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The effect of sodium ascorbate on the mechanical properties of hyaluronan-based vascular constructs
ARTICLE IN PRESS
Biomaterials 27 (2006) 623–630 www.elsevier.com/locate/biomaterials
The effect of sodium ascorbate on the mechanical properties of hyaluronan-based vascular constructs Chiara Arrigonia, Davide Camozzia, Barbara Imbertia, Sara Manterob, Andrea Remuzzia, a
Department of Biomedical Engineering, Mario Negri Institute for Pharmacological Research, Via Gavazzeni, 11-24125 Bergamo, Italy b Department of Biomedical Engineering, Politecnico di Milano, Piazza Leonardo Da Vinci, 32-20133 Milano, Italy Received 23 March 2005; accepted 20 June 2005 Available online 26 July 2005
Abstract Esterified hyaluronic acid (HYAFF) is routinely used for clinical tissue engineering applications such as skin and cartilage. In a previous study we developed a technique for in vitro generation of cylindrical constructs from cellularized HYAFF flat sheets. In the present investigation we studied the possibility to improve mechanical properties of this vascular construct by the addition of sodium ascorbate (SA). Non-woven HYAFF flat sheets were seeded with porcine aortic smooth muscle cells (SMCs) and cultured for 14 or 28 days with standard medium or medium additioned with SA. In selected experiments HYAFF sheets seeded with SMCs were wrapped to obtain cylindrical shape and then cultured in control medium or SA additioned medium for up to 28 days. We estimated cell viability for flat sheets, and performed histological examination, analysis of extracellular matrix (ECM) deposition and mechanical tests on tubular constructs. The number of viable cells and ECM deposition increased with time in constructs cultured in the presence of SA, as compared to control group. Moreover, SA improved mechanical properties of the vascular construct lowering material stiffness and increasing tensile strength as compared to untreated controls. The addition of SA to the medium improved cell proliferation and ECM synthesis on this biodegradable material, which leads to the formation of well organized, mechanical resistant tissue-engineered structure. r 2005 Elsevier Ltd. All rights reserved. Keywords: Tissue engineering; Hyaluronic acid; Smooth muscle cell; Sodium ascorbate; Mechanical properties
1. Introduction The main aim of vascular tissue engineering is to generate functional blood vessels in vitro to be used as vascular grafts, since synthetic small caliber vascular prosthesis cannot be used to replace diseased arterial segments with diameter less than 6 mm. Many challenges remain to be solved before a blood vessel replacement will be available, including achievement of adequate mechanical strength, non-thrombogenic and long-term function [1,2]. In the last years several techniques have been developed for in vitro generation of artificial blood vessels [3–5]. Despite promising results Corresponding author. Tel.: +39 035 319888; fax: +39 035 319331.
E-mail address: [email protected] (A. Remuzzi). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.06.009
obtained so far in this area of investigation, a crucial factor for further development of a tissue-engineered blood vessel is the biodegradable material used as scaffold. A number of biodegradable materials have been used to this purpose, from synthetic polymers to natural extracellular matrix structures. We have recently investigated adhesion and proliferation of aortic smooth muscle cells (SMCs) seeded on esterified hyaluronic acid (HYAFF-11s) scaffolds and successfully developed a simple technique to obtain cellularized tubular constructs from HYAFF flat sheets in vitro [6]. HYAFF is a biodegradable material currently used for tissue engineering of skin and cartilage [7,8]. This material is highly compatible with cells and matrix and its degradation products induce extracellular matrix production and neoformation of blood capillaries [9].
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Since the extracellular matrix (ECM) within native arteries is a determinant of mechanical properties of these blood vessels, we reasoned that enhancing ECM protein production by SMCs seeded on the biodegradable scaffold by biochemical stimulation [2,10] could improve tissue organization with higher mechanical resistance and better elastic properties. Ascorbic acid (AA) and its derivatives have been shown to stimulate collagen synthesis in monolayers of SMCs [11]. It has been shown that AA influences hydroxylation of proline and lysine necessary for molecular stabilization of collagen. Moreover, it has been also demonstrated that AA stimulates the production of smooth muscle-specific proteins, including smooth muscle myosin heavy chain-1 (SM1) and calponin-1, in cultured rat SMCs [12]. These findings suggest that AA may also sustain dedifferentiation of SMCs. The first aim of this work was to investigate whether AA may influence the proliferation of SMCs cultured on flat sheets of non-woven HYAFF scaffold. The second aim was to study the effects of AA on HYAFF tubular vascular constructs investigating ECM production and distribution into the vessel wall, and evaluate mechanical properties of these constructs. We also performed evaluation of protein expression by immunofluorescence staining to study phenotypic drift of SMCs on this threedimensional scaffold.
2. Materials and methods 2.1. Cell culture techniques Primary porcine aortic SMCs were isolated from normal 2–4 week-old pigs by a collagenase digestion method described previously [6], and were used between passages 4 and 6. Cells were characterized as smooth muscle based on their positive immuno-reaction to antibodies against a-smooth muscle actin (aSMA, Sigma-Aldrich Corporation, St Louis, MO, US) and calponin (Sigma). Monolayer cultures were fed with Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 4.5 g/l glucose, supplemented with 10% fetal bovine serum (FBS, Invitrogen Ltd, Paisley, UK), 10% porcine serum (PS; Invitrogen), 4 mM L-glutamine (Invitrogen), 100 U/ml penicillin G sodium (Invitrogen), 100 mg/ml streptomycin sulphate (Invitrogen), and 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, Sigma). Culture medium was changed three-times a week. 2.2. Seeding and culture on biomaterial The biodegradable scaffold used for cell seeding was a 100% benzyl-ester of hyaluronic acid (HYAFF-11) configured as sheets of non-woven mesh. The material was kindly provided by Fidia Advanced Biopolymers (Abano Terme, Italy). Scaffolds were treated before cell seeding with a solution of human fibronectin (BD Bioscience, Bedford, MA, US) in water with a density of 3.2 mg/cm2 of non-woven mesh. SMCs
were dispensed over the HYAFF scaffold at a concentration of 3 106 cells/cm2 then were allowed to attach to the scaffold for 3 h in a cell incubator before adding complete culture medium. Initially, cell proliferation was studied by seeding SMCs on 1 1 cm flat sheets of non-woven HYAFF and keeping them in culture for 14 and 28 days with standard medium or medium supplemented with 50 mg/ml of sodium ascorbate (SA, Sigma). To obtain tubular constructs, 4 2 cm HYAFF sheets precoated with fibronectin were seeded with 24 106 PSMC, cultured as flat sheets for 7 days and subsequently wrapped around glass cylindrical mandrels with an outside diameter of 3.5 mm.To maintain the tubular shape a suture wire (Ethicon SpA, Pomezia, Italy) was wrapped around the construct to avoid unfolding from the mandrel. Tubular constructs obtained with this technique were cultured for additional 14 or 28 days in culture medium without or supplemented with SA. At the end the mandrel was withdrawn, sections of the tubular constructs of 5 mm in length were then cut and used for histological analysis and mechanical testing. 2.3. Cell viability test Cell proliferation was evaluated by 3-[4,5-dimethylthiazol-2]2,5-dipheniltetrazolium bromide (MTT) viability assay as previously described [6,13]. Briefly, cell seeded scaffolds were rinsed with phosphate-buffered saline (PBS, Invitrogen) and incubated for 3 h at 371C with 0.5 mg/ml MTT (Sigma) in sterile DMEM medium w/o phenol red. After incubation, un-reacted MTT was removed by rinsing with PBS. The reaction product was extracted for 3 h with 5 ml of extraction solution (90% isopropanol/10% dimethyl sulphoxide, Sigma). The absorbance of each sample was measured by a spectrophotometer (Beckman Instruments Inc, Fullerton, CA, US) at 570 nm with 690 nm as reference wavelength. To relate measured absorbance with cell number per unit surface area of the scaffold, we used a calibration curve [14] obtained measuring the absorbance of HYAFF samples seeded with known number of cells ranging from 2.5 105 to 1.5 106 cells/cm2. 2.4. Morphometric analysis of ECM volume For histological analysis, representative samples for each experimental condition were fixed in Bouin solution (BioOptica, Milan, Italy) for 4 h, dehydrated in alcohol, and embedded in paraffin. Sections (5 mm thick) were stained with haematoxylin and eosin and used for optical microscopy. The presence of ECM was evaluated on sections stained with Masson’s trichrome and subsequent morphometric analysis, as previously described [6]. To this purpose 15–20 images of each construct section were systematically sampled along radial position and across the construct wall thickness, digitized using a digital camera connected to the microscope and digitally overlaid with an orthogonal grid composed of 154 points. The fraction of available volume within the scaffold effectively occupied by ECM was estimated as the ratio of grid points falling in ECM positively stained areas and grid points falling within the construct but outside HYAFF fibers. The number of images used for this quantification allowed to obtain an estimated probable error smaller than 3%, as reported in literature [15].
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2.5. Immunofluorescence staining
3.1. Results
To assess the presence of SMC proteins, tissue sections were stained with a mouse monoclonal anti-aSMA antibody (Sigma) and a mouse monoclonal anti-calponin antibody (Sigma). Tissue specimens were infiltrated by immersion in 30% sucrose/PBS for at least 1 h at room temperature, embedded in OCT medium, and frozen in liquid nitrogen. Tissue sections were cut at 3 mm thickness. Following fixation with ice-cold acetone (Carlo Erba, Milan, Italy), non-specific binding of antibodies was blocked with 1% bovine serum albumin (Sigma) in PBS. Sections were then incubated with primary antibodies for 1 h at room temperature (1A4, 1:200; hCP, 1:1000). After washing in PBS, sections were incubated with FITC-conjugated goat anti-mouse IgG antibodies (1:40, Sigma) for 1 hour at room temperature. Counter-staining with DAPI (Sigma) was performed to label cell nuclei for 30 min at room temperature. Slides were then mounted and examined with an inverted light microscope (Olympus Mod. IX70, Hamburg, Germany) equipped with epifluorescence.
3.1. Cell proliferation and distribution on flat HYAFF sheets
2.6. Mechanical testing To test mechanical properties of the tissue-engineered constructs, we followed a specific protocol [6]. Briefly, ringshaped specimens with a reproducible length of 5 mm were cut from each tubular construct and mounted with suitable holders on a computer controlled testing machine (MTS Synergie 200 H, Eden Prairie, MN, US). Specimens of coronary arteries from pig hearts, taken within 4 h of cold ischemia, have been cut from native vessels with the same procedure. Initial sample length was calculated from the displacement reached when a reference pre-load of 0.015 N was applied. Each sample was pre-conditioned, with loading speed of 1.0 mm/min and load endpoint of 0.4 N. The sample was left to recover for 120 s and a relaxation test was then performed. The test was stopped after 500 s and the sample left to recover for additional 120 s. A failure tensile test was finally carried out until complete sample rupture. Due to non-homogeneous thickness of the vascular constructs, constitutive parameters could not be directly calculated. To overcome this limitation, we evaluated the following structural parameters for each sample: 1) ultimate force per unit axial length (Tmax) defined as the maximum force reached during the failure tensile test divided by sample axial length; (2) ultimate strain (emax) as the strain level corresponding to Tmax; (3) stiffness (K) calculated as the slope of the loading ramp of the relaxation test between the end of the toe region (at T1 ¼ 0.04 N/mm) and the end of the loading ramp itself (at T0 ¼ 0.08 N/mm); (4) the first time constant (t) defined as the intersection between the time axis and the line tangent to the initial portion of the reduced relaxation curve. 2.7. Statistical analysis Results are expressed as the mean7standard deviation (SD). Statistical analysis was carried out using the analysis of variance or the unpaired Student t-test as appropriate. Specific comparisons between groups were calculated using the Bonferroni correction [16].
SMCs isolated by enzymatic digestion from porcine aorta exhibited a spindle-shaped phenotype with a classic ‘‘hill-and-valley’’ growth pattern at confluence, as shown in Fig. 1, and stained positively for aSMA and calponin with extended filaments across cell bodies (see Fig. 1B and 1C). The number of viable cells on 1 1 cm HYAFF sheets 14 days after seeding was significantly higher in the presence of SA as compared to untreated controls averaging 6.6070.48 106 vs. 3.3470.47 106 (po0.01, n ¼ 6 and 7, respectively). Due to this increase in viable cell number, sheets cultured with SA were more compact than untreated sheets until the end of culture. At 4 weeks after seeding, the SMC number was still significantly higher in the presence of SA, averaging 5.9770.49 106 vs 2.6370.88 106 (po0.01, n ¼ 6 and 4 respectively). Histological analysis of these samples (data not shown) confirmed higher proliferation of SMCs in constructs treated with SA and better ingrowth of seeded cells into HYAFF cultured with SA. 3.2. Tubular construct generation We compared tubular constructs obtained using control medium and SA-additioned medium at 14 and 28 days, respectively. Most of the samples of the control group did not last in culture until 28 days. Around day 20 they began to unwrap from the mandrel loosing tubular structure. Constructs cultured in the presence of SA for 28 days were even more compact and regular in shape than control constructs at 14 days. At 28 days of culture, SA-treated constructs appeared cellularized for the entire length of the scaffold. At light microscopy, SA-treated constructs showed cells more uniformly distributed along HYAFF layers in the entire construct wall, while in control constructs cells were less uniformly distributed. In the presence of SA, HYAFF layers were more adherent one to the other resulting in a more compact wall of the construct, likely due to increased cell proliferation, adhesion to the scaffold and extracellular matrix deposition, especially on the outer surface of each individual layer. 3.3. Morphometrical analysis of ECM Deposition of ECM was observed predominantly at the outer surface of the scaffold, both in control and in treated constructs. We used morphometric analysis to quantify the presence of ECM in both experimental conditions. At 14 days of culture the construct wall volume occupied by ECM was slightly higher with SA than in control constructs, averaging 17.175.1 vs.
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Fig. 1. Representative cell morphology at phase-contrast light microscopy of porcine aortic SMC monolayer (A, bar represents 100 mm). Expression of aSMA (B) and calponin (C) by porcine SMCs monolayers (bars represent 50 mm).
14.473.1%, respectively. With time ECM volume significantly increased in SA-treated constructs, averaging 28.977.7% (po0.05 vs. SA construct at 14 days). 3.4. Immunofluorescence staining As observed for cell distribution across the construct thickness, also the pattern of protein expression was more pronounced at the edge of individual HYAFF layers, likely reflecting higher cell density in these regions. Representative images of fluorescent antibody staining performed to identify the presence and distribution of aSMA and calponin, two specific proteins of SMC cytoskeleton, are reported in Fig. 2. At 14 days aSMA expression was similar in control and SA-treated constructs. SMCs appeared elongated and the protein was present across the whole cell body with its characteristic filaments. At 28 days aSMA expression decreased in control constructs, while in SA-treated constructs there was a strong increase in fluorescence signal. As shown in Fig. 2, in SA-treated constructs more nuclei were present (see DAPI blue staining) and extended aSMA filaments (stained in green) as compared to untreated samples, with less cell density and weaker protein expression. In SA-treated constructs cells completely surrounded HYAFF fibers, as shown in Fig. 2, with positively stained filaments. Calponin
labeling showed a punctate pattern in the whole cell bodies that sometimes resulted in filaments (see Fig. 2). Protein expression with time followed the same pattern of aSMA, with differences between control and SAtreated constructs even more evident. Control constructs at day 14 showed a downregulation of calponin expression that was still present on day 28. In contrast, addition of SA induced a stronger expression of the protein by SMCs at day 14 that further increased at day 28. At this time point we observed once again SMCs with strong calponin filaments surrounding completely the HYAFF fibers. 3.5. Mechanical testing Calculated parameters from mechanical tests are reported in Fig. 3. Control and SA-treated scaffolds showed a different behavior during failure test. Control constructs underwent frequently unwrapping of individual layers, while failure of SA-treated construct samples was consistently due to rupture of the tubular cross section. The failure tensile resistance Tmax, the stiffness K and the first time constant t all were only numerically different in SA-treated constructs at 14 days as compared to control constructs, but the differences did not reach statistical significance. With culture time, Tmax only numerically increased in SA-treated
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Fig. 2. aSMA and calponin expression (green staining) of control and SA-treated constructs at 14 and 28 days of culture. Cell nuclei are labeled by blue fluorescence with DAPI. Asterisks represent HYAFF fibers.
constructs, while K significantly decreased, and t significantly increased comparing values calculated at 14 and 28 days. We also compared the parameters
calculated from mechanical tests of cellularized constructs in the presence of SA with those of samples of native blood vessels, as shown in Fig. 3. While
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mechanical resistance of SA constructs was significantly lower than that of coronary arteries (Tmax averaged 0.4170.11 N/mm in SA constructs and 1.1270.42 N/mm in coronary artery specimens, po0.01), the elastic properties of constructs cultured with SA approached those of native vessels. Thus, while control constructs showed value of K and t, respectively, significantly higher and lower than those of native vessels, SA constructs showed value of these parameters comparable to those of native vessels.
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The biodegradable scaffold we investigated is made of non-woven flat sheets of HYAFF-11s fibers. During manufacturing, the sheets are compressed to obtain a compact uniform meshwork with 250 mm thickness. We have recently reported that HYAFF scaffold can be used for in vitro reconstruction of small caliber blood vessel [6]. We seeded vascular SMCs on flat HYAFF sheets and generated a tubular construct by wrapping sheets around a mandrel. Despite good cellularization within the scaffold thickness, the mechanical properties of these tubular constructs were still far from those of native coronary arteries being less resistant and less elastic than native vessels [6]. The content and organization of ECM proteins within native arteries determine their complex mechanical properties [17,18], however, it has been shown that isolated SMCs in vitro do not secrete significant amounts of ECM proteins in the absence of biochemical stimulation [10]. In addition, in our previous study we observed an increasing number of cells undergoing apoptosis during in vitro culture on HYAFF scaffold in the absence of specific growth factors [6]. To improve these experimental conditions, we used SA to increase ECM protein production by SMCs seeded on the scaffold. Davidson and coworkers [11] have shown that ascorbate (a derivative of vitamin C) induces exogenous collagen synthesis in cultured SMCs, acting as a cofactor for the hydrolyzation of proline and lysine. Moreover, they reported that ascorbate-treated cultures showed slightly faster growth rate as compared to untreated cells. Our present results show that the number of viable SMCs seeded on HYAFF sheets and stimulated with SA was almost double than that measured on control unstimulated scaffolds at 14 and 28 days. These data demonstrate that SA-induced cell proliferation previously observed in monolayer cultures [11] is also even more pronounced in three-dimensional conditions, such as on our scaffold. The three-dimensional structure of scaffold itself may allow more efficient accumulation of ECM proteins and consecutive migration and proliferation of SMCs within the HYAFF fibers.
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4. Discussion
40
20
0
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Fig. 3. Mechanical properties of control and SA-treated constructs, as defined in the text, compared with those of native porcine coronary artery. (A) Tensile resistance as maximum load per unit width (Tmax) of control constructs cultured for 14 days (black bar, n ¼ 5), SA treated constructs (white bars) cultured for 14 days (n ¼ 4) and for 28 days (n ¼ 7) and coronary artery samples (gray bar, n ¼ 7). (B) Stiffness parameter K. (C) First time constant t. *po 0.05 vs. SAtreated constructs at 14 days, **po0.01 vs. SA-treated constructs at 14 days, 1po0.05 vs. control and SA-treated constructs cultured for 14 days, 11po0.01 vs. all control and SA-treated constructs.
These data suggested us to use SA in culture medium to obtain a better performance of the cylindrical constructs made from flat sheets of HYAFF and we obtained a significant improvement of the vascular
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construct in terms of structure and mechanical properties. The presence of SA until the end of culture allowed SMCs to proliferate within the three-layered wall improving the adhesion between layers. Actually, the layers of SA-treated constructs were completely adherent while in untreated samples some areas within wall thickness showed cluster colonization and poor adherence (data not shown). That SA increased ECM production by SMCs is also indicated by the morphometrical analysis we performed on histological sections of the vascular constructs. The higher amount of cells and matrix induced by SA within the scaffold allowed improving also mechanical properties of tubular constructs. Thus, some improvement was observed in tensile resistance of the constructs cultured with SA, while important changes have been obtained on elastic properties of the constructs. The material stiffness decreased using SA reaching that of native arterial vessels and the time constant was comparable to that of the coronary artery segments. These results may be explained by the SA-induced increase in cells and matrix within construct thickness with stronger binding of scaffold fibers at their intersections, in such a way to offer more adequate compactness and elasticity. Since the scaffold itself does not show elastic properties [6] the more elastic behavior observed in SA-treated constructs must derive from increased amount of ECM and possibly from different composition of ECM. That increased cell and matrix deposition within a three-dimensional scaffold allows to improve mechanical properties of vascular constructs has been also reported by Hoerstrup and coworkers [19]. Arakawa and coworkers recently reported [12] that SA stimulates the production of SMC-specific proteins in cultured SMCs, including smooth muscle myosin heavy chain-1 (SM1) and calponin 1. These findings suggest that dedifferentiated SMCs in monolayer cultures can be induced to differentiate in vitro by addition of SA in the medium. In our vascular construct we also observed that addition of SA improved differentiation of SMCs, with upregulation of two SMC-specific proteins, aSMA and calponin. This upregulation of SMC markers is time dependent, with the two proteins being more expressed at 28 days of culture as compared to 14 days. It is important to note that expression of calponin was maintained in SMC only in the presence of SA. Since calponin is expressed only in differentiated SMCs [20], these data indicate that SA had predominant contribution on contractile phenotype differentiation, the phenotype of SMCs into the arterial vessel. We can not rule out the possibility that SAinduced differentiation of SMCs may derive from the enhanced production of ECM that can itself constitute a stimulus for the expression of SMC specific markers. However, despite the mechanisms responsible for this effect, our finding is relevant for the development of
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tissue-engineered vascular constructs since an engineered blood vessel substitute beside adequate mechanical properties must be characterized by cellular components that respond to hemodynamic stimulation in a physiological way.
5. Conclusion In conclusion, our present study shows that addition of SA to SMC cultures on a three-dimensional scaffold allowed to improve cell viability, ECM production and helped to maintain SMC phenotype. These cellular effects allowed to improve the structure and mechanical properties of a vascular construct based on HYAFF non-woven scaffold. Further studies on the possibility to induce in vitro final maturation of these constructs may allow to obtain a new type of bioartificial vascular graft.
Acknowledgments HYAFF material used in this study was kindly provided by Fidia Advanced Biopolymers S.r.l., Abano Terme, Padova, Italy. This study was supported in part by FIRB (Italian Fund for Basic Research) through the Contract N. RBNE01EBES. References [1] Nerem RM, Seliktar D. Vascular tissue engineering. Annu Rev Biomed Eng 2001;3:225–43. [2] Ratcliffe A. Tissue engineering of vascular grafts. Matrix Biol 2000;19(4):353–7. [3] L’Heureux N, Paquet S, Labbe R, Germain L, Auger FA. A completely biological tissue-engineered human blood vessel. FASEB J 1998;12(1):47–56. [4] Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Functional arteries grown in vitro. Science 1999; 284(5413):489–93. [5] Hoerstrup SP, Zund G, Sodian R, Schnell AM, Grunenfelder J, Turina MI. Tissue engineering of small caliber vascular grafts. Eur J Cardiothorac Surg 2001;20(1):164–9. [6] Remuzzi A, Mantero S, Colombo M, Morigi M, Binda E, Camozzi D, et al. Vascular smooth muscle cells on hyaluronic acid: culture and mechanical characterization of an engineered vascular construct. Tissue Eng 2004;10(5–6):699–710. [7] Galassi G, Brun P, Radice M, Cortivo R, Zanon GF, Genovese P, et al. In vitro reconstructed dermis implanted in human wounds: degradation studies of the HA-based supporting scaffold. Biomaterials 2000;21(21):2183–91. [8] Gao J, Dennis JE, Solchaga LA, Goldberg VM, Caplan AI. Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge. Tissue Eng 2002;8(5):827–37. [9] Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair Regen 1999;7(2):79–89. [10] Mitchell SL, Niklason LE. Requirements for growing tissueengineered vascular grafts. Cardiovasc Pathol 2003;12(2):59–64.
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[11] Davidson JM, LuValle PA, Zoia O, Quaglino Jr. D, Giro M. Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms. J Biol Chem 1997;272(1):345–52. [12] Arakawa E, Hasegawa K, Irie J, Ide S, Ushiki J, Yamaguchi K, et al. L-ascorbic acid stimulates expression of smooth musclespecific markers in smooth muscle cells both in vitro and in vivo. J Cardiovasc Pharmacol 2003;42(6):745–51. [13] Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Meth 1986;89(2): 271–7. [14] van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Meth 1994;174(1–2):311–20.
[15] Weibel ER. Pratical methods for biological morphometry. London: Academic Press Inc.; 1979. [16] Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res 1980;47(1):1–9. [17] Armentano RL, Levenson J, Barra JG, Fischer EI, Breitbart GJ, Pichel RH, et al. Assessment of elastin and collagen contribution to aortic elasticity in conscious dogs. Am J Physiol 1991;260 (6 Pt 2):H1870–7. [18] Bank AJ, Wang H, Holte JE, Mullen K, Shammas R, Kubo SH. Contribution of collagen, elastin, and smooth muscle to in vivo human brachial artery wall stress and elastic modulus. Circulation 1996;94(12):3263–70. [19] Hoerstrup SP. Living, autologous pulmonary artery conduits tissue engineered from human humbilical cord cells. Ann Thorac Surg 2002;74:46–52. [20] Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 1995;75:487–517.
Biomaterials 27 (2006) 623–630 www.elsevier.com/locate/biomaterials
The effect of sodium ascorbate on the mechanical properties of hyaluronan-based vascular constructs Chiara Arrigonia, Davide Camozzia, Barbara Imbertia, Sara Manterob, Andrea Remuzzia, a
Department of Biomedical Engineering, Mario Negri Institute for Pharmacological Research, Via Gavazzeni, 11-24125 Bergamo, Italy b Department of Biomedical Engineering, Politecnico di Milano, Piazza Leonardo Da Vinci, 32-20133 Milano, Italy Received 23 March 2005; accepted 20 June 2005 Available online 26 July 2005
Abstract Esterified hyaluronic acid (HYAFF) is routinely used for clinical tissue engineering applications such as skin and cartilage. In a previous study we developed a technique for in vitro generation of cylindrical constructs from cellularized HYAFF flat sheets. In the present investigation we studied the possibility to improve mechanical properties of this vascular construct by the addition of sodium ascorbate (SA). Non-woven HYAFF flat sheets were seeded with porcine aortic smooth muscle cells (SMCs) and cultured for 14 or 28 days with standard medium or medium additioned with SA. In selected experiments HYAFF sheets seeded with SMCs were wrapped to obtain cylindrical shape and then cultured in control medium or SA additioned medium for up to 28 days. We estimated cell viability for flat sheets, and performed histological examination, analysis of extracellular matrix (ECM) deposition and mechanical tests on tubular constructs. The number of viable cells and ECM deposition increased with time in constructs cultured in the presence of SA, as compared to control group. Moreover, SA improved mechanical properties of the vascular construct lowering material stiffness and increasing tensile strength as compared to untreated controls. The addition of SA to the medium improved cell proliferation and ECM synthesis on this biodegradable material, which leads to the formation of well organized, mechanical resistant tissue-engineered structure. r 2005 Elsevier Ltd. All rights reserved. Keywords: Tissue engineering; Hyaluronic acid; Smooth muscle cell; Sodium ascorbate; Mechanical properties
1. Introduction The main aim of vascular tissue engineering is to generate functional blood vessels in vitro to be used as vascular grafts, since synthetic small caliber vascular prosthesis cannot be used to replace diseased arterial segments with diameter less than 6 mm. Many challenges remain to be solved before a blood vessel replacement will be available, including achievement of adequate mechanical strength, non-thrombogenic and long-term function [1,2]. In the last years several techniques have been developed for in vitro generation of artificial blood vessels [3–5]. Despite promising results Corresponding author. Tel.: +39 035 319888; fax: +39 035 319331.
E-mail address: [email protected] (A. Remuzzi). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.06.009
obtained so far in this area of investigation, a crucial factor for further development of a tissue-engineered blood vessel is the biodegradable material used as scaffold. A number of biodegradable materials have been used to this purpose, from synthetic polymers to natural extracellular matrix structures. We have recently investigated adhesion and proliferation of aortic smooth muscle cells (SMCs) seeded on esterified hyaluronic acid (HYAFF-11s) scaffolds and successfully developed a simple technique to obtain cellularized tubular constructs from HYAFF flat sheets in vitro [6]. HYAFF is a biodegradable material currently used for tissue engineering of skin and cartilage [7,8]. This material is highly compatible with cells and matrix and its degradation products induce extracellular matrix production and neoformation of blood capillaries [9].
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Since the extracellular matrix (ECM) within native arteries is a determinant of mechanical properties of these blood vessels, we reasoned that enhancing ECM protein production by SMCs seeded on the biodegradable scaffold by biochemical stimulation [2,10] could improve tissue organization with higher mechanical resistance and better elastic properties. Ascorbic acid (AA) and its derivatives have been shown to stimulate collagen synthesis in monolayers of SMCs [11]. It has been shown that AA influences hydroxylation of proline and lysine necessary for molecular stabilization of collagen. Moreover, it has been also demonstrated that AA stimulates the production of smooth muscle-specific proteins, including smooth muscle myosin heavy chain-1 (SM1) and calponin-1, in cultured rat SMCs [12]. These findings suggest that AA may also sustain dedifferentiation of SMCs. The first aim of this work was to investigate whether AA may influence the proliferation of SMCs cultured on flat sheets of non-woven HYAFF scaffold. The second aim was to study the effects of AA on HYAFF tubular vascular constructs investigating ECM production and distribution into the vessel wall, and evaluate mechanical properties of these constructs. We also performed evaluation of protein expression by immunofluorescence staining to study phenotypic drift of SMCs on this threedimensional scaffold.
2. Materials and methods 2.1. Cell culture techniques Primary porcine aortic SMCs were isolated from normal 2–4 week-old pigs by a collagenase digestion method described previously [6], and were used between passages 4 and 6. Cells were characterized as smooth muscle based on their positive immuno-reaction to antibodies against a-smooth muscle actin (aSMA, Sigma-Aldrich Corporation, St Louis, MO, US) and calponin (Sigma). Monolayer cultures were fed with Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 4.5 g/l glucose, supplemented with 10% fetal bovine serum (FBS, Invitrogen Ltd, Paisley, UK), 10% porcine serum (PS; Invitrogen), 4 mM L-glutamine (Invitrogen), 100 U/ml penicillin G sodium (Invitrogen), 100 mg/ml streptomycin sulphate (Invitrogen), and 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, Sigma). Culture medium was changed three-times a week. 2.2. Seeding and culture on biomaterial The biodegradable scaffold used for cell seeding was a 100% benzyl-ester of hyaluronic acid (HYAFF-11) configured as sheets of non-woven mesh. The material was kindly provided by Fidia Advanced Biopolymers (Abano Terme, Italy). Scaffolds were treated before cell seeding with a solution of human fibronectin (BD Bioscience, Bedford, MA, US) in water with a density of 3.2 mg/cm2 of non-woven mesh. SMCs
were dispensed over the HYAFF scaffold at a concentration of 3 106 cells/cm2 then were allowed to attach to the scaffold for 3 h in a cell incubator before adding complete culture medium. Initially, cell proliferation was studied by seeding SMCs on 1 1 cm flat sheets of non-woven HYAFF and keeping them in culture for 14 and 28 days with standard medium or medium supplemented with 50 mg/ml of sodium ascorbate (SA, Sigma). To obtain tubular constructs, 4 2 cm HYAFF sheets precoated with fibronectin were seeded with 24 106 PSMC, cultured as flat sheets for 7 days and subsequently wrapped around glass cylindrical mandrels with an outside diameter of 3.5 mm.To maintain the tubular shape a suture wire (Ethicon SpA, Pomezia, Italy) was wrapped around the construct to avoid unfolding from the mandrel. Tubular constructs obtained with this technique were cultured for additional 14 or 28 days in culture medium without or supplemented with SA. At the end the mandrel was withdrawn, sections of the tubular constructs of 5 mm in length were then cut and used for histological analysis and mechanical testing. 2.3. Cell viability test Cell proliferation was evaluated by 3-[4,5-dimethylthiazol-2]2,5-dipheniltetrazolium bromide (MTT) viability assay as previously described [6,13]. Briefly, cell seeded scaffolds were rinsed with phosphate-buffered saline (PBS, Invitrogen) and incubated for 3 h at 371C with 0.5 mg/ml MTT (Sigma) in sterile DMEM medium w/o phenol red. After incubation, un-reacted MTT was removed by rinsing with PBS. The reaction product was extracted for 3 h with 5 ml of extraction solution (90% isopropanol/10% dimethyl sulphoxide, Sigma). The absorbance of each sample was measured by a spectrophotometer (Beckman Instruments Inc, Fullerton, CA, US) at 570 nm with 690 nm as reference wavelength. To relate measured absorbance with cell number per unit surface area of the scaffold, we used a calibration curve [14] obtained measuring the absorbance of HYAFF samples seeded with known number of cells ranging from 2.5 105 to 1.5 106 cells/cm2. 2.4. Morphometric analysis of ECM volume For histological analysis, representative samples for each experimental condition were fixed in Bouin solution (BioOptica, Milan, Italy) for 4 h, dehydrated in alcohol, and embedded in paraffin. Sections (5 mm thick) were stained with haematoxylin and eosin and used for optical microscopy. The presence of ECM was evaluated on sections stained with Masson’s trichrome and subsequent morphometric analysis, as previously described [6]. To this purpose 15–20 images of each construct section were systematically sampled along radial position and across the construct wall thickness, digitized using a digital camera connected to the microscope and digitally overlaid with an orthogonal grid composed of 154 points. The fraction of available volume within the scaffold effectively occupied by ECM was estimated as the ratio of grid points falling in ECM positively stained areas and grid points falling within the construct but outside HYAFF fibers. The number of images used for this quantification allowed to obtain an estimated probable error smaller than 3%, as reported in literature [15].
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2.5. Immunofluorescence staining
3.1. Results
To assess the presence of SMC proteins, tissue sections were stained with a mouse monoclonal anti-aSMA antibody (Sigma) and a mouse monoclonal anti-calponin antibody (Sigma). Tissue specimens were infiltrated by immersion in 30% sucrose/PBS for at least 1 h at room temperature, embedded in OCT medium, and frozen in liquid nitrogen. Tissue sections were cut at 3 mm thickness. Following fixation with ice-cold acetone (Carlo Erba, Milan, Italy), non-specific binding of antibodies was blocked with 1% bovine serum albumin (Sigma) in PBS. Sections were then incubated with primary antibodies for 1 h at room temperature (1A4, 1:200; hCP, 1:1000). After washing in PBS, sections were incubated with FITC-conjugated goat anti-mouse IgG antibodies (1:40, Sigma) for 1 hour at room temperature. Counter-staining with DAPI (Sigma) was performed to label cell nuclei for 30 min at room temperature. Slides were then mounted and examined with an inverted light microscope (Olympus Mod. IX70, Hamburg, Germany) equipped with epifluorescence.
3.1. Cell proliferation and distribution on flat HYAFF sheets
2.6. Mechanical testing To test mechanical properties of the tissue-engineered constructs, we followed a specific protocol [6]. Briefly, ringshaped specimens with a reproducible length of 5 mm were cut from each tubular construct and mounted with suitable holders on a computer controlled testing machine (MTS Synergie 200 H, Eden Prairie, MN, US). Specimens of coronary arteries from pig hearts, taken within 4 h of cold ischemia, have been cut from native vessels with the same procedure. Initial sample length was calculated from the displacement reached when a reference pre-load of 0.015 N was applied. Each sample was pre-conditioned, with loading speed of 1.0 mm/min and load endpoint of 0.4 N. The sample was left to recover for 120 s and a relaxation test was then performed. The test was stopped after 500 s and the sample left to recover for additional 120 s. A failure tensile test was finally carried out until complete sample rupture. Due to non-homogeneous thickness of the vascular constructs, constitutive parameters could not be directly calculated. To overcome this limitation, we evaluated the following structural parameters for each sample: 1) ultimate force per unit axial length (Tmax) defined as the maximum force reached during the failure tensile test divided by sample axial length; (2) ultimate strain (emax) as the strain level corresponding to Tmax; (3) stiffness (K) calculated as the slope of the loading ramp of the relaxation test between the end of the toe region (at T1 ¼ 0.04 N/mm) and the end of the loading ramp itself (at T0 ¼ 0.08 N/mm); (4) the first time constant (t) defined as the intersection between the time axis and the line tangent to the initial portion of the reduced relaxation curve. 2.7. Statistical analysis Results are expressed as the mean7standard deviation (SD). Statistical analysis was carried out using the analysis of variance or the unpaired Student t-test as appropriate. Specific comparisons between groups were calculated using the Bonferroni correction [16].
SMCs isolated by enzymatic digestion from porcine aorta exhibited a spindle-shaped phenotype with a classic ‘‘hill-and-valley’’ growth pattern at confluence, as shown in Fig. 1, and stained positively for aSMA and calponin with extended filaments across cell bodies (see Fig. 1B and 1C). The number of viable cells on 1 1 cm HYAFF sheets 14 days after seeding was significantly higher in the presence of SA as compared to untreated controls averaging 6.6070.48 106 vs. 3.3470.47 106 (po0.01, n ¼ 6 and 7, respectively). Due to this increase in viable cell number, sheets cultured with SA were more compact than untreated sheets until the end of culture. At 4 weeks after seeding, the SMC number was still significantly higher in the presence of SA, averaging 5.9770.49 106 vs 2.6370.88 106 (po0.01, n ¼ 6 and 4 respectively). Histological analysis of these samples (data not shown) confirmed higher proliferation of SMCs in constructs treated with SA and better ingrowth of seeded cells into HYAFF cultured with SA. 3.2. Tubular construct generation We compared tubular constructs obtained using control medium and SA-additioned medium at 14 and 28 days, respectively. Most of the samples of the control group did not last in culture until 28 days. Around day 20 they began to unwrap from the mandrel loosing tubular structure. Constructs cultured in the presence of SA for 28 days were even more compact and regular in shape than control constructs at 14 days. At 28 days of culture, SA-treated constructs appeared cellularized for the entire length of the scaffold. At light microscopy, SA-treated constructs showed cells more uniformly distributed along HYAFF layers in the entire construct wall, while in control constructs cells were less uniformly distributed. In the presence of SA, HYAFF layers were more adherent one to the other resulting in a more compact wall of the construct, likely due to increased cell proliferation, adhesion to the scaffold and extracellular matrix deposition, especially on the outer surface of each individual layer. 3.3. Morphometrical analysis of ECM Deposition of ECM was observed predominantly at the outer surface of the scaffold, both in control and in treated constructs. We used morphometric analysis to quantify the presence of ECM in both experimental conditions. At 14 days of culture the construct wall volume occupied by ECM was slightly higher with SA than in control constructs, averaging 17.175.1 vs.
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Fig. 1. Representative cell morphology at phase-contrast light microscopy of porcine aortic SMC monolayer (A, bar represents 100 mm). Expression of aSMA (B) and calponin (C) by porcine SMCs monolayers (bars represent 50 mm).
14.473.1%, respectively. With time ECM volume significantly increased in SA-treated constructs, averaging 28.977.7% (po0.05 vs. SA construct at 14 days). 3.4. Immunofluorescence staining As observed for cell distribution across the construct thickness, also the pattern of protein expression was more pronounced at the edge of individual HYAFF layers, likely reflecting higher cell density in these regions. Representative images of fluorescent antibody staining performed to identify the presence and distribution of aSMA and calponin, two specific proteins of SMC cytoskeleton, are reported in Fig. 2. At 14 days aSMA expression was similar in control and SA-treated constructs. SMCs appeared elongated and the protein was present across the whole cell body with its characteristic filaments. At 28 days aSMA expression decreased in control constructs, while in SA-treated constructs there was a strong increase in fluorescence signal. As shown in Fig. 2, in SA-treated constructs more nuclei were present (see DAPI blue staining) and extended aSMA filaments (stained in green) as compared to untreated samples, with less cell density and weaker protein expression. In SA-treated constructs cells completely surrounded HYAFF fibers, as shown in Fig. 2, with positively stained filaments. Calponin
labeling showed a punctate pattern in the whole cell bodies that sometimes resulted in filaments (see Fig. 2). Protein expression with time followed the same pattern of aSMA, with differences between control and SAtreated constructs even more evident. Control constructs at day 14 showed a downregulation of calponin expression that was still present on day 28. In contrast, addition of SA induced a stronger expression of the protein by SMCs at day 14 that further increased at day 28. At this time point we observed once again SMCs with strong calponin filaments surrounding completely the HYAFF fibers. 3.5. Mechanical testing Calculated parameters from mechanical tests are reported in Fig. 3. Control and SA-treated scaffolds showed a different behavior during failure test. Control constructs underwent frequently unwrapping of individual layers, while failure of SA-treated construct samples was consistently due to rupture of the tubular cross section. The failure tensile resistance Tmax, the stiffness K and the first time constant t all were only numerically different in SA-treated constructs at 14 days as compared to control constructs, but the differences did not reach statistical significance. With culture time, Tmax only numerically increased in SA-treated
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Fig. 2. aSMA and calponin expression (green staining) of control and SA-treated constructs at 14 and 28 days of culture. Cell nuclei are labeled by blue fluorescence with DAPI. Asterisks represent HYAFF fibers.
constructs, while K significantly decreased, and t significantly increased comparing values calculated at 14 and 28 days. We also compared the parameters
calculated from mechanical tests of cellularized constructs in the presence of SA with those of samples of native blood vessels, as shown in Fig. 3. While
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mechanical resistance of SA constructs was significantly lower than that of coronary arteries (Tmax averaged 0.4170.11 N/mm in SA constructs and 1.1270.42 N/mm in coronary artery specimens, po0.01), the elastic properties of constructs cultured with SA approached those of native vessels. Thus, while control constructs showed value of K and t, respectively, significantly higher and lower than those of native vessels, SA constructs showed value of these parameters comparable to those of native vessels.
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The biodegradable scaffold we investigated is made of non-woven flat sheets of HYAFF-11s fibers. During manufacturing, the sheets are compressed to obtain a compact uniform meshwork with 250 mm thickness. We have recently reported that HYAFF scaffold can be used for in vitro reconstruction of small caliber blood vessel [6]. We seeded vascular SMCs on flat HYAFF sheets and generated a tubular construct by wrapping sheets around a mandrel. Despite good cellularization within the scaffold thickness, the mechanical properties of these tubular constructs were still far from those of native coronary arteries being less resistant and less elastic than native vessels [6]. The content and organization of ECM proteins within native arteries determine their complex mechanical properties [17,18], however, it has been shown that isolated SMCs in vitro do not secrete significant amounts of ECM proteins in the absence of biochemical stimulation [10]. In addition, in our previous study we observed an increasing number of cells undergoing apoptosis during in vitro culture on HYAFF scaffold in the absence of specific growth factors [6]. To improve these experimental conditions, we used SA to increase ECM protein production by SMCs seeded on the scaffold. Davidson and coworkers [11] have shown that ascorbate (a derivative of vitamin C) induces exogenous collagen synthesis in cultured SMCs, acting as a cofactor for the hydrolyzation of proline and lysine. Moreover, they reported that ascorbate-treated cultures showed slightly faster growth rate as compared to untreated cells. Our present results show that the number of viable SMCs seeded on HYAFF sheets and stimulated with SA was almost double than that measured on control unstimulated scaffolds at 14 and 28 days. These data demonstrate that SA-induced cell proliferation previously observed in monolayer cultures [11] is also even more pronounced in three-dimensional conditions, such as on our scaffold. The three-dimensional structure of scaffold itself may allow more efficient accumulation of ECM proteins and consecutive migration and proliferation of SMCs within the HYAFF fibers.
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40
20
0
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Fig. 3. Mechanical properties of control and SA-treated constructs, as defined in the text, compared with those of native porcine coronary artery. (A) Tensile resistance as maximum load per unit width (Tmax) of control constructs cultured for 14 days (black bar, n ¼ 5), SA treated constructs (white bars) cultured for 14 days (n ¼ 4) and for 28 days (n ¼ 7) and coronary artery samples (gray bar, n ¼ 7). (B) Stiffness parameter K. (C) First time constant t. *po 0.05 vs. SAtreated constructs at 14 days, **po0.01 vs. SA-treated constructs at 14 days, 1po0.05 vs. control and SA-treated constructs cultured for 14 days, 11po0.01 vs. all control and SA-treated constructs.
These data suggested us to use SA in culture medium to obtain a better performance of the cylindrical constructs made from flat sheets of HYAFF and we obtained a significant improvement of the vascular
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construct in terms of structure and mechanical properties. The presence of SA until the end of culture allowed SMCs to proliferate within the three-layered wall improving the adhesion between layers. Actually, the layers of SA-treated constructs were completely adherent while in untreated samples some areas within wall thickness showed cluster colonization and poor adherence (data not shown). That SA increased ECM production by SMCs is also indicated by the morphometrical analysis we performed on histological sections of the vascular constructs. The higher amount of cells and matrix induced by SA within the scaffold allowed improving also mechanical properties of tubular constructs. Thus, some improvement was observed in tensile resistance of the constructs cultured with SA, while important changes have been obtained on elastic properties of the constructs. The material stiffness decreased using SA reaching that of native arterial vessels and the time constant was comparable to that of the coronary artery segments. These results may be explained by the SA-induced increase in cells and matrix within construct thickness with stronger binding of scaffold fibers at their intersections, in such a way to offer more adequate compactness and elasticity. Since the scaffold itself does not show elastic properties [6] the more elastic behavior observed in SA-treated constructs must derive from increased amount of ECM and possibly from different composition of ECM. That increased cell and matrix deposition within a three-dimensional scaffold allows to improve mechanical properties of vascular constructs has been also reported by Hoerstrup and coworkers [19]. Arakawa and coworkers recently reported [12] that SA stimulates the production of SMC-specific proteins in cultured SMCs, including smooth muscle myosin heavy chain-1 (SM1) and calponin 1. These findings suggest that dedifferentiated SMCs in monolayer cultures can be induced to differentiate in vitro by addition of SA in the medium. In our vascular construct we also observed that addition of SA improved differentiation of SMCs, with upregulation of two SMC-specific proteins, aSMA and calponin. This upregulation of SMC markers is time dependent, with the two proteins being more expressed at 28 days of culture as compared to 14 days. It is important to note that expression of calponin was maintained in SMC only in the presence of SA. Since calponin is expressed only in differentiated SMCs [20], these data indicate that SA had predominant contribution on contractile phenotype differentiation, the phenotype of SMCs into the arterial vessel. We can not rule out the possibility that SAinduced differentiation of SMCs may derive from the enhanced production of ECM that can itself constitute a stimulus for the expression of SMC specific markers. However, despite the mechanisms responsible for this effect, our finding is relevant for the development of
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tissue-engineered vascular constructs since an engineered blood vessel substitute beside adequate mechanical properties must be characterized by cellular components that respond to hemodynamic stimulation in a physiological way.
5. Conclusion In conclusion, our present study shows that addition of SA to SMC cultures on a three-dimensional scaffold allowed to improve cell viability, ECM production and helped to maintain SMC phenotype. These cellular effects allowed to improve the structure and mechanical properties of a vascular construct based on HYAFF non-woven scaffold. Further studies on the possibility to induce in vitro final maturation of these constructs may allow to obtain a new type of bioartificial vascular graft.
Acknowledgments HYAFF material used in this study was kindly provided by Fidia Advanced Biopolymers S.r.l., Abano Terme, Padova, Italy. This study was supported in part by FIRB (Italian Fund for Basic Research) through the Contract N. RBNE01EBES. References [1] Nerem RM, Seliktar D. Vascular tissue engineering. Annu Rev Biomed Eng 2001;3:225–43. [2] Ratcliffe A. Tissue engineering of vascular grafts. Matrix Biol 2000;19(4):353–7. [3] L’Heureux N, Paquet S, Labbe R, Germain L, Auger FA. A completely biological tissue-engineered human blood vessel. FASEB J 1998;12(1):47–56. [4] Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Functional arteries grown in vitro. Science 1999; 284(5413):489–93. [5] Hoerstrup SP, Zund G, Sodian R, Schnell AM, Grunenfelder J, Turina MI. Tissue engineering of small caliber vascular grafts. Eur J Cardiothorac Surg 2001;20(1):164–9. [6] Remuzzi A, Mantero S, Colombo M, Morigi M, Binda E, Camozzi D, et al. Vascular smooth muscle cells on hyaluronic acid: culture and mechanical characterization of an engineered vascular construct. Tissue Eng 2004;10(5–6):699–710. [7] Galassi G, Brun P, Radice M, Cortivo R, Zanon GF, Genovese P, et al. In vitro reconstructed dermis implanted in human wounds: degradation studies of the HA-based supporting scaffold. Biomaterials 2000;21(21):2183–91. [8] Gao J, Dennis JE, Solchaga LA, Goldberg VM, Caplan AI. Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge. Tissue Eng 2002;8(5):827–37. [9] Chen WY, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair Regen 1999;7(2):79–89. [10] Mitchell SL, Niklason LE. Requirements for growing tissueengineered vascular grafts. Cardiovasc Pathol 2003;12(2):59–64.
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