Mithieux, Rasko et al. 2004; Rnjak, Li et al. 2009; Annabi, Mithieux et al. 2010). Increases in hydrogel porosity using high pressure CO2 or the incorporation of glycosaminoglycans improve cell infiltration into hydrogels (Annabi, Mithieux et al. 2010; Tu, Mithieux et al. 2010) where the maintenance of fibroblasts within these scaffolds present them as candidate dermal substitutes.
Electrospun synthetic elastin allows for the formation of highly organized biomaterials with tunable mechanical biological properties. Electrospun synthetic elastin is formed by the electrospinning and chemical cross-linking of rhTE to yield ribbon-like microfibers (Figure 5) whose dimensions match those of native elastin fibers (Nivison-Smith, Rnjak et al. 2010). Highly porous electrospun synthetic elastin scaffolds, generated by using high flow rates facilitate the infiltration of dermal fibroblasts in vitro and present an alternative to synthetic elastin hydrogels as a dermal replacement (Rnjak, Li et al. 2009).
As a potential vascular material, electrospun synthetic elastin shows attractive characteristics including internal mammary artery-matched elastic mechanical properties, low platelet adhesion (Wise, Byrom et al. 2011) and support of growing human vascular cells including SMCs, endothelial cells (Figure 6) and embryonic palatal mesenchymal stem cells (Li, Mondrinos et al. 2005; Nivison-Smith, Rnjak et al. 2010). Synthetic human elastin fibers can also direct cell spreading to resemble cell organization in vivo. For example, the radial alignment of SMC in the arterial media is mimicked by culture of these cells on parallel synthetic elastin fibers (Nivison-Smith & Weiss 2011, submitted). Blended conduits of synthetic elastin and silk or polycaprolactone display elasticity and cell adhesion properties courtesy of the rhTE component while the composite component confers additional mechanical strength (Hu, Wang et al. 2010; Wise, Byrom et al. 2011).
Elastin is an essential matrix protein, so it is logical that biomaterials designed for elastic tissues should incorporated elastin. Difficulties in sourcing pure intact elastin preparations, particularly those that reflect human sequences, has limited the generation of these materials. Synthetic human elastin that is made from rhTE presents a versatile and stable component of vascular and dermal materials. Elastin-based constructs demonstrate mechanical and biological properties consistent with native elastin and have potential for a wider range of applications.
We acknowledge grant support from the Australian Research Council and the National Health and Medical Research Council. We thank Dr. Anna Waterhouse for the images related to elastin hydrogels. We acknowledge the contributions of the staff and the facilities made available at the AMMRF (Australian Microscopy & Microanalysis Research Facility) at the Australian Center for Microscopy and Microanalysis, The University of Sydney.