Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis Francesca Boccafoschi,1 Martina Ramella,1 Teresa Sibillano,2 Liberato De Caro,2 Cinzia Giannini,2 Roberto Comparelli,3 Antonella Bandiera,4 Mario Cannas1 1

Department of Health Sciences, University of Piemonte Orientale “A. Avogadro”, 28100 Novara, Italy Institute of Crystallography, National Research Council, 70126 Bari, Italy 3 CNR-IPCF, U.O.S. Bari, c/o Dip. Chimica, 70126 Bari, Italy 4 Department of Life Sciences, University of Trieste, 34127 Trieste, Italy 2

Received 6 May 2014; revised 27 May 2014; accepted 4 June 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35257 Abstract: The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices. In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen–HELP matrices, even showing an improved structural remodeling through the involvement of proteins

to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modC 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: ulus. V 00A:000–000, 2014.

Key Words: human elastin-like polypeptide, extracellular matrix remodeling, collagen scaffold, tissue engineering, C2C12

How to cite this article: Boccafoschi F, Ramella M, Sibillano T, De Caro L, Giannini C, Comparelli R, Bandiera A, Cannas M. 2014. Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Three-dimensional (3D) biomimetic scaffolds have recently found extensive applications in biomedical tissue engineering because of several features: (a) water retention capacity; (b) porosity to allow cells to grow and arrange in a 3D environment; (c) extracellular matrix (ECM) remodeling to maintain adequate biomechanical characteristics of the scaffold; and (d) cell phenotype maintaining.1,2 For these reasons, natural polymers, such as collagen and elastin, represent a promising alternative in creating 3D scaffolds for vascular cell tissue engineering. Collagen type I, in particular, has suitable biomechanical properties showing a low antigenicity and low inflammatory response and, thus, can be used for tissue engineering applications.3 Moreover, it possesses desirable biological and hematological properties, provides a suitable substrate for endothelial cell adhesion,4

and it can be remodeled by the host and integrated into existing tissue.5 The collagen family is represented by more than 20 different types with a common molecular structure: a righthanded triple helix composed of three left-handed twists of a-chains where each a-chain is a repeated aminoacidic sequence (Gly-X-Y) where X is typically proline and Y is hydroxyproline.6 Intra- and intermolecular hydrogen bonds are responsible for the stability of the triple helix. The great strength of the collagen fibers, however, originates from the stable intermolecular covalent bonds between adjacent tropocollagen molecules.7,8 Due to the highly stable structure, collagen represents the major stress-bearing component of the fibrous matrix of blood vessels.9 In particular collagen types I and III are fibril-forming collagens and are the main components of blood vessels wall.10

Correspondence to: F. Boccafoschi; e-mail: [email protected] Contract grant sponsor: FIRB 2009/2010 project Rete integrata per la Nano Medicina (RINAME); contract grant number: RBAP114AMK_006

C 2014 WILEY PERIODICALS, INC. V

1

Today, the recombinant DNA technology offers the opportunity to synthesize biomimetic macromolecules inspired to ECM components. The recombinant DNA technology approach results in a reproducible composition, sequence and molecular mass of the protein biopolymer mimicking the naturally occurring protein, overcoming the potential pathogens transmission related to natural polymers.11 The recombinant elastin like polymers are modeled after the aminoacidic sequence of the native elastin, another abundant ECM protein, which plays a pivotal role in tissue biomechanical properties and modulates a variety of cellular responses.10,12 Elastin is a potent autocrine regulator of vascular smooth muscle cells (VSMCs) activity; this regulation is important in preventing fibrocellular pathology. Elastin induces actin stress fiber organization, inhibits proliferation and regulates migration avoiding vessel restenosis due to VSMCs proliferation.13 Also elastin-derived peptides possess several biological activities as cell migration, differentiation, proliferation, chemotaxis, and upregulation of metalloproteinases (MMPs) and for this reason there are also known as “matrikines.”14 Literature reports the use of elastin-like polypeptides, coated on synthetic polymers, for vascular applications. As expected, elastin surface modified materials demonstrated enhanced smooth muscle cells adhesion and exhibited a spindle-shape morphology, actin filament organization, and smooth muscle myosin heavy chain (MHC) expression showing elastin-like polypeptides as promising surface modifiers for scaffolds in the engineering of contractile vascular tissues.15,16 As a novelty with respect to the cited works, in our research the use of recombinant elastin-like polypeptides, made by a short repetitive monomeric sequence (exons 23 and 24 of native tropoelastin), was not limited as surface coating but it is used as co-polymer in a 3D matrix. As few papers reported in literature, an osteogenic effect of elastin was found, which may be en undesirable effect in this context.17,18 Most of these studies used the whole protein or different peptides sequences, while only one demonstrated the osteogenic effect of human elastin-like polypeptide (HELP) on mesenchymal stem cell19 in a specifically enriched medium. Anyway, the over expression of contractile proteins, as shown in our work, inhibits the osteogenic effect and vice versa.20 The HELP employed here is a bioinspired component that mimics human elastin. The backbone of HELP consists of eight peptide units corresponding to the most regularly repeated domain of human elastin coded by exon 24. The primary structure is composed by two different alternating structural domains, the hydrophobic hexa-peptidic residue, that characterizes human elastin and the alanine and lysine aminoacids–rich hydrophilic domains that, in the native protein, are responsible for the crosslinking among the chains and for the formation of the elastin network that confers elasticity to tissues.21 Interestingly, the hexa-peptidic motif that characterizes the hydrophobic domains of the HELP macromolecule has been described also as a functional domain with several biological activities.22 The HELP retains several peculiar biophysical features, proper of the native tropoelastin, such as,

2

BOCCAFOSCHI ET AL.

for example, the reversible inverse phase transition, responsible for the change in solubility that results in the temperature activated self-aggregation of the elastin polypeptides.23 The functionality of the recombinant HELP macromolecules can be even extended by addition of further functional, bioactive domains, improving the potential of the resulting product. The smart nature of this class of compounds makes them attractive for many applications in the biomedical and biotechnological fields, in particular for biomaterial development. Recent results suggest that this kind of compounds can be successfully employed in combination with other ECM components to obtain new composites.19 The approach to use hybrid matrices should overcome the limit of the weak mechanical properties of natural polymers. In fact, the presence of collagen and elastin in native blood vessels wall may supply strength and elasticity to the engineered vascular tissue and is essential for adequate mechanical properties.24 In the present study, we realized and compared the properties of 3D scaffolds made of collagen alone and HELP-enriched collagen. The biological and biomechanical properties of scaffolds have been evaluated in terms of mechanical properties (tensile stress and rupture), ECM composition and cellular contractile phenotype maintaining. In addition, the 3D scaffolds with and without HELP have been also analyzed with small-angle X-ray scattering (SAXS) to investigate the scaffold structural properties at the nanostructure level, by comparing the features extracted from the diffraction patterns with those observed for the native aorta vessels, used as reference model for tissue regeneration. SAXS is a powerful technique extensively used for structural/morphological analysis of a wide range of materials, going from metals or polymers to porous materials, nanoparticles, and so forth.25,26 This X-ray based technique is able to yield morphological information on size and shape of the scattering objects,27,28 but it also allows to reveal any structural nanoscale periodicity in case of ordered systems.29 The method has recently gained an important role in the study of biological macromolecules in solution25 as well as of artificial scaffolds for tissue engineering applications.30 MATERIALS AND METHODS

Expression and purification of HELPs Escherichia coli competent cells were transformed with the expression construct bearing HELPs synthetic gene expression, as detailed elsewhere.31 The purification from the crude bacterial extract was obtained exploiting the inverse transition properties of the recombinant HELPs. The purity of recombinant polymers was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and HPLC confirmed that samples did not contain contaminants.23 After purification, the material was dialyzed in cold water and then lyophilized for long-term storage. Recombinant HELPs sequences are HELP [AAAAAAKAAAKAAQF GLVPGVG VAPGVG VAPGVG VAPGVG LAPGVG VAPGVG VAPGVG VAPGIAP] HELP1 [AA GLVPGVG VAPGVG VAPGVG VAPGVG LAPGVG VAPGVG VAPGVG VAPGIGPGAP].

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

As lacking of the crosslinking regions, HELP1 has been used as control for mechanical properties characterization, determining an imperfect structuring a-helix domains that favor the b-turn sequences along the chain, thus, generating a weaker matrix.

pure collagen, pure HELP and native porcine aorta placed into Lindemann glass capillary tubes of 0.7 mm diameter, permanently immersed in formaldehyde 4% water solution. Three different patterns have been collected for each samples in different points.

3D tissue-engineering scaffold Briefly, 1 g of air-died and ultraviolet-sterilized collagen type I tendons extracted from rat tails were solubilized in 300 mL 0.1% acetic acid, obtaining a collagen acid solution at a concentration of 2 mg/mL, as quantified with BCA assay.32 Collagen gels were processed by mixing the collagen acid solution with a suspension of 1 3 106/mL C2C12 murine myoblast (ATCC CRL-1772). Collagen acidity was neutralized by adding NaHCO3 (0.26 M) and NaOH (1 M) to the collagen. 50 mg/mL of HELPs were added to collagen blend. After 30 min at 37 C the collagen acid solution was jellified in 24-well, with cells trapped within, and Dulbecco’s modified Eagle’s medium 10% fetal bovine serum was placed as a nutrient supplement for the cells (all from Lonza, Belgium). Samples diameters were measured by the use of a gauge. Measures are expressed as percentage of contraction [100 2 (diameterday x/diameterday 0)%].

Mechanical testing The Instron 5564 testing Instrument (Instron Corporation, Canton, MA) was used to inspect the mechanical properties of the scaffolds. The device comprised: an electronic control console and a loading frame capable of testing up to 2.5 N in tension; a drive system that induced tension on the sample until the rupture of the sample. Samples were loaded on the testing system and mechanical tests were performed. System control and data analyses were accomplished using InstronV BluehillTM2 material-testing software (Instron Corporation). Sample dimensions (length, width, thickness, and shape) were inserted into a software set-up as starting parameters before proceeding with mechanical testing. Uniaxial strain application at a rate of 0.5 mm/min was provided. Tensile stress and engineering stress–strain were considered in the present study on collagen and collagen–HELPs scaffolds after 3, 7, and 21 days of culture. Stress–strain data were used to obtain the average elastic modulus (k). As k is defined as the slope of the stress–strain curve in the elastic deformation region, the elastic modulus has been calculated as:

Native aorta and mammary artery samples Native porcine aorta has been used as reference model for SAXS analyses, while native porcine mammary has been used for mechanical testing. Vessels have been isolated from young healthy pigs not subjected to any pharmacological treatment and used as reference for native blood vessel wall. SAXS setup A Fr-E1 SuperBright rotating copper anode microsource (45 kV/55 mA; Cu Ka, k 5 0.15405 nm, 2475 W) was focused by a multilayer focusing optics (Confocal Max-Flux; CMF 15– 105) and then collimated by a three pinholes SAXS camera (diameters of 300, 150, and 500 mm). The TritonTM20 gasfilled proportional counter SAXS detector, 20 cm in diameter, and 200 mm effective pixel size, was positioned for the present experiment at a distance of 2150 mm from the sample to collect several diffractions orders across different pieces of the collagen-based materials. The collected 2D images, once beam centred, were calibrated with a silver behenate standard33 and azimuthally integrated into 1D radial profiles. This laboratory source shows unique features thanks to a X-ray microsource brilliance comparable to a bending-magnet synchrotron light source, and then able to perform measurements also for biological samples. Moreover, the use of highbrilliance X-ray microsource,34 together with the application of a novel restoration algorithm35 has further improved the laboratory performances as demonstrated by results showed on the nanoscale structural characterization of air-dried rattail tendon.36 In the present work, samples were fixed in formaldehyde 4% water solution and maintained at 4 C until the analyses has been performed. SAXS diffraction patterns have been collected from samples of collagen artificial vessels,

R

l 5 stress =strain where stress is the force causing the deformation, divided by the area to which the force is applied, and strain is the deformation of the sample caused by the stress to the original state of the object. All tests were performed in triplicate in wet condition. Scanning electron microscopy To evaluate the matrix reorganization, scanning electron microscopy (SEM) was performed according to the following procedure: samples were rinsed twice with PBS and fixed in Karnowsky solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.2–7.4) for 48 h. Following fixation, samples were treated for 30 min with 1% osmium tetroxide in 0.15 M cacodylate buffer solution. Samples were then dehydrated with graded ethanol (from 50 to 100%), soaked for 30 min in hexamethyldisilazane, dried, and sputter-coated with gold–palladium. Images were collected using a SEM at different magnifications. Fieldemission scanning electron microscopy (FE-SEM) was performed by a Zeiss Sigma microscope operated at 5 kV and equipped with in-lens secondary electron detector. FE-SEM samples were dehydrated and deposited onto silicon slides and dried under vacuum. Samples were mounted onto stainless steel sample holders by double-sided carbon tape and gold sputtered prior to analysis. Immunohistochemical staining Samples have been fixed in formaldehyde 4%. For elastin staining, samples have been dehydrated by soaking in

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

3

FIGURE 1. Cell-induced scaffold contraction. Three-dimensional scaffolds contraction has been measured over time as a percentage with respect to T0. n value 5 3. * indicates statistical significance with p  0.05. Figure in squares are representative of matrices at 21 days.

increasing ethanol content solutions (from 50 to 100%), followed by a final incubation in xylene. The specimens were embedded in paraffin and serial sections were cut with a microtome (Leica, mod. Histoslide 2000R, Germany) in consecutive 5-mm thick sections. Rehydrated sections were processed with primary antielastin antibodies (DBS, clone BA-49), followed by a secondary anti-mouse antibody (Vector, CA), incubated and observed at fluorescent microscope (Leica, DM2500, Italy). For cellular staining, after 3, 7, and 21 days samples have been fixed overnight in 4% formalin at 4 C. For actin staining, rhodamin-phalloidin (Sigma, Italy) has been used, while 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, Italy) has been used for nuclear staining. Cell morphology has been observed at fluorescent microscope (Leica, DM 2500, Italy).

After rinsing with PBS 0.1% Tween, the membranes were incubated with secondary anti-mouse or anti-rabbit antibody-peroxidase conjugates (Perkin Elemer) for 1 h at room temperature. Proteins bands were visualized using ECL detection reagent (Perkin Elmer) in a chemisensitive visualization (VersaDoc, BioRad, Italy). The images were acquired and semi-quantitative analyses were performed (Quantity One image analysis software, BioRad, Italy). Statistical analysis Results are expressed as mean 6 standard deviation (SD). Statistical significance was determined using Student’s t-test and statistical analysis of variance were used taking p < 0.05 as the minimum level of significance. RESULTS

Western blot Samples were lysed in SDS buffer (1%, w/v SDS, 100 mM Tris-HCl, pH 7.0, 95 C). Protein concentration was determined using the BCA assay (Pierce, Rockford IL). Proteins were separated using SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. Blotted proteins were blocked with 5% non-fat dried milk in PBS, pH 7.4, for 1 h at room temperature and incubated overnight with primary antimyogenin (cod. MAB3876, Millipore, Italy), anti-MHC (cod. 05–716, Millipore, Italy), antielastin (DBS, clone: BA4), antilectin-like receptor (anti-LOX; cod. ABT112, Millipore, Italy), anti-MMP2 (IM53 Calbiochem, Italy), fibrillin1 (Santa Cruz), collagen III (Sigma, Italy), and tubulin (cod. 05–661, Millipore, Italy) antibodies at a ratio of 1:500 in PBS.

4

BOCCAFOSCHI ET AL.

Scaffold mechanical properties Scaffold contraction has been measured over time up to 21 days in presence and absence of cells seeded in the 3D matrices (Fig. 1). Results showed that in all cases, in absence of cells, the scaffold diameters remained unaltered, while cells contraction caused the matrices shrinkage. Moreover, in presence of HELP, matrix shrinkage occurred rapidly after 24 h and increased reaching the maximum contraction after 7 days of culturing. Contraction remained constant until 21 days of culture. Shrinkage of collagen matrices increased progressively getting a plateau after 7 days. In all cases, matrix shrinkage appeared more significant in presence of HELP. Mechanical properties have been evaluated by comparing tensile stress measures and stress–strain curves (Fig. 2).

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 2. Mechanical properties of the collagen and HELPs enriched collagen scaffolds. A: Tensile stress (kPa); (B) elastic modulus. Data are expressed as mean 6 SD of three different experiments. * indicates statistical significance with respect to corresponding 3 and 7 days with p  0.05. ** indicates statistical significance with respect to collagen with p  0.05 C: Stress–strain curve, tensile stress and elastic modulus of native porcine mammary artery. D: Stress–strain curves representative of all the experimental data obtained (collagen, collagen–HELP, and collagen–HELP1) at 3, 7, and 21 days culturing. n value 5 3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2(A) shows tensile stress results obtained on collagen, collagen–HELP, and collagen–HELP1 scaffold after 3, 7, and 21 days culturing. In all cases, an extended culturing period (21 days) improved at least fourfold the loading capacity before rupture. Moreover, in presence of HELP a significant difference has been observed starting from 7 days and compared to pure collagen and collagen–HELP1 matrices. This significant difference has been maintained at 21 days, where collagen matrix displayed a maximum tensile stress at 0.7 6 0.1 kPa, collagen–HELP1 reached 0.8 6 0.1 kPa while collagen–HELP reached 2.4 6 0.1 kPa before rupture. Elastic modulus has been calculated from stress–strain curves and results have been shown in Figure 2(B). At 7 days all matrices displayed no significant differences of elastic modulus. A substantial change has been shown in stiffness after 21 days. In fact, collagen–HELP reached a fourfold higher stiffness with respect to collagen and collagen–HELP1. Results prove that the mechanical properties improvement was due to an optimized crosslink between the matrix components. In fact, HELP1 did not include the crosslinking sequences. In the same culturing

condition, the significant changes observed with HELP were not shown. No statistically significant differences were observed between collagen–HELP1 and pure collagen scaffolds. Stress–strain curves [Fig. 2(C,D)] show the relation between stress, derived from measuring the load applied to the sample, and strain, derived from measuring the deformation of the sample as percentage. Figure 2(C) shows results obtained with respect to native tissues (mammary arteries). These showed a tensile stress of roughly 120 6 33 kPa and an elastic modulus of 555 6 89 kPa. As the stress– strain curves where not clearly distinguishable in the same graph, results obtained from the collagenous matrices have been indicated in Figure 2(D). At 3 and 7 days, collagen showed a very low resistance to stress (roughly 0.2 kPa). The presence of a composite matrices (collagen–HELP) improved the resistance up to approximately 0.6 kPa. The two curves evidenced a different behavior, in fact, collagen presented a linear trend where the elastic and the plastic region were indistinguishable. On HELP-enriched scaffolds the curve changed and two regions were evidenced: a first

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

5

FIGURE 3. One-dimensional SAXS profiles after background subtraction and deconvolution comparison between (A) the collagen scaffolds and native porcine vessel, (B) the collagen–HELP scaffolds and native porcine vessel, (C) the collagen scaffolds and pure collagen sample, and (D) the collagen–HELP scaffold and pure HELP sample. All the samples were placed in 0.7 mm glass capillary permanently immersed in formaldehyde 4% water solution. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

region, corresponding to a low loading stress, where the curve showed an elastic behavior, and a second region, exposing the sample to a higher load, where a plastic deformation was evidenced. Thus, a yield point should be considered as corresponding to the initial fracture. A dramatic change was observed after 21 days, in fact, even collagen stress–strain curve showed a nonlinear trend, with a region where a slight elasticity was evidenced, reaching a yield point at roughly 0.7 kPa. Collagen–HELP matrix differed substantially, displaying a non-linear trend, a strong yield point increasing (2.4 kPa), together with a small deformation (20%) before achieving a plastic deformation. These results have been confirmed by western blot analyses where elastin expression increased over time, especially in presence of HELP (see following sections). Even in this case, the stress–strain curves obtained with collagen–HELP1 matrices did not significantly differ with respect to pure collagen scaffold, confirming the importance of the crosslinking sequences in order to obtain a mechanically improved matrix.

6

BOCCAFOSCHI ET AL.

Structural characterization of 3D scaffolds by SAXS analyses and SEM analysis SAXS profiles, shown in Figure 3, were collected from collagen scaffolds, maintained in culture for 3, 7, and 21 days, with and without HELP. Samples were compared to a native aorta [Fig. 3(A,B)], used as structural reference model, besides pure collagen [Fig. 3(C)] and pure HELP samples [Fig. 3(D)]. The numbers in the upper side of figures indicate the diffraction orders. Figure 3(A,B) shows that collagen and collagen–HELP matrices profiles show many common features and almost the same periodicity of the porcine vessel’s one. From a qualitative point of view, it is clearly evident that SAXS profiles of 3D scaffolds made of collagen alone and HELP-enriched collagen appear dissimilar from those acquired from samples of pure collagen and pure HELP, while they resemble much more to the SAXS data recorded on native porcine aorta. In order to provide also a quantitative evaluation of the degree of similarity between SAXS patterns, the correlation

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 4. Correlation coefficient between porcine aorta and collagen (black points) and collagen–HELP (red points) scaffolds diffraction patterns as a function of the days of culture. *indicates statistical significance with p  0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

coefficient between each profile acquired from the collagen scaffolds, with and without HELP, and the SAXS profile of porcine aorta vessel, pure collagen and pure HELP has been estimated. The correlation coefficient cij has been calculated over a set of N 5 180 data points, by comparing SAXS patterns over an interval of q ranging from 0.026 to 0.100 Å21. The value of cij lies in the range between 21 and 1, where cij 5 1 indicates positive correlated variables, whereas cij 5 21 indicates anticorrelated variables; cij 5 0 indicates an uncorrelation between patterns. The statistical significance of the correlation coefficient depends on the number of data points used for its computation. Considering that in our case N 5 180, correlation coefficients of 0.3–0.45 correspond to a 95–99% level of significance. Figure 4 shows the correlation coefficient between the reference models (porcine aorta) and the engineered scaffolds (black points for collagen, red points for collagen– HELP) as a function of the days of culture. The value and the relative error of the correlation coefficient, reported in Figure 4, have been estimated by averaging the value over a

set of three measurements for each sample, collected in different points. The results show that the correlation between collagen scaffolds and porcine vessel increases with culturing time reaching its maximum value after 21 days. This finding demonstrates a progressive increase in the degree of similarity between SAXS patterns registered on the artificial scaffolds and the porcine aorta reference model. The highest degree of similarity with native porcine aorta model is observed at the longest culturing time (21 days) in presence of HELP, indicating how sample’s structure changes in presence of both components and how it become increasingly similar to the desired material. On the contrary, the correlation between artificial collagen vessels and pure collagen tissues consistently decreases with the days of culture (not shown). SEM analysis (Fig. 5) shows the ECM organization in porcine aorta, collagen and collagen–HELP samples. Aorta nicely shows a complex ECM network, mainly constituted by collagen fibers (black arrows). Collagen matrix mostly consisted of thin uniform collagen fibrils gathered in bundles or layers. Finally, on HELP-enriched samples, collagen fibrils appear quite regularly covered by a grainy substance, composed of beads up to 20 nm thick and reminiscent of the elastic fibers.37 Biological properties: cells phenotype characterization and ECM remodeling 3D matrices remodeling has been studied in order to detect any difference between pure collagen and HELP-enriched matrices, in particular focusing on elastin synthesis and collagen-elastin reorganization. We evaluated the samples using an antielastin antibody that did not cross-react with HELP, as shown in Figure 6(A), while it specifically recognizes insoluble elastin, alpha-elastin, soluble noncrosslinked precursor of elastin (tropoelastin). Immunostaining evidenced that endogenous elastin has been synthetized by cells grown in all 3D matrices. Notably, the presence of HELP had an additive effect on endogenous elastin expression, since the HELP enriched samples showed a higher densitometric value with respect to pure collagen matrix [Fig. 6(B)]. This effect was maintained over time until 21 days [Fig. 6(C)].

FIGURE 5. SEM microscopy on porcine aorta, collagen and collagen–HELP scaffolds after 21 days of culture. Images are representative of three different experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

7

FIGURE 6. Endogenous elastin expression. A: Western blot analyses with antielastin on HELP. The antibody do not recognize the recombinant peptide. B: Western blot analyses with antielastin on cell lysates obtained after 24 h with and without cells growing into the matrices. The antibody specifically recognizes endogenous elastin synthetized by the cells. C: Elastin expression after 3, 7, and 21 days culturing into collagen and collagen–HELP matrices. n value 5 3. *indicates statistical significance with p  0.05. D: Immunohistochemical analyses performed with antielastin antibody and revealed with fluorescence on collagen and collagen–HELP matrices at 7 and 21 days. DAPI has been used for nuclear counterstaining (high magnification images 1003). The small picture shows the analyses performed on a native porcine aorta and it has been used as model reference. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Elastin has been visualized by immunofluorescence [Fig. 6(D)]. A faint fluorescence signal has been detected on pure collagen scaffold which weakly increased up to 21 days. On collagen–HELP matrix, a spot-like fluorescence signal was

8

BOCCAFOSCHI ET AL.

detected, suggesting the presence of microfibril-rich elastin globules close to the cell surface. This was also suggested by the fact that fluorescence is merged with DAPI staining. Interestingly, after 21 days collagen–HELP showed a very

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 7. ECM rearrangement. Western blot analyses on collagen and collagen–HELP matrices with antifibrillin1 (A), anticollagen type III (B), and anti-LOX (C) at 3, 7, and 21 days culturing. Relative densitometry values obtained from three different experiments are expressed as mean 6 SD. n value 5 3. *indicates statistical significance with p  0.05.

intense and homogeneous fluorescence, also evidencing fibrillar structure. Anyway, a well-organized vascular elastin lamina, as shown in details in the small picture, was still not recognized. Figure 7 shows ECM components rearrangement over time. Fibrillin 1 [Fig. 7(A)] expression resulted increased in HELP enriched matrices after 3, 7, and 21 days, while in collagen matrix fibrillin 1 synthesis was not significantly upregulated. These results are consistent with elastin increased expression which resulted slightly delayed, considering that fibrillin forms the substrate for elastin deposition. Collagen type III expression resulted significantly increased in collagen–HELP matrices after 3, 7, and 21 days of culture [Fig. 7(B)]. LOX expression [Fig. 7(C)] has been evaluated, being an endogenous enzyme that crosslinks soluble ECM molecules, such as elastin and collagen. Results showed a significant increase after 21 days only in the case cells are cultured on collagen–HELP matrix, confirming a chemical– physical ECM remodeling as previously demonstrated by SAXS analyses. ECM remodeling has been confirmed also by MMP2 synthesis which cleaved protein resulted persistently enhanced in HELP enriched samples after 3, 7, and 21 days as well (Fig. 8). All results have been normalized with respect to tubulin expression, which resulted equally expressed and comparable in all cases.

In order to evaluate the ability of collagen–HELP matrices to induce a switching to a differentiating phenotype, myogenin, and MHC were investigated. Moreover, cell phenotype has been characterized by fluorescence images and Western blot analyses. After 3 days, myogenin expression resulted enhanced in collagen–HELP matrix and the difference significantly increased after 7 days of culture. A substantial enhancement has been finally shown after 21 days [Fig. 9(A)]. Results obtained from MHC expression confirmed these data, showing a slight enhancement after 3 days, reaching a statistically significant difference after 7 days and maintained up to 21 days [Fig. 9(B)]. Figure 10 showed cell morphology after cytoskeletal and nuclear staining with rhodamin–phalloidin and DAPI. After 3, 7, and 21 days of culture, cells seeded into collagen scaffold showed a fibroblast-like morphology. After 3 days, C2C12 cultured into collagen–HELP scaffold showed a spindle-shaped morphology even if multinucleated myotubes were still not present. Instead, after 7 days of culture, multinucleated myotubes were shown. We also observed the nucleus by DAPI staining. Nuclei in the undifferentiated myoblasts (collagen scaffold) displayed a round morphology whereas in the differentiated cells (collagen–HELP) an elongated shape was revealed [Fig. 10(A)]. Fusion index (FI) [Fig. 10(B)] increased between days 7 and 21 in collagen– HELP matrices. Figure 9(C) showed that, after 7 and

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

9

FIGURE 8. ECM remodeling. Western blot analyses on collagen and collagen–HELP scaffold with MMP2 at 3, 7, and 21 days culturing. Relative densitometry values obtained from three different experiments are expressed as mean 6 SD. n value 5 3. *indicates statistical significance with p  0.05.

21 days of culture in collagen–HELP matrices, the differences in terms of myotube length significantly differed.

DISCUSSION

Due to the excellent biocompatibility, collagen has been widely used as natural polymer. Unfortunately, from a mechanical point of view, it completely lacks of elasticity, reaching a plastic deformation when subjected to weak mechanical stresses. This issue should be overcome by the

use of elastic polymers, such as elastin, which use is normally limited by the insolubility. Native elastin’s intrinsic insolubility has restricted its ability to be purified and processed into forms suitable for biomedical and industrial applications.38 Proteinaceous material and, in particular, the class of elastin-like proteins modeled after the sequence of naturally occurring tropoelastin have emerged as a promising alternative to native ECM derived materials for biomedical applications because they show high cytocompatibility, low-

FIGURE 9. Biological characterization. Western blot analyses on collagen and collagen–HELP matrices with antimyogenin (A) and antimyosin heavy chain (B) at 3, 7, and 21 days culturing. Relative densitometry values obtained from three different experiments are expressed as mean 6 SD. n value 5 3. *indicates statistical significance with p  0.05.

10

BOCCAFOSCHI ET AL.

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 10. Cell morphology. A: Fluorescent microscopy images of C2C12 growing into different matrices. Rhodamin-phalloidin has been used for actin filaments and DAPI for nuclear staining. Cells were observed after 3, 7, and 21 days. Figures are representative of three different experiments. B: Fusion index calculated as the fraction of total nuclei present inside myotubes. *indicates statistical significance with p  0.05. C: Myotubes length. *indicates statistical significance with p  0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

immunogenicity, biodegradation properties and low toxicity. The biomimetic approach based on the expression from synthetic genes artificially built by recombinant methods allows to overcome several hurdles that limit the use of traditional biomaterials. This methodology ensures a tight control on the sequence and composition of the final products, avoiding the inherent variability and batch-to-batch variation typical of product purified from natural sources. Thus, the employment of artificially expressed products guarantees the reproducibility and standardization of the starting material and elimination of the biological risk related to the delivery of pathogens. The use of HELP has several advantages. The simplified primary structure, with respect to the native protein allows to establish a link between the sequence and any function observed in a biological context. Moreover, this polypeptide has been designed to be even modified by addition of a bioactive domain that can confer further functionality to the final product.31 The thermosensitive behavior and the self-assembling properties can be exploited for its purification and can give

thermoresponsive properties to the biomaterial derived from this compound. Due to these properties, this recombinant product results easily water soluble, thus, the use is certainly pursuable in several tissue engineering applications. With the characteristic high stability, fiber-former capability, low immunogenicity, low stimulation of both platelets activation and smooth muscle cells proliferation, elastin, and elastin-derived materials are excellent candidates for use in vascular tissue engineering. In literature, it has been reported that native elastin inhibited smooth muscle cells migration without an effect on endothelial cells motility, which suggested that this might inhibit the formation of anastomotic intimal hyperplasia.39 Furthermore, Defife et al.40 demonstrated reduced fibrinogen and immunoglobulin G adsorption in vitro as well as decreased release of proinflammatory cytokines by monocytes when in presence of elastin. Our results strengthen these findings, adding some important characteristics to collagen–HELP scaffolds. In fact, results demonstrated that, in presence of recombinant

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

11

biomimetic elastin, cells acquired an enhanced contractile phenotype, forming a microtubular network entrapped into the 3D matrix. This contributed to improve also the mechanical properties of the scaffold, together with the complementary effects of elastin on ECM. Stress–strain curves showed a great increase in tensile stress of HELP enriched collagen matrices, probably due to both cells contraction and elastin neosynthesis, which might consequently influence collagen fibers orientation. Moreover, a huge difference was due to the presence of very different amount of water at 7 versus 21 days. In fact, as the cells contract, and this is shown by the enhancement of the contractile cell phenotype and by the improved matrix remodeling process (as will be discussed later on), leading to a more compressed matrix, at the same time the squeezing effect leads to a loss of water contents. The matrices appeared definitely more compact in presence of HELP, as shown even by contraction, and this feature is indicative of a loss in water content which resulted in greatly improved stress resistance capability. Indeed, the morphological and structural analysis confirm a strong similarity between the HELP enriched collagen scaffolds and the native porcine vessel referenced model at the longest culturing period (21 days), as evidenced from the quantitative comparison between the SAXS patterns. Cells also contribute to the matrix remodeling through synthetizing important proteins and enzymes involved in ECM reorganization. In fact, lysyl oxidase catalyses the formation of covalent crosslinks between lysine residues of two adjacent tropoelastin molecules. This amino acid crosslink consolidates the scaffold, confers elastic function and ensures resistance.41 In fact, the crosslinking is essential to strengthen the growing elastic fibers. When the lysyl oxidase activity is inhibited, the level of crosslinked amino acids strongly decreases and it is followed by a reduced elastin accumulation in tissues.42,43 Also in native vessels, medial smooth muscle cells produce ECM with specific architecture which provides elastic recoil (elastic fibers) and tensile strength (collagen fibers) necessary to maintain adequate structural mechanical properties. In particular, the major components of elastic fibers consist of a crosslinked elastin core surrounded by a mantle of fibrillin microfibrils. Assembly of fibrillin into microfibrils and the association of microfibrils with tropoelastin (soluble secreted form of elastin) to form elastic fibers is a highly organized process. Secreted profibrillin is processed and assembled into pericellular microfibrils and microfibril bundles. Tropoelastin globules which have assembled at the cell surface coalesce on the microfibril fibrillin template.44 Once deposited on the fibrillin microfibril template, tropoelastin is crosslinked by lysyl oxidase to form the insoluble core of mature elastic fibers.45 This process clearly occurred only on collagen– HELP matrices, as evidenced by fluorescent elastin immunostaining. Moreover, collagen III together with type I are the most represented collagens in vascular wall. Due to the fibril-forming ability, these two collagen types are mainly responsible for conferring strength to the vessel wall.46 All these findings were confirmed by our data, where the ECM components modification resulted in a direct effect on

12

BOCCAFOSCHI ET AL.

mechanical properties, with a dramatic improvement in the scaffold’ stiffness. In particular, we can argue that the presence of collagen type III in collagen–HELP scaffolds supports the view that our constructs closely resemble a native vessel wall. Moreover, remodeling and homeostasis of the ECM is mediated primarily by a large group of zincdependent endopeptidases, the matrix MMPs. The effect of gel contraction may be mediated in part through MMPs. Creemers et al.47 demonstrated that MMP-2 is primarily responsible for collagen remodeling in the soft connective tissue of the rabbit periosteum, specifically MMP-2 plays an important role in the digestion of collagen types I, II, IV, and V. In literature, it is reported that insoluble elastin and fibrillin microfibrils are catabolized by specific MMPs, such as MMP248 and it has been demonstrated that fibrillin enhances matrix metalloprotease expression.49 The enduring changes in the composition of matrices allow adaptation and repair. For instance, the proteolytic effects of MMPs play an important role in vascular remodeling, cellular migration and the processing of ECM proteins and adhesion molecules.50,51 Finally, literature reports that elastin can influence SMC morphology, proliferation and phenotype.13 In our case, this effect has been driven by HELP, in terms of microtubes formation and myogenetic markers expression, reinforcing the biomechanical characteristics of the scaffold. In conclusion, the addition of recombinant HELP macromolecule improves the biomechanical properties of collagen-based matrices, suitable in particular for vascular tissue engineering applications. Future studies will consider the use of vascular primary cells, together with a maturation in presence of a mechanical stimulation in order to achieve a hierarchical organization of the matrix which supports optimal biomechanical properties of the scaffold. ACKNOWLEDGMENTS

Authors are grateful to Prof. Anthony Weiss, School of Molecular Bioscience, University of Sidney, for his precious comments and for the critical revision of the results. Authors also thank Dr. Elena Grossini from Physiology Laboratory, University of Piemonte Orientale, who kindly provided the native porcine vessels. Rocco Lassandro is acknowledged for his technical support in the XMI-LAB. REFERENCES 1. Dutta CR, Dutta AK. Cell-interactive 3D-scaffold; advances and applications. Biotechnol Adv 2009;27:334–339. 2. Kim BS, Nikolovski J, Bonadio J, Smiley E, Mooney DJ. Engineered smooth muscle tissue: Regulating cell phenotype with the scaffold. Exp Cell Res 1999;251:318–328. ^ nio RA, Lia RC, Cancian 3. Goissis G, Marcantonio E Jr, Marcanto DC, de Carvalho WM. Biocompatibility studies of anionic collagen membranes with different degree of glutaraldehyde cross-linking. Biomaterials 1999;20:27–34. 4. Boccafoschi F, Habermehl J, Vesentini S, Mantovani D. Biological performances of collagen-based scaffolds for vascular tissue engineering. Biomaterials 2005;26:7410–7417. 5. Berglund JD, Mohseni MM, Nerem RM, Sambanis A. A biological hybrid model for collagen-based tissue engineered vascular constructs. Biomaterials 2003;24:1241–1254. 6. Berisio R, Vitagliano L, Mazzarella L, Zagari A. Recent progress on collagen triple helix structure, stability and assembly. Protein Pept Lett 2002;9:107–116.

HELPs IMPROVE THE BIOMECHANICAL PROPERTIES OF COLLAGEN SCAFFOLDS

ORIGINAL ARTICLE

7. Chang MC, Tanaka J. XPS study for the microstructure development of hydroxyapatite-collagen nanocomposites cross-linked using glutaraldehyde. Biomaterials 2002;23:3879–3885. 8. Habermehl J, Skopinska J, Boccafoschi F, Sionkowska A, Kaczmarek H, Laroche G, Mantovani D. Preparation of ready-touse, stockable and reconstituted collagen. Macromol Biosci 2005; 5:821–828. 9. Weber KT, Sun Y, Tyagi SC, Cleutjens. Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. J Mol Cell Cardiol 1994;26:279–292. 10. Urry DW, Pattanaik A. Elastic protein-based materials in tissue reconstruction. Ann N Y Acad Sci 1997;831:32–46. 11. MacEwan SR, Chilkoti A. Elastin-like polypeptides: Biomedical applications of tunable biopolymers. Biopolymers 2010;94:60–77. 12. Chilkoti A, Christensen T, MacKay JA. Stimulus responsive elastin biopolymers: Applications in medicine and biotechnology. Curr Opin Chem Biol 2006;10:652–657. 13. Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, Schwartz RS, Keating MT, Li DY. A critical role for elastin signaling in vascular morphogenesis and disease. Development 2003; 130:411–423. 14. Duca L, Floquet N, Alix AJ, Haye B, Debelle L. Elastin as a matrikine. Crit Rev Oncol Hematol 2004;49:235–244. 15. Blit PH, Battiston KG, Yang M, Paul Santerre J, Woodhouse KA. Electrospun elastin-like polypeptide enriched polyurethanes and their interactions with vascular smooth muscle cells. Acta Biomater 2012;8:2493–2503. 16. Srokowski EM, Woodhouse KA. Evaluation of the bulk platelet response and fibrinogen interaction to elastin-like polypeptide coatings. J Biomed Mater Res A 2014;102:540–51. 17. Simionescu A, Philips K, Vyavahare N. Elastin-derived peptides and TGF-b1 induce osteogenic responses in smooth muscle cells. Biochem Biophys Res Commun 2005;334:524–532. 18. Sinha A, Vyavahare N. High-glucose levels and elastin degradation products accelerate osteogenesis in vascular smooth muscle cells. Diab Vasc Dis Res 2013;10:410–419. 19. Celebi B, Cloutier M, Rabelo RB, Mantovani D, Bandiera A. Human elastin-based recombinant biopolymers improve mesenchymal stem cell differentiation. Macromol Biosci 2012;12:1546– 1554. 20. Murray SS, Murray EJ, Glackin CA, Urist MR. Bone morphogenetic protein inhibits differentiation and affects expression of helix-loop–helix regulatory molecules in myoblastic cells. J Cell Biochem 1993;53:51–60. 21. Bandiera A, Taglienti A, Micali F, Pani B, Tamaro M, Crescenzi V, Manzini G. Expression and characterization of human-elastinrepeat-based temperature-responsive protein polymers for biotechnological purposes. Biotechnol Appl Biochem 2005;42:247– 256. 22. Lombard C, Arzel L, Bouchu D, Wallach J, Saulnier J. Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24. Biochimie 2006;88:1915–1921. 23. Bandiera A, Sist P, Urbani R. Comparison of thermal behavior of two recombinantly expressed human elastin-like polypeptides for cell culture applications. Biomacromolecules 2010;11:3256–3265. 24. Lin S, Mequanint K. The role of Ras-ERK-IL-1b signaling pathway in upregulation of elastin expression by human coronary artery smooth muscle cells cultured in 3D scaffolds. Biomaterials 2012; 33:7047–7056. 25. Petoukhov MV, Svergun DI. Applications of small-angle X-ray scattering to biomacromolecular solutions. Int J Biochem Cell Biol 2013;45:429–437. 26. Blanchet CE, Svergun DI. Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution. Annu Rev Phys Chem 2013;64:37–54. 27. Svergun DI, Shtykova EV, Volkov VV, Feigin LA. Small-angle Xray scattering, synchrotron radiation, and the structure of bioand nanosystems. Crystallogr Rep 2011;56:725–750. 28. Dorfs D, Krahne R, Giannini C, Falqui A, Zanchet D, Manna L. Quantum dots: Synthesis and characterization. Compr Nanosci Technol 2011;1:219–270. 29. Guinier A, Fournet G. Small Angle Scattering of X-Rays. New York: Wiley; 1955.

30. Frisman I, Seliktar D, Bianco-Peled H. Nanostructuring biosynthetic hydrogels for tissue engineering: A cellular and structural analysis. Acta Biomater 2013;8:51–60. 31. Bandiera A. Assembly and optimization of expression of synthetic genes derived from the human elastin repeated motif. Prep Biochem Biotechnol 2010;40:198–212. 32. Rajan N, Habermehl J, Cote` MF, Doillon CJ, Mantovani D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc 2006;1:2753–2758. 33. Blanton TN, Huang TC, Toraya H, Hubbard CR, Robie SB, Loue D, Go HE, Will G, Gilles R, Raftery T. A possible low-angle X-ray diffraction calibration standard. Powder Diffr 1995;10:91–95. 34. Altamura D, Lassandro R, Vittoria FA, De Caro L, Siliqi D, Ladisa M, Giannini C. X-ray microimaging laboratory (XMI-LAB). J Appl Cryst 2012;45:869–873. 35. De Caro L, Altamura D, Vittoria FA, Carbone G, Qiao F, Manna L, Giannini C. A superbright X-ray laboratory microsource empowered by a novel restoration algorithm. J Appl Cryst 2012;45:1228– 1235. 36. De Caro L, Altamura D, Sibillano T, Siliqi D, Filograsso G, Bunk O, Giannini C. Rat-tail tendon fiber SAXS high-order diffraction peaks recovered by a superbright laboratory source and a novel restoration algorithm. J App Cryst 2013;46:672–678. 37. Raspantia M, Protasonia M, Manellia A, Guizzardib S, Mantovanic V, Salac A. The extracellular matrix of the human aortic wall: Ultrastructural observations by FEG-SEM and by tapping-mode AFM. Micron 2006;37:81–86. 38. Jordan SW, Haller CA, Sallach RE, Apkarian RP, Hanson SR, Chaikof EL. The effect of a recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a baboon arteriovenous shunt. Biomaterials 2007;28:1191–1197. 39. Ito S, Ishimaru S, Wilson SE. Application of coacervated alphaelastin to arterial prostheses for inhibition of anastomotic intimal hyperplasia. ASAIO J 1998;44:M501–505. 40. Defife KM, Hagen KM, Clapper DL, Anderson JM. Photochemically immobilized polymer coatings: Effects on protein adsorption, cell adhesion, and leukocyte activation. J Biomater Sci Polym Ed 1999;10:1063–1074. 41. Debelle L, Tamburro AM. Elastin: Molecular description and function. Int J Biochem Cell Biol 1999;31:261–272. 42. Tinker D, Romero-Chapman N, Reiser K, Hyde D, Rucker R. Elastin metabolism during recovery from impaired crosslink formation. Arch Biochem Biophys 1990;278:326–332. 43. Tinker D, Rucker RB. Role of selected nutrients in synthesis, accumulation, and chemical modification of connective tissue proteins. Physiol Rev 1985;65:607–657. 44. Sherratt MJ. Tissue elasticity and the ageing elastic fibre. Age 2009;31:305–325. 45. Stephan S, Ball SG, Williamson M, Bax DV, Lomas A, Shuttleworth CA, Kielty CM. Cell-matrix biology in vascular tissue engineering. J Anat 2006;209:495–502. 46. Wagenseil JE, Mecham RP. Vascular extracellular matrix and arterial mechanics. Physiol Rev 2009;89:957–989. 47. Creemers LB, Jansen IDC, Docherty AJP, Reynolds JJ, Beertsen W, Everts V. Gelatinase a (mmp-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue. Matrix Biol 1998;17:35–46. 48. Ashworth JL, Murphy G, Rock MJ, Sherratt MJ, Shapiro SD, Shuttleworth CA, Kielty CM. Fibrillin degradation by matrix metalloproteinases: Implication for connective tissue remodeling. Biochem J 1999;340:11–181. 49. Booms P, Pregla R, Ney A, Barthel F, Reinhardt DP, Pletschacher A, Mundlos S, Robinson PN. RGD-containing fibrillin-1 fragments upregulate matrix metalloproteinase expression in cell culture: A potential factor in the pathogenesis of the Marfan syndrome. Hum Genet 2005;116:51–61. 50. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry. Circ Res 2003;92:827–839. 51. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: The good, the bad, and the ugly. Circ Res 2002;90:251–262.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

13