Elastic Fiber Production in Cardiovascular Tissue-Equivalents
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Matrix Biology 22 (2003) 339–350 Elastic fiber production in cardiovascular tissue-equivalents Jennifer L. Long, Robert T. Tranquillo* Department of Chemical Engineering & Materials Science and Department of Biomedical Engineering, 7-114 BSBE, 312 Church St SE, University of Minnesota, Minneapolis, MN 55455, USA Received 10 January 2003; received in revised form 30 April 2003; accepted 30 April 2003 Abstract Elastic fiber incorporation is critical to the success of tissue-engineered arteries and heart valves. Elastic fibers have not yet been observed in tissue-engineered replacements fabricated in vitro with smooth muscle cells. Here, rat smooth muscle cells (SMC) or human dermal fibroblasts (HDF) remodeled collagen or fibrin gels for 4 weeks as the basis for a completely biological cardiovascular tissue replacement. Immunolabeling, alkaline extraction and amino acid analysis identified and quantified elastin. Organized elastic fibers formed when neonatal SMC were cultured in fibrin gel. Fibrillin-1 deposition occurred but elastin was detected in regions without fibrillin-1, indicating that a microfibril template is not required for elastic fiber formation within fibrin. Collagen did not support substantial elastogenesis by SMC. The quantity of crosslinked elastic fibers was enhanced by treatment with TGF-b1 and insulin, concomitant with increased collagen production. These additives overcame ascorbate’s inhibition of elastogenesis in fibrin. The elasticfibers that formed in fibrin treated with TGF- b1 and insulin contained crosslinks, as evidenced by the presence of desmosine and an altered elastin labeling pattern when b-aminopropionitrile (BAPN) was added. These findings indicate that in vitro elastogenesis can be achieved in tissue engineering applications, and they suggest a physiologically relevant model system for the study of three-dimensional elastic structures. ᮊ 2003 Elsevier B.V.yInternational Society of Matrix Biology. All rights reserved. Keywords: Elastin; Fibrillin; Tissue engineering; Fibrin; Smooth muscle cell 1. Introduction component. Approaches to date have included building tissue replacements on an elastin scaffold isolated from Elastic fibers are a critical structural component in cadaveric tissues (Berglund et al., 2001), supplying cardiovascular tissues. Present in arteries and heart soluble tropoelastin to a cell culture (Stone et al., 2001), valves, they dictate mechanics at low strains before and designing biocompatible synthetic elastic polymers stiffer collagen fibers are engaged (Bank et al., 1996; (Urry and Pattanaik, 1997). Evaluation of spontaneous Vesely, 1998). They also confer elasticity, preventing elastogenesis in tissue-engineered constructs has been dynamic tissue creep by stretching under load then limited. A rolled tube of vascular cell-derived matrix recoiling to their original configurations after load demonstrated elastin immunolabeling after three months release (Ross and Bornstein, 1971). Elastin knock-out of culture, but in the internal interfaces of the fibroblastic studies and clinical observations have revealed an essen- adventitial layer rather than within the muscular medial tial regulatory function during artery development. In layer (L’Heureux et al., 1998). Smooth muscle cells the absence of extracellular elastin accumulation, smooth produced significantly less elastin mRNA in a non- muscle proliferation stenoses arteries (Karnik et al., fibrillar collagen sponge than in a poly-glycolic acid 2003; Urban et al., 2002). scaffold (Kim et al., 1999). Transplanted or host cells To ensure appropriate mechanical function and pre- may deposit elasticfibers after tissue-engineered con- vent these serious complications, successful cardiovas- struct implantation (Berthod et al., 2001; Shum-Tim et cular tissue replacements must incorporate an elastic al., 1999; Stock et al., 2001), but this necessitates in *Corresponding author. Tel.: q1-612-625-6868; fax: q1-612-626- vivo maturation and precludes in vitro study of elasto- 6583. genesis. Elastic fiber formation by vascular cells E-mail address: [email protected] (R.T. Tranquillo). entrapped within three-dimensional fibrillar collagen or 0945-053X/03/$30.00 ᮊ 2003 Elsevier B.V.yInternational Society of Matrix Biology. All rights reserved. doi:10.1016/S0945-053XŽ03.00052-0 340 J.L. Long, R.T. Tranquillo / Matrix Biology 22 (2003) 339–350 fibrin culture has not been shown to our knowledge. SMC (Merrilees et al., 2002). Beneficial proteoglycans Those two biopolymers are studied here as the basis for may protect elastin binding protein function as it shuttles a completely biological arterial media substitute. tropoelastin to nascent elastic fibers. Disruption of this Traditional monolayer cell culture studies have iden- system interferes with elastogenesis in multiple clinical tified several factors that influence elastogenesis. Tro- conditions (Hinek et al., 2000; Hinek and Wilson, poelastin expression in the strongly elastogenicaortic 2000). The recent studies illustrate the complexity of smooth muscle cell (SMC) is developmentally regulat- elastogenesis, which is now understood to encompass ed, with maximal secretion by cells freshly isolated from gene transcription, post-transcriptional regulation, and the neonate and virtually none by adult cells (Johnson coordinated assembly of multiple molecules within a et al., 1995; McMahon et al., 1985). Inhibitors include receptive extracellular milieu. Despite the increasing bFGF, which decreases elastin mRNA transcription appreciation of extracellular elastogenesis modulation, (Carreras et al., 2002), and ascorbate, which destabilizes the basic question of which environmental scaffolds elastin mRNA while improving collagen mRNA stability promote elastogenesis has not been previously (Davidson et al., 1997). TGF-b1 and insulin-like growth addressed. We compare collagen and fibrin scaffolds, factor-1 both increase tropoelastin mRNA and protein measuring elasticfibers directly, rather than intermedi- by various cultured cells (Davidson et al., 1993; Sauvage ates such as RNA, to account for the many steps in et al., 1998; Wolfe et al., 1993). TGF-b1 has also been mature elasticfiber production. found to upregulate the elastin gene promoter in genet- Monolayer cell culture has been informative, and can ically modified mice (Katchman et al., 1994) and to produce structures many cell layers thick. However, increase lysyl oxidase activity in cultured cells (Shanley reproducing the organization of native tissues is chal- et al., 1997). Lysyl oxidase crosslinks tropoelastin into lenging, if not impossible, with the monolayer approach. its mature fully functional form, but can be inhibited by This work demonstrates and quantifies elastogenesis in nitricoxide donors suchas b-aminopropionitrile (Smith- a totally biological tissue replacement that can mimic Mungo and Kagan, 1998). the characteristic alignment of native tissues. This align- Dermal fibroblast elastogenesis has also been studied ment confers the mechanical strength of tissue and extensively due to elastin’s importance in skin. Its provides a template for organized extracellular matrix regulation by soluble molecules is similar to that of deposition. We have previously reported that SMC SMC (Kahari et al., 1992). Unlike SMC, dermal fibro- compact tubes of fibrin gel around a non-adhesive blasts maintain elastin mRNA synthesis until mid-adult- mandrel, producing strong circumferential alignment of hood (Fazio et al., 1988) but deposit little insoluble both the initial fibrin and the subsequent cell-produced elastin (Narayanan et al., 1976). This discrepancy may collagen. The alignment in that case matches that of the be due to fibroblast incompetence in depositing elastic arterial medial layer (Grassl et al., 2002). Constructs fibers. A skin substitute co-culturing keratinocytes and prepared by constrained cell-induced compaction of fibroblasts on a collagen-GAG-chitin sponge produced highly hydrated and entangled native protein fibril net- elastin and fibrillin-1, while cultures of fibroblasts alone works (as depicted in Fig. 1) are termed ‘tissue-equiv- did not (Duplan-Perrat et al., 2000). Because of their alents’ (Tranquillo, 1999). While this study uses accessibility and robust collagen production, dermal disc-shaped tissue-equivalents for simplicity, the method fibroblasts have also been used in cardiovascular tissue can be extended to more relevant geometries for arteries engineering including heart valve-equivalents (Neidert and valves as has been reported by this laboratory et al., 2003). Elasticfiber productionin biopolymer (Grassl et al., 2002, 2003; Neidert et al., 2003).We culture without keratinocytes is critical for the valve show here that the organized elasticfibers are produced application, so is examined here. in this system in addition to substantial collagen, thus Extracellular matrix (ECM) likely influences elasto- approaching the ECM composition of small diameter genesis since the vasculature develops within a three- arteries. dimensional matrix rather than from a monolayer of Important regulators identified in the traditional cell cells. In one series of studies, cells cultured on decel- culture studies described above are examined in the lularized elastin-containing tissue layers incorporated three-dimensional gel culture system for tissue-equiva- new elastin into the existing matrix, producing more lent formation. Neonatal and adult SMC are compared, than on tissue-culture plastic alone (Mecham, 1981;