- Email: [email protected]
Collagen-based layer-by-layer coating on electrospun polymer scaffolds
Biomaterials 33 (2012) 9198e9204
Contents lists available at SciVerse ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Collagen-based layer-by-layer coating on electrospun polymer scaffolds Yen B. Truong a, Veronica Glattauer a, Kelsey L. Briggs b, Stefan Zappe b, John A.M. Ramshaw a, * a b
CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3169, Australia Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15219, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2012 Accepted 9 September 2012 Available online 1 October 2012
Preparation of microfibre constructs of collagen by electrospinning has been problematic due to the instability of collagen in volatile solvents, such as 1,1,1,3,3,3-hexafluoro-2-propanol, so that electrospinning leads to a substantial amount of gelatin fibres. In the present study we have demonstrated the production of collagen-based microfibre constructs by use of a layer-by-layer coating process onto a preformed synthetic polymer microfibre base. Soluble native collagen, which has a basic isoelectric point, has been used with modified triple-helical collagens that have acidic isoelectric points. These modified collagens have been prepared as deamidated, succinylated, maleylated and citraconylated derivatives. Together, the acidic and basic collagens have successfully coated polyacrylonitrile and poly(DL-lactide-co-glycolide) fibres, as shown by spectroscopy and microscopy. These coatings allow good cell attachment and spreading on the fibres. The native, triple helical form of the collagen has been confirmed through use of a conformation dependent monoclonal antibody. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Collagen Polyacrylonitrile Poly(lactide-co-glycolide) Electrospinning Cell adhesion Biocompatibility
1. Introduction Collagen is an effective and widely used material for biomedical applications and has gained broad clinical and consumer acceptance as a safe material [1,2]. Its properties can be adapted to meet a range of different clinical applications, so that reliable commercial products are now readily available for use in a variety of medical disciplines. As a commercial medical product, collagen can be part of natural, stabilised tissue that is used in the device, such as in a bioprosthetic heart valve, or it can be fabricated as a reconstituted, purified product from animal sources, such as in wound dressings [1,2]. An important area for further development is, for example, in tissue engineering, where a collagen-based scaffold for cell and stem cell expansion on an accurate, functional matrix is important. Collagens are characterised by the presence of a triple-helix structure that is supercoiled from three polyproline-II-like helices [3]. The steric constraints for this structure lead to the presence of glycine as every third amino acid in the sequence to allow for close packing of the three polypeptide chains, while a high content of the imino acids proline (Pro) and hydroxyproline (Hyp) promote the polyproline-II-like conformation of individual chains and provide stability [3]. When native collagen is denatured it forms gelatin; the individual chains in gelatin do not readily reform into the original * Corresponding author. Tel.: þ61 3 9545 8111; fax: þ61 3 9545 8101. E-mail address: [email protected] (J.A.M. Ramshaw).
aligned structure, although shorter segments of triple-helix lacking the native alignment can assemble, providing junctional domains that allow the gel network of gelatin to form [4]. Current technologies for designing tissue engineered constructs have difficulty reproducing the complexity of native tissues and organs. Electrospinning has become an increasingly popular method for fabricating nano- to micrometre diameter fibres and scaffolds for various tissue engineering applications [5e7]. Electrospun collagen would be an ideal tissue engineering scaffold as it could promote cell attachment and growth and allow penetration of cells into the fibre matrix. Electrospinning approaches have been described for collagen [8,9] using, for example, 1,1,1,3,3,3hexafluoro-2-propanol (HFIP) as the solvent for the collagen. However, concerns have been raised that this approach using HFIP leads to significant denaturation of the collagen, resulting in a loss of the triple helical structure to give gelatine or a gelatin/collagen composite. The extent of this denaturation is probably large, at least 50% [10], and possibly complete [11] leading to loss of reproducibility and of certain positive properties that native collagen can provide. Various other solvent systems have subsequently been examined. In some cases collagen has been electrospun with a copolymer in a single phase, including poly(3-caprolactone) [12] or poly(ethylene oxide) [13]. Alternatively, other solvent systems have been examined, of which binary mixtures of phosphate buffered saline (PBS) and EtOH have emerged as the most promising [14]. In the present study, we have examined a method that limits the risk of collagen denaturation through production of collagen
0142-9612/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.09.012
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
microfibres by layer-by-layer coating of both stable and resorbable electrospun polymer fibres. Layer-by-layer coating gives a thicker and more durable coating compared to grafted single layer coatings [15e17]. Overlapping layers of collagen having alternating positive and negative charges are assembled layer-by-layer to create a multilayer coating of collagen on the fibre. Native collagen, such as type I collagen, has a basic isoelectric point, greater than pH 9 [18]. Previously, forming layer-by layer coatings of collagen has been limited by that lack of an acidic collagen. Hence other negatively charged polymer such poly(styrene) sulfonate [19] or natural polymers such as hyaluronic acid [20] have been used, giving heterologous coatings. In the present study, however, we have used collagens with acidic isoelectric points, obtained through both reversible and irreversible chemical modifications to collagen, so that the assembled coating layer is homologous, consisting solely of collagen. 2. Materials and methods 2.1. Collagen preparation For layer-by-layer coating, porcine type I and III collagens were prepared from de-fatted, minced porcine skin by pepsin digestion and NaCl precipitation at [21,22]. Tissue was digested using 1 mg/ml pepsin in 100 mM acetic acid adjusted to pH 2.4 with hydrochloric acid for 48 h. Soluble collagen was precipitated by addition of NaCl to 0.7 M. The collagen precipitate was further purified at pH 7.4 in 50 mM Tris/ HCl by removal of type III collagen by precipitation with 1.7 M NaCl, followed by precipitation of type I collagen by 2.4 M NaCl. Type III collagen was further enriched by rapid (NH4)2SO4 precipitation [22]. Collagen purity was assessed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) [23] using NuPAGE 4e12% Bis-Tris gels with MES running gel buffer (Invitrogen) at 150 V for 3 h, and using delayed reduction for type III collagen samples [24]. 2.2. Modification of collagen Deamidated collagen was prepared from purified pepsin soluble collagen by treatment with 5% w/v NaOH in saturated Na2SO4 at 20 C for 4 d [25]. Deamidated collagen was recovered by neutralising, dialysis and freeze drying. Succinylated collagen was prepared from purified pepsin soluble collagen at pH 8 by reacting with powdered succinic anhydride, with the pH maintained at pH 8 by NaOH addition [25]. Succinylated collagen was recovered by dialysis and freeze drying. Maleylated and citraconylated collagens were prepared in a similar manner using maleic anhydride [26] and citraconic anhydride [27] on 2 mg/ml solutions and were used from these solutions. Reversing this modification was by adjusting to pH 3.0 with acetic acid and HCl and holding at room temperature for 48 h [26,27].
9199
2.5. Layer-by-layer coating of fibres Un-modified collagen was prepared as a 1 mg/ml solution in 50 mM acetic acid, taken to 0.2 M NaCl and adjusted to between pH 7.0 and pH 7.3 with Tris. Deamidated and succinylated collagens were prepared as 1 mg/ml solutions in 50 mM Tris with subsequent addition of acetic acid to pH 7.4 and then NaCl to give 0.2 M concentration. Maleylated and citraconylated collagens were diluted to 1 mg/ml and made 0.2 M in NaCl, then adjusted to pH 7.4 with acetic acid. All solutions were maintained at 4 C and centrifuged prior to use at 3250 g for 15 min. Surface modified PAN and PLGA fibres were coated by immersion in alternating solutions of native and modified collagen (deamidated/succinylated/citraconylated/ maleylated) solutions at 4 C to give 7, 11, or 17 collagen layers. Between each coating step the samples were rinsed with 10 mM sodium phosphate buffer, pH 7.4 containing 0.2 M NaCl. Collagen layers were stabilised during the process or at the end of the coating process by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking in EtOH after samples had been taken through a graded series of aqueous EtOH up to 100% EtOH. The amount of EDC used was 1% (w/w) of the original weight of the polymeric fibre sheet. After EDC treatment overnight, the final material was washed extensively from EtOH into deionised water and dried at room temperature prior to use. 2.6. Characterisation of coated fibres For SEM, iridium coated membranes were examined as described above (Section 2.4). FITR was performed on membranes as described above (Section 2.4). The presence of native triple-helical collagen was demonstrated by using a conformation dependent monoclonal antibody (MAb) that reacts solely with native collagen and not with gelatin [30]. Membranes were examined using MAb 5D8-G9/Col1, which is specific for type I collagen [30] and using a purified Alexa FluorÒ 488 goat antimouse IgG (H þ L) (Molecular Probes, Life Technologies) secondary antibody diluted 1/500 in PBS, followed by 3 washes each for 5 min in PBS. Samples were viewed using an Olympus BX-61 microscope. 2.7. Cell adhesion Native, modified and collagen-coated PAN and PLGA fibres were examined for cell attachment and spreading using L929 mouse lung fibroblasts (Cell line ATCCCCL-1, Rockville, MD). Samples were seeded with 6.5 104 L929 cells per cm2 and cultured in minimum essential medium (MEM) containing 10% v/v foetal bovine serum and 1% non-essential amino acids (Invitrogen, USA) at 37 C, with 5% CO2 in air for 16 h. For some experiments, pre-stained cells were used to assist in visualisation. A confluent cell layer on a T75 cell culture flask (Nunc, Denmark) was incubated with a fluorescent DilC12(3) membrane stain (BD Biosciences) at 1 mg mL1 in media for 1 h at 37 C. This was followed with a 10 ml wash with PBS and the cells were then harvested with TrypLEÔ Express (GibcoÒ) and washed
2.3. Electrospun polymers Synthetic polymer microfibers were prepared by electrospinning [28]. For polyacrylonitrile (PAN)(Aldrich, CAS25014-49-8, Tg 85.0 C), fibres were spun from a 10% (w/v) solution of PAN in dimethyl formamide using a flow rate of 0.2 ml/h through a 25 gauge needle at 200 mm distance from tip to collector and a 10 kV potential difference. For poly acid terminated 50:50 poly(DL-lactide-co-glycolide) (PLGA), inherent viscosity 0.55e0.75 dL/g (Lactel, Durect Corp., AL), fibres were spun from a 40% (w/v) solution in N,N-dimethylacetamide (Aldrich) using a 23G needle at 150 mm distance from tip to collector and a 20 kV potential. After electrospinning, samples were placed in an oven (50 C, >16 h) to complete removal of solvent and then stored over desiccant until used. 2.4. Surface modification of synthetic polymer microfibres Surface modification of the PAN nanofibres was carried out by alkaline hydrolysis [29]. Samples were soaked in 7.5% (w/v) NaOH for times up to 180 min at 50 C. After removal from the NaOH, samples were washed extensively in deionised water. After washing, samples were dried at 50 C overnight. Modification was shown by Fourier transform infra-red spectroscopy (FTIR), using a Nicolet 6700 instrument (Thermo Scientific), and by contact angle analysis using a goniometer, model PG-3 (Fibrosystem AB, Sweden) with a 4 ml drop. The electrospun fibre mats were examined before and after surface modification by scanning electron microscopy (SEM). Dry membranes were coated using a Polaron SC5750 sputter coater using iridium with a setting at 50 mA for 15 s resulting in approximately a 5 nm coat thickness. Coated membranes were imaged at 2 kV using a Philips XL30 field emission scanning electron microscope. Average fibre diameter was determined from SEM images [28]. PLGA, with free acid groups, was used with no further surface modification and was analysed by the same methods.
Fig. 1. SDS -gel electrophoresis of porcine collagen preparations. Lane 1, type I collagen; Lane 2, deamidated type I collagen; Lane 3, succinylated type I collagen; Lane 4, maleylated type I collagen; Lane 5, citraconylated type I collagen; Lane 6, acid treated maleylated type I collagen; Lane 7, acid treated citraconylated type I collagen; Lane 8, type III collagen; Lane 9, succinylated type III collagen.
9200
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
Fig. 2. SEM examination of electrospun fibre mats. (A) PAN fibres, (B) PLGA fibres. Bars ¼ 5 mm.
twice in 30 ml media. For other experiments, a Live/DeadÔ cell viability assay was used (Molecular Probes, Invitrogen, USA) following the manufacturer’s instructions. This shows live calcein AM stained (green) cells and any dead, ethidium homodimer1 stained (red) cells. Prior to adding the reagents scaffolds were rinsed in phosphate buffered saline (PBS) to remove any non-adherent cells. All samples were assessed using a fluorescent microscope (Eclipse TE2000-U, Nikon, Australia). For examination of cells by SEM, membranes were rinsed with PBS and fixed with 3% glutaraldehyde for 1 h before washing and dehydrating through a graded series of EtOH and into hexamethyldisilazane prior to drying. Coating and examination was as described above (Section 2.4).
3. Results and discussion 3.1. Collagen preparation and modification The porcine type I collagen was of high purity (Fig. 1, Lane 1), and contained some b-components and other higher oligomers. The type III collagen was of good purity, but contained small quantities of type I collagen. It is difficult to fully remove type I collagen during
Fig. 3. SEM examination of PAN fibres after treatment with NaOH for various times. (A) unmodified fibres; (B) 30 min; (C) 60 min; (D) 180 min. Bars ¼ 1 mm.
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
9201
purification of type III collagen [31] (Fig. 1, Lane 8). The effectiveness of the deamidation was seen by a reduced mobility for the modified collagen (Fig. 1, Lane 2). Similarly, the mobility of types I and III collagen were reduced by succinylation (Fig. 1, Lanes 3 & 9), consistent with previous reports of succinylation slowing electrophoretic mobility [32]. Also, maleylated and citraconylated type I collagens had reduced mobilities (Fig. 1, Lanes 4 & 5). These later modification reactions also provide negatively charged collagen, but in these two cases the modifying group can be readily removed. Thus, maleyl groups can be removed from the protein at a pH of less than 5 [26]; this study used 50 mM acetic acid adjusted to pH 3.0 with HCl. Citraconyl groups are more readily removed, with a half life of around 1.5 h at pH 3.5 [27]. In the present case, with collagen, the reversibility of these modifications by acid treatment of the previously modified samples was shown by the return of a migration that was similar to that of unmodified type I collagen (Fig. 1, Lanes 6 & 7). In the present study, collagens type I and type III were used, but the layer-by-layer coating method is suitable for other collagen types that are available in sufficient quantity, and for combinations of different collagen types. This could include recombinant collagens [33] and also bacterial collagen-like proteins [34], which are available with basic and acidic isoelectric points [35] and so can be used directly without the need for any chemical modification. 3.2. Polymer electrospinning and modification Uniform mats of both PAN and PLGA fibres were readily obtained using the preferred electrospinning conditions, giving fibre diameters that were typically 270e730 nm for the PAN fibres (Fig. 2A), and 415e1030 nm for PLGA fibres (Fig. 2B). For PAN the fibre network was distinct, whereas with the PLGA some fusion between fibres was evident. The fibrous network provides good strength, flexibility and dimensional stability to the layer-by-layer coated materials. In the present study, the focus has been on the potential for using layer-by layer coating of fibres. So that while electrospinning is very versatile, variations in the electrospun mat have not been examined in this study. Thus, variations in fibre diameter from nanometres up to more typically less than 1 micron are typically available, which, along with variations in the organisation and structure of the fibres mats, can give rise to a range of other product configurations and variations in performance. It is known, for example, that fibroblast adhesion is decreased, while proliferation increased on aligned, collagen-based (possibly gelatine) scaffolds compared to non-aligned, random scaffolds [9], while the variations in texture that can be produced [28,36] can also influence cell proliferation and morphology [36]. This study has used two polymer examples, one of which, PLGA, is readily resorbable. A wide range of other synthetic polymers could also be used. In addition, various biological polymers have been used successfully for electrospinning, including silk [37], gelatin [38], elastin [39] and hence would be suitable as carrier fibres for the collagen. The coating technology is not limited to fibres, but could be used on a range of surfaces. 3.3. Evaluation of modified PAN fibres SEM was used to evaluate the structural integrity of the fibres after the NaOH treatments for various times. These data (Fig. 3) showed that the fibres remained intact after 180 min incubation in NaOH, but some surface etching had occurred. FTIR was used to examine if chemical changes had occurred to the PAN fibres. These data showed little change at 60 min, but by 120 min clear changes in the FTIR spectra were evident (Fig. 4) indicating that successful modification of the PAN surface had occurred, with little further change during the 180 min of soaking in NaOH, with a preferred
Fig. 4. FTIR spectroscopy of PAN samples. (A) Unmodified PAN fibres, (B) PAN fibres modified by 7.5% (w/v) NaOH at 50 C for 60 min and (C), similarly, for 120 min.
time of 120 min. After 120 min, compared to native PAN fibres, there was a small decrease in the peak at 2243 cm1, while new peaks were observed at 1407 cm1, 1568 cm1,1667 cm1 and 3344 cm1, suggesting an increase in carboxylate groups that could assist collagen attachment. 3.4. Layer-by-layer collagen coating of modified polymer fibres The layer-by-layer coating process successfully placed a collagen coating onto both the PAN and PLGA fibre mats. SEM examination showed that the coatings were uniform, and for the PAN fibres conformed to the topological changes that had been
9202
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
Fig. 5. SEM examination of (A) PAN and (B) PLGA, after coating with seven alternating layers of native type I collagen I and succinylated type I collagen. Bars ¼ 1 mm.
introduced by the NaOH treatment (Fig. 5). After 7 alternating single layers, the collagen coating was leading to small sheet like linkages between adjacent fibres (Fig. 5). In initial studies, it was found that with 11 and 17 single layer coatings the extent of these inter-fibre sheets was more extensive (data not shown), and so subsequent studies used 7 alternating single layers with the final layer being the native un-modified collagen, rather than modified collagen. In this study, the collagen was treated by EDC mediated crosslinking, to increase its persistence and stability, although the use of cross-linking is optional. However, if an acid treatment is to follow, to reverse the maleyl or citraconyl modifications, then crosslinking would be necessary to prevent the acid solubilisation of the collagen coating. Several other types of cross-links could also be used, including chemical treatments, such as glutaraldehyde, UV treatment and glycosyl cross-links formed via non-enzymatic glycation [40]. The changes in contact angle as a result of the coatings were measured. The PAN sample had an average contact angle of 108
that did not change over a period of 1 min, and the drop did not appear to be absorbed over a longer period. After the coating, the sample was immediately wetted and a contact angle could not be determined. FTIR also showed the presence of the protein coating (data not shown). The presence of native, triple-helical structure of the collagen in the coating was shown by immunohistology using of a conformation dependent MAb, that does not react with denatured collagen (gelatin). These data, shown for PLGA (Fig. 6), showed that collagen on the surface of the coated fibres was present in its native, triplehelical conformation (Fig. 6B). Control samples, without both primary and secondary antibodies (Fig. 6A) or without the primary antibody (Fig. 6C) showed no response. This method, or other methods such as electron microscopy, do not show small amounts of denatured collagen (gelatin) if present. However, in this case the conditions used for the coatings and the chemical modifications have previously been shown to be mild [41] and do not denature the collagens [25], unlike the organic solvents, such as HFIP, that have been used in electrospinning [8e11].
Fig. 6. Examination of collagen coatings using collagen type specific MAb’s that react with collagen in a native triple-helical conformation. (A) Control PLGA fibres coated with collagen type I/succinylated collagen type I, (B) PLGA fibres coated with collagen type I/succinylated collagen type I stained with anti-collagen type I MAb and Alexa FluorÒ 488 labelled goat anti-mouse secondary antibody, (C) PLGA fibres coated with collagen type I/succinylated collagen type I stained with only Alexa FluorÒ 488 labelled goat anti-mouse secondary antibody Bars ¼ 100 mm.
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
9203
Fig. 7. Examination of L-929 fibroblasts growing on PAN fibres coated with 7 alternating layers of various collagen combinations. (A) Pre-stained cells (DilC12(3) membrane stain) on fibres with no collagen coating, (B) Pre-stained cells on fibres coated with type I collagen and succinylated type I collagen, (C) Pre-stained cells on fibres coated with type III collagen and succinylated type III collagen. (D) SEM of cells on fibres with no collagen coating, (E) SEM of cells on fibres coated with type I collagen and succinylated type I collagen, (F) SEM of cells on fibres coated with type III collagen and succinylated type III collagen. Bars (AeC) ¼ 100 mm. Bars (DeF) ¼ 50 mm.
3.5. Cell culture on collagen-coated fibres PAN mats coated with either collagen type I or collagen type III both showed good attachment and significant spreading of cells (Fig. 7B, C). Similar results were also obtained with PLGA fibres where coating with collagen, including reversibly modified collagens also led to good attachment and significant cell spreading with good cell viability (Fig. 8B,C). In the present study cell binding to PAN and NaOH modified PAN mats and to unmodified PLGA was poor (Figs. 7A,8A). However, for some core fibres, such as gelatin, the cell binding could be as good as, or better than with collagen [42]. These cell culture data were further confirmed by scanning electron microscopy of the cells after 16 h (Figs. 7 and 8). Cells
grown on non-collagen-coated surfaces all had a rounded morphology. On the other hand, cells grown on collagen-modified materials typically showed an elongated and frequently flattened morphology. This was the case equally for samples with only a few or many collagen coating layers and for both PAN and PLGAbased constructs (Figs. 7 and 8). These data show that the modified collagens do not lead to any cytotoxicity. Previously, anionic collagen, also prepared by alkaline deamidation, had been shown to perform acceptably in bone healing [43]. In the present study, the polymer fibres have been used without additives. The progress of cell growth on the collagen-coated fibres could be modulated if required through incorporation of growth factors or low molecular weight compounds within the electrospun fibre base [44,45].
Fig. 8. Examination of L-929 fibroblasts growing on PLGA fibres coated with 7 alternating layers of various collagen combinations. (A) Live/dead staining of cells on fibres with no collagen coating, (B) Live/dead staining of cells on fibres coated with type I collagen and succinylated type I collagen, (C) Live/dead staining of cells on fibres coated with type I collagen and citraconylated type I collagen. (D) SEM of cells on fibres coated with type I collagen and succinylated type I collagen, (E) SEM of cells on fibres coated with type I collagen and citraconylated type I collagen, (F) SEM of cells on fibres coated with type I collagen and maleylated type I collagen. Bars (AeC) ¼ 100 mm. Bars (DeF) ¼ 20 mm.
9204
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
4. Conclusion The present study has shown that layer-by-layer coating of an electrospun fibre network with alternating layers of triple-helical anionic and cationic collagens provides an effective method for producing homologous collagen coatings. The approach uses nondenaturing conditions, so the collagen retains its native triple helical structure and is not converted to gelatin. The electrospun base can be made from various materials, including both resorbable and non-resorbable polymers. The collagen coated fibres in this study were found to provide a good substrate for cell adhesion and spreading, while maintaining dimensional stability. The approach has the potential to be used on other surface types. This system provides an alternative approach for producing collagen-based microfibre materials for biomedical applications, such as wound dressings or adhesion barriers, and for use in tissue engineering applications. Acknowledgements K.B. was in part supported through a Research Experience for Undergraduates (REU) supplement to the NSF CAREER award CBET0748062 (S.Z.). References [1] Ramshaw JA, Werkmeister JA, Glattauer V. Collagen-based biomaterials. Biotechnol Genet Eng Rev 1996;13:335e82. [2] Ramshaw JA, Peng YY, Glattauer V, Werkmeister JA. Collagens as biomaterials. J Mater Sci Mater Med 2009;20(Suppl. 1):S3e8. [3] Brodsky B, Ramshaw JA. The collagen triple-helix structure. Matrix Biol 1997; 15:545e54. [4] Benguigui L, Busnel JP, Durand D. Study of junction zones in gelatin gels through selective enzymatic digestion. Polymer 1991;32:2680e5. [5] Reneker DH, Chun I. Nanometre diameter fibres of polymer, produced by electrospinning. Nanotechnology 1996;7:216e23. [6] Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989e2006. [7] Holzwarth JM, Ma PX. Biomimetic nanofibrous scaffolds for bone tissue engineering. Biomaterials 2011;32:9622e9. [8] Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules 2002;3:232e8. [9] Zhong S, Teo WE, Zhu X, Beuerman RW, Ramakrishna S, Yung LY. An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture. J Biomed Mater Res A 2006;79:456e63. [10] Yang L, Fitié CF, van der Werf KO, Bennink ML, Dijkstra PJ, Feijen J. Mechanical properties of single electrospun collagen type I fibers. Biomaterials 2008;29: 955e62. [11] Zeugolis DI, Khew ST, Yew ES, Ekaputra AK, Tong YW, Yung LY, et al. Biomaterials 2008;29:2293e305. [12] Tillman BW, Yazdani SK, Lee SJ, Geary RL, Atala A, Yoo JJ. The in vivo stability of electrospun polycaprolactoneecollagen scaffolds in vascular reconstruction. Biomaterials 2009;30:583e8. [13] Buttafoco L, Kolkman NG, Engbers-Buijtenhuijs P, Poot AA, Dijkstra PJ, Vermes I, et al. Electrospinning of collagen and elastin for tissue engineering applications. Biomaterials 2006;27:724e34. [14] Dong B, Arnoult O, Smith ME, Wnek GE. Electrospinning of collagen nanofiber scaffolds from benign solvents. Macromol Rapid Commun 2009;30:539e42. [15] Feng ZQ, Lu HJ, Leach MK, Huang NP, Wang YC, Liu CJ, et al. The influence of type-I collagen-coated PLLA aligned nanofibers on growth of blood outgrowth endothelial cells. Biomed Mater 2010;5:065011. [16] Chen JP, Li SF, Chiang YP. Bioactive collagen-grafted poly-L-lactic acid nanofibrous membrane for cartilage tissue engineering. J Nanosci Nanotechnol 2010;10:5393e8. [17] Duan Y, Wang Z, Yan W, Wang S, Zhang S, Jia J. Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties. J Biomater Sci Polym Ed 2007;18:1153e64. [18] Peng YY, Glattauer V, Werkmeister JA, Ramshaw JAM. Evaluation of collagen products for cosmetic application. J Cosmet Sci 2004;55:327e41.
[19] Grant GGS, Koktysh DS, Yun B, Matts RL, Kotov RL. Layer-by-layer assembly of collagen thin films: controlled thickness and biocompatibility. Biomed Microdevices 2001;3:301e6. [20] Zhang J, Senger B, Vautier D, Picart C, Schaaf P, Voegel JC, et al. Natural polyelectrolyte films based on layer-by layer deposition of collagen and hyaluronic acid. Biomaterials 2005;26:3353e61. [21] Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol 1982;82(Pt A):33e64. [22] Trelstad RL, Catanese VM, Rubin DF. Collagen fractionation: separation of native types I, II and III by differential precipitation. Anal Biochem 1976;71: 114e8. [23] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680e5. [24] Sykes B, Puddle B, Francis M, Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 1976;72:1472e80. [25] Rauterberg J, Kühn K. The renaturation behaviour of modified collagen molecules. Hoppe Seylers Z Physiol Chem 1968;349:611e22. [26] Butler PJ, Harris JI, Hartley BS, Leberman R. Reversible blocking of peptide amino groups by maleic anhydride. Biochem J 1967;103:78Pe9P. [27] Dixon HB, Perham RN. Reversible blocking of amino groups with citraconic anhydride. Biochem J 1968;109:312e4. [28] Truong YB, Glattauer V, Lang G, Hands K, Kyratzis IL, Werkmeister JA, et al. A comparison of the effects of fibre alignment of smooth and textured fibres in electrospun membranes on fibroblast cell adhesion. Biomed Mater 2010;5: 25005. [29] Gupta ML, Gupta B, Oppermann W, Hardtmann G. Surface modification of polyacrylonitrile staple fibers via alkaline hydrolysis for superabsorbent applications. J Appl Polym Sci 2004;91:3127e33. [30] Werkmeister JA, Ramshaw JA, Ellender G. Characterisation of a monoclonal antibody against native human type I collagen. Eur J Biochem 1990;187: 439e43. [31] ChandraRajan J. Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation. Biochem Biophys Res Commun 1978;83:180e6. [32] Zhang Z, Liu W, Li D, Li G. Physicochemical properties of succinylated calfskin pepsin-solubilized collagen. Biosci Biotechnol Biochem 2007;71: 2057e60. [33] Werkmeister JA, Ramshaw JA. Recombinant protein scaffolds for tissue engineering. Biomed Mater 2012;7:012002. [34] Rasmussen M, Jacobsson M, Björck L. Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins. J Biol Chem 2003;278:32313e6. [35] Xu C, Yu Z, Inouye M, Brodsky B, Mirochnitchenko O. Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability. Biomacromolecules 2010;11: 348e56. [36] Moroni L, Licht R, de Boer J, de Wijin JR, van Blitterwijk CA. Fiber diameter and texture of electrospun PEOT/PBT scaffolds influence human mesenchymal stem cell proliferation and morphology, and the release of incorporated compounds. Biomaterials 2006;27:4911e22. [37] Jin HJ, Fridrikh SV, Rutledge GC, Kaplan DL. Electrospinning Bombyx mori silk with poly(ethylene oxide). Biomacromolecules 2002;3:1233e9. [38] Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang ZM. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res B Appl Biomater 2005;72:156e65. [39] Rnjak-Kovacina J, Wise SG, Li Z, Maitz PK, Young CJ, Wang Y, et al. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. Biomaterials 2011;32:6729e36. [40] Ramshaw JAM, Glattauer V, Werkmeister JA. Stabilisation of collagen in clinical applications. In: Wise DL, editor. Handbook of biomaterials engineering. New York: Marcel Dekker Inc; 2000. p. 717e38. [41] Hartley BS. Strategy and tactics in protein chemistry. Biochem J 1970;119: 805e22. [42] Abraham LC, Zuena E, Perez-Ramirez B, Kaplan DL. Guide to collagen characterization for biomaterial studies. J Biomed Mater Res B Appl Biomater 2008;87:264e85. [43] Rocha LB, Goissis G, Rossi MA. Biocompatibility of anionic collagen matrix as scaffold for bone healing. Biomaterials 2002;23:449e56. [44] Ji W, Sun Y, Yang F, van den Beucken JJ, Fan M, Chen Z, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm Res 2011;28:1259e72. [45] Meinel AJ, Germershaus O, Luhmann T, Merkle HP, Meinel L. Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications. Eur J Pharm Biopharm 2012;81:1e13.
Contents lists available at SciVerse ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Collagen-based layer-by-layer coating on electrospun polymer scaffolds Yen B. Truong a, Veronica Glattauer a, Kelsey L. Briggs b, Stefan Zappe b, John A.M. Ramshaw a, * a b
CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3169, Australia Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15219, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2012 Accepted 9 September 2012 Available online 1 October 2012
Preparation of microfibre constructs of collagen by electrospinning has been problematic due to the instability of collagen in volatile solvents, such as 1,1,1,3,3,3-hexafluoro-2-propanol, so that electrospinning leads to a substantial amount of gelatin fibres. In the present study we have demonstrated the production of collagen-based microfibre constructs by use of a layer-by-layer coating process onto a preformed synthetic polymer microfibre base. Soluble native collagen, which has a basic isoelectric point, has been used with modified triple-helical collagens that have acidic isoelectric points. These modified collagens have been prepared as deamidated, succinylated, maleylated and citraconylated derivatives. Together, the acidic and basic collagens have successfully coated polyacrylonitrile and poly(DL-lactide-co-glycolide) fibres, as shown by spectroscopy and microscopy. These coatings allow good cell attachment and spreading on the fibres. The native, triple helical form of the collagen has been confirmed through use of a conformation dependent monoclonal antibody. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Collagen Polyacrylonitrile Poly(lactide-co-glycolide) Electrospinning Cell adhesion Biocompatibility
1. Introduction Collagen is an effective and widely used material for biomedical applications and has gained broad clinical and consumer acceptance as a safe material [1,2]. Its properties can be adapted to meet a range of different clinical applications, so that reliable commercial products are now readily available for use in a variety of medical disciplines. As a commercial medical product, collagen can be part of natural, stabilised tissue that is used in the device, such as in a bioprosthetic heart valve, or it can be fabricated as a reconstituted, purified product from animal sources, such as in wound dressings [1,2]. An important area for further development is, for example, in tissue engineering, where a collagen-based scaffold for cell and stem cell expansion on an accurate, functional matrix is important. Collagens are characterised by the presence of a triple-helix structure that is supercoiled from three polyproline-II-like helices [3]. The steric constraints for this structure lead to the presence of glycine as every third amino acid in the sequence to allow for close packing of the three polypeptide chains, while a high content of the imino acids proline (Pro) and hydroxyproline (Hyp) promote the polyproline-II-like conformation of individual chains and provide stability [3]. When native collagen is denatured it forms gelatin; the individual chains in gelatin do not readily reform into the original * Corresponding author. Tel.: þ61 3 9545 8111; fax: þ61 3 9545 8101. E-mail address: [email protected] (J.A.M. Ramshaw).
aligned structure, although shorter segments of triple-helix lacking the native alignment can assemble, providing junctional domains that allow the gel network of gelatin to form [4]. Current technologies for designing tissue engineered constructs have difficulty reproducing the complexity of native tissues and organs. Electrospinning has become an increasingly popular method for fabricating nano- to micrometre diameter fibres and scaffolds for various tissue engineering applications [5e7]. Electrospun collagen would be an ideal tissue engineering scaffold as it could promote cell attachment and growth and allow penetration of cells into the fibre matrix. Electrospinning approaches have been described for collagen [8,9] using, for example, 1,1,1,3,3,3hexafluoro-2-propanol (HFIP) as the solvent for the collagen. However, concerns have been raised that this approach using HFIP leads to significant denaturation of the collagen, resulting in a loss of the triple helical structure to give gelatine or a gelatin/collagen composite. The extent of this denaturation is probably large, at least 50% [10], and possibly complete [11] leading to loss of reproducibility and of certain positive properties that native collagen can provide. Various other solvent systems have subsequently been examined. In some cases collagen has been electrospun with a copolymer in a single phase, including poly(3-caprolactone) [12] or poly(ethylene oxide) [13]. Alternatively, other solvent systems have been examined, of which binary mixtures of phosphate buffered saline (PBS) and EtOH have emerged as the most promising [14]. In the present study, we have examined a method that limits the risk of collagen denaturation through production of collagen
0142-9612/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.09.012
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
microfibres by layer-by-layer coating of both stable and resorbable electrospun polymer fibres. Layer-by-layer coating gives a thicker and more durable coating compared to grafted single layer coatings [15e17]. Overlapping layers of collagen having alternating positive and negative charges are assembled layer-by-layer to create a multilayer coating of collagen on the fibre. Native collagen, such as type I collagen, has a basic isoelectric point, greater than pH 9 [18]. Previously, forming layer-by layer coatings of collagen has been limited by that lack of an acidic collagen. Hence other negatively charged polymer such poly(styrene) sulfonate [19] or natural polymers such as hyaluronic acid [20] have been used, giving heterologous coatings. In the present study, however, we have used collagens with acidic isoelectric points, obtained through both reversible and irreversible chemical modifications to collagen, so that the assembled coating layer is homologous, consisting solely of collagen. 2. Materials and methods 2.1. Collagen preparation For layer-by-layer coating, porcine type I and III collagens were prepared from de-fatted, minced porcine skin by pepsin digestion and NaCl precipitation at [21,22]. Tissue was digested using 1 mg/ml pepsin in 100 mM acetic acid adjusted to pH 2.4 with hydrochloric acid for 48 h. Soluble collagen was precipitated by addition of NaCl to 0.7 M. The collagen precipitate was further purified at pH 7.4 in 50 mM Tris/ HCl by removal of type III collagen by precipitation with 1.7 M NaCl, followed by precipitation of type I collagen by 2.4 M NaCl. Type III collagen was further enriched by rapid (NH4)2SO4 precipitation [22]. Collagen purity was assessed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) [23] using NuPAGE 4e12% Bis-Tris gels with MES running gel buffer (Invitrogen) at 150 V for 3 h, and using delayed reduction for type III collagen samples [24]. 2.2. Modification of collagen Deamidated collagen was prepared from purified pepsin soluble collagen by treatment with 5% w/v NaOH in saturated Na2SO4 at 20 C for 4 d [25]. Deamidated collagen was recovered by neutralising, dialysis and freeze drying. Succinylated collagen was prepared from purified pepsin soluble collagen at pH 8 by reacting with powdered succinic anhydride, with the pH maintained at pH 8 by NaOH addition [25]. Succinylated collagen was recovered by dialysis and freeze drying. Maleylated and citraconylated collagens were prepared in a similar manner using maleic anhydride [26] and citraconic anhydride [27] on 2 mg/ml solutions and were used from these solutions. Reversing this modification was by adjusting to pH 3.0 with acetic acid and HCl and holding at room temperature for 48 h [26,27].
9199
2.5. Layer-by-layer coating of fibres Un-modified collagen was prepared as a 1 mg/ml solution in 50 mM acetic acid, taken to 0.2 M NaCl and adjusted to between pH 7.0 and pH 7.3 with Tris. Deamidated and succinylated collagens were prepared as 1 mg/ml solutions in 50 mM Tris with subsequent addition of acetic acid to pH 7.4 and then NaCl to give 0.2 M concentration. Maleylated and citraconylated collagens were diluted to 1 mg/ml and made 0.2 M in NaCl, then adjusted to pH 7.4 with acetic acid. All solutions were maintained at 4 C and centrifuged prior to use at 3250 g for 15 min. Surface modified PAN and PLGA fibres were coated by immersion in alternating solutions of native and modified collagen (deamidated/succinylated/citraconylated/ maleylated) solutions at 4 C to give 7, 11, or 17 collagen layers. Between each coating step the samples were rinsed with 10 mM sodium phosphate buffer, pH 7.4 containing 0.2 M NaCl. Collagen layers were stabilised during the process or at the end of the coating process by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking in EtOH after samples had been taken through a graded series of aqueous EtOH up to 100% EtOH. The amount of EDC used was 1% (w/w) of the original weight of the polymeric fibre sheet. After EDC treatment overnight, the final material was washed extensively from EtOH into deionised water and dried at room temperature prior to use. 2.6. Characterisation of coated fibres For SEM, iridium coated membranes were examined as described above (Section 2.4). FITR was performed on membranes as described above (Section 2.4). The presence of native triple-helical collagen was demonstrated by using a conformation dependent monoclonal antibody (MAb) that reacts solely with native collagen and not with gelatin [30]. Membranes were examined using MAb 5D8-G9/Col1, which is specific for type I collagen [30] and using a purified Alexa FluorÒ 488 goat antimouse IgG (H þ L) (Molecular Probes, Life Technologies) secondary antibody diluted 1/500 in PBS, followed by 3 washes each for 5 min in PBS. Samples were viewed using an Olympus BX-61 microscope. 2.7. Cell adhesion Native, modified and collagen-coated PAN and PLGA fibres were examined for cell attachment and spreading using L929 mouse lung fibroblasts (Cell line ATCCCCL-1, Rockville, MD). Samples were seeded with 6.5 104 L929 cells per cm2 and cultured in minimum essential medium (MEM) containing 10% v/v foetal bovine serum and 1% non-essential amino acids (Invitrogen, USA) at 37 C, with 5% CO2 in air for 16 h. For some experiments, pre-stained cells were used to assist in visualisation. A confluent cell layer on a T75 cell culture flask (Nunc, Denmark) was incubated with a fluorescent DilC12(3) membrane stain (BD Biosciences) at 1 mg mL1 in media for 1 h at 37 C. This was followed with a 10 ml wash with PBS and the cells were then harvested with TrypLEÔ Express (GibcoÒ) and washed
2.3. Electrospun polymers Synthetic polymer microfibers were prepared by electrospinning [28]. For polyacrylonitrile (PAN)(Aldrich, CAS25014-49-8, Tg 85.0 C), fibres were spun from a 10% (w/v) solution of PAN in dimethyl formamide using a flow rate of 0.2 ml/h through a 25 gauge needle at 200 mm distance from tip to collector and a 10 kV potential difference. For poly acid terminated 50:50 poly(DL-lactide-co-glycolide) (PLGA), inherent viscosity 0.55e0.75 dL/g (Lactel, Durect Corp., AL), fibres were spun from a 40% (w/v) solution in N,N-dimethylacetamide (Aldrich) using a 23G needle at 150 mm distance from tip to collector and a 20 kV potential. After electrospinning, samples were placed in an oven (50 C, >16 h) to complete removal of solvent and then stored over desiccant until used. 2.4. Surface modification of synthetic polymer microfibres Surface modification of the PAN nanofibres was carried out by alkaline hydrolysis [29]. Samples were soaked in 7.5% (w/v) NaOH for times up to 180 min at 50 C. After removal from the NaOH, samples were washed extensively in deionised water. After washing, samples were dried at 50 C overnight. Modification was shown by Fourier transform infra-red spectroscopy (FTIR), using a Nicolet 6700 instrument (Thermo Scientific), and by contact angle analysis using a goniometer, model PG-3 (Fibrosystem AB, Sweden) with a 4 ml drop. The electrospun fibre mats were examined before and after surface modification by scanning electron microscopy (SEM). Dry membranes were coated using a Polaron SC5750 sputter coater using iridium with a setting at 50 mA for 15 s resulting in approximately a 5 nm coat thickness. Coated membranes were imaged at 2 kV using a Philips XL30 field emission scanning electron microscope. Average fibre diameter was determined from SEM images [28]. PLGA, with free acid groups, was used with no further surface modification and was analysed by the same methods.
Fig. 1. SDS -gel electrophoresis of porcine collagen preparations. Lane 1, type I collagen; Lane 2, deamidated type I collagen; Lane 3, succinylated type I collagen; Lane 4, maleylated type I collagen; Lane 5, citraconylated type I collagen; Lane 6, acid treated maleylated type I collagen; Lane 7, acid treated citraconylated type I collagen; Lane 8, type III collagen; Lane 9, succinylated type III collagen.
9200
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
Fig. 2. SEM examination of electrospun fibre mats. (A) PAN fibres, (B) PLGA fibres. Bars ¼ 5 mm.
twice in 30 ml media. For other experiments, a Live/DeadÔ cell viability assay was used (Molecular Probes, Invitrogen, USA) following the manufacturer’s instructions. This shows live calcein AM stained (green) cells and any dead, ethidium homodimer1 stained (red) cells. Prior to adding the reagents scaffolds were rinsed in phosphate buffered saline (PBS) to remove any non-adherent cells. All samples were assessed using a fluorescent microscope (Eclipse TE2000-U, Nikon, Australia). For examination of cells by SEM, membranes were rinsed with PBS and fixed with 3% glutaraldehyde for 1 h before washing and dehydrating through a graded series of EtOH and into hexamethyldisilazane prior to drying. Coating and examination was as described above (Section 2.4).
3. Results and discussion 3.1. Collagen preparation and modification The porcine type I collagen was of high purity (Fig. 1, Lane 1), and contained some b-components and other higher oligomers. The type III collagen was of good purity, but contained small quantities of type I collagen. It is difficult to fully remove type I collagen during
Fig. 3. SEM examination of PAN fibres after treatment with NaOH for various times. (A) unmodified fibres; (B) 30 min; (C) 60 min; (D) 180 min. Bars ¼ 1 mm.
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
9201
purification of type III collagen [31] (Fig. 1, Lane 8). The effectiveness of the deamidation was seen by a reduced mobility for the modified collagen (Fig. 1, Lane 2). Similarly, the mobility of types I and III collagen were reduced by succinylation (Fig. 1, Lanes 3 & 9), consistent with previous reports of succinylation slowing electrophoretic mobility [32]. Also, maleylated and citraconylated type I collagens had reduced mobilities (Fig. 1, Lanes 4 & 5). These later modification reactions also provide negatively charged collagen, but in these two cases the modifying group can be readily removed. Thus, maleyl groups can be removed from the protein at a pH of less than 5 [26]; this study used 50 mM acetic acid adjusted to pH 3.0 with HCl. Citraconyl groups are more readily removed, with a half life of around 1.5 h at pH 3.5 [27]. In the present case, with collagen, the reversibility of these modifications by acid treatment of the previously modified samples was shown by the return of a migration that was similar to that of unmodified type I collagen (Fig. 1, Lanes 6 & 7). In the present study, collagens type I and type III were used, but the layer-by-layer coating method is suitable for other collagen types that are available in sufficient quantity, and for combinations of different collagen types. This could include recombinant collagens [33] and also bacterial collagen-like proteins [34], which are available with basic and acidic isoelectric points [35] and so can be used directly without the need for any chemical modification. 3.2. Polymer electrospinning and modification Uniform mats of both PAN and PLGA fibres were readily obtained using the preferred electrospinning conditions, giving fibre diameters that were typically 270e730 nm for the PAN fibres (Fig. 2A), and 415e1030 nm for PLGA fibres (Fig. 2B). For PAN the fibre network was distinct, whereas with the PLGA some fusion between fibres was evident. The fibrous network provides good strength, flexibility and dimensional stability to the layer-by-layer coated materials. In the present study, the focus has been on the potential for using layer-by layer coating of fibres. So that while electrospinning is very versatile, variations in the electrospun mat have not been examined in this study. Thus, variations in fibre diameter from nanometres up to more typically less than 1 micron are typically available, which, along with variations in the organisation and structure of the fibres mats, can give rise to a range of other product configurations and variations in performance. It is known, for example, that fibroblast adhesion is decreased, while proliferation increased on aligned, collagen-based (possibly gelatine) scaffolds compared to non-aligned, random scaffolds [9], while the variations in texture that can be produced [28,36] can also influence cell proliferation and morphology [36]. This study has used two polymer examples, one of which, PLGA, is readily resorbable. A wide range of other synthetic polymers could also be used. In addition, various biological polymers have been used successfully for electrospinning, including silk [37], gelatin [38], elastin [39] and hence would be suitable as carrier fibres for the collagen. The coating technology is not limited to fibres, but could be used on a range of surfaces. 3.3. Evaluation of modified PAN fibres SEM was used to evaluate the structural integrity of the fibres after the NaOH treatments for various times. These data (Fig. 3) showed that the fibres remained intact after 180 min incubation in NaOH, but some surface etching had occurred. FTIR was used to examine if chemical changes had occurred to the PAN fibres. These data showed little change at 60 min, but by 120 min clear changes in the FTIR spectra were evident (Fig. 4) indicating that successful modification of the PAN surface had occurred, with little further change during the 180 min of soaking in NaOH, with a preferred
Fig. 4. FTIR spectroscopy of PAN samples. (A) Unmodified PAN fibres, (B) PAN fibres modified by 7.5% (w/v) NaOH at 50 C for 60 min and (C), similarly, for 120 min.
time of 120 min. After 120 min, compared to native PAN fibres, there was a small decrease in the peak at 2243 cm1, while new peaks were observed at 1407 cm1, 1568 cm1,1667 cm1 and 3344 cm1, suggesting an increase in carboxylate groups that could assist collagen attachment. 3.4. Layer-by-layer collagen coating of modified polymer fibres The layer-by-layer coating process successfully placed a collagen coating onto both the PAN and PLGA fibre mats. SEM examination showed that the coatings were uniform, and for the PAN fibres conformed to the topological changes that had been
9202
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
Fig. 5. SEM examination of (A) PAN and (B) PLGA, after coating with seven alternating layers of native type I collagen I and succinylated type I collagen. Bars ¼ 1 mm.
introduced by the NaOH treatment (Fig. 5). After 7 alternating single layers, the collagen coating was leading to small sheet like linkages between adjacent fibres (Fig. 5). In initial studies, it was found that with 11 and 17 single layer coatings the extent of these inter-fibre sheets was more extensive (data not shown), and so subsequent studies used 7 alternating single layers with the final layer being the native un-modified collagen, rather than modified collagen. In this study, the collagen was treated by EDC mediated crosslinking, to increase its persistence and stability, although the use of cross-linking is optional. However, if an acid treatment is to follow, to reverse the maleyl or citraconyl modifications, then crosslinking would be necessary to prevent the acid solubilisation of the collagen coating. Several other types of cross-links could also be used, including chemical treatments, such as glutaraldehyde, UV treatment and glycosyl cross-links formed via non-enzymatic glycation [40]. The changes in contact angle as a result of the coatings were measured. The PAN sample had an average contact angle of 108
that did not change over a period of 1 min, and the drop did not appear to be absorbed over a longer period. After the coating, the sample was immediately wetted and a contact angle could not be determined. FTIR also showed the presence of the protein coating (data not shown). The presence of native, triple-helical structure of the collagen in the coating was shown by immunohistology using of a conformation dependent MAb, that does not react with denatured collagen (gelatin). These data, shown for PLGA (Fig. 6), showed that collagen on the surface of the coated fibres was present in its native, triplehelical conformation (Fig. 6B). Control samples, without both primary and secondary antibodies (Fig. 6A) or without the primary antibody (Fig. 6C) showed no response. This method, or other methods such as electron microscopy, do not show small amounts of denatured collagen (gelatin) if present. However, in this case the conditions used for the coatings and the chemical modifications have previously been shown to be mild [41] and do not denature the collagens [25], unlike the organic solvents, such as HFIP, that have been used in electrospinning [8e11].
Fig. 6. Examination of collagen coatings using collagen type specific MAb’s that react with collagen in a native triple-helical conformation. (A) Control PLGA fibres coated with collagen type I/succinylated collagen type I, (B) PLGA fibres coated with collagen type I/succinylated collagen type I stained with anti-collagen type I MAb and Alexa FluorÒ 488 labelled goat anti-mouse secondary antibody, (C) PLGA fibres coated with collagen type I/succinylated collagen type I stained with only Alexa FluorÒ 488 labelled goat anti-mouse secondary antibody Bars ¼ 100 mm.
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
9203
Fig. 7. Examination of L-929 fibroblasts growing on PAN fibres coated with 7 alternating layers of various collagen combinations. (A) Pre-stained cells (DilC12(3) membrane stain) on fibres with no collagen coating, (B) Pre-stained cells on fibres coated with type I collagen and succinylated type I collagen, (C) Pre-stained cells on fibres coated with type III collagen and succinylated type III collagen. (D) SEM of cells on fibres with no collagen coating, (E) SEM of cells on fibres coated with type I collagen and succinylated type I collagen, (F) SEM of cells on fibres coated with type III collagen and succinylated type III collagen. Bars (AeC) ¼ 100 mm. Bars (DeF) ¼ 50 mm.
3.5. Cell culture on collagen-coated fibres PAN mats coated with either collagen type I or collagen type III both showed good attachment and significant spreading of cells (Fig. 7B, C). Similar results were also obtained with PLGA fibres where coating with collagen, including reversibly modified collagens also led to good attachment and significant cell spreading with good cell viability (Fig. 8B,C). In the present study cell binding to PAN and NaOH modified PAN mats and to unmodified PLGA was poor (Figs. 7A,8A). However, for some core fibres, such as gelatin, the cell binding could be as good as, or better than with collagen [42]. These cell culture data were further confirmed by scanning electron microscopy of the cells after 16 h (Figs. 7 and 8). Cells
grown on non-collagen-coated surfaces all had a rounded morphology. On the other hand, cells grown on collagen-modified materials typically showed an elongated and frequently flattened morphology. This was the case equally for samples with only a few or many collagen coating layers and for both PAN and PLGAbased constructs (Figs. 7 and 8). These data show that the modified collagens do not lead to any cytotoxicity. Previously, anionic collagen, also prepared by alkaline deamidation, had been shown to perform acceptably in bone healing [43]. In the present study, the polymer fibres have been used without additives. The progress of cell growth on the collagen-coated fibres could be modulated if required through incorporation of growth factors or low molecular weight compounds within the electrospun fibre base [44,45].
Fig. 8. Examination of L-929 fibroblasts growing on PLGA fibres coated with 7 alternating layers of various collagen combinations. (A) Live/dead staining of cells on fibres with no collagen coating, (B) Live/dead staining of cells on fibres coated with type I collagen and succinylated type I collagen, (C) Live/dead staining of cells on fibres coated with type I collagen and citraconylated type I collagen. (D) SEM of cells on fibres coated with type I collagen and succinylated type I collagen, (E) SEM of cells on fibres coated with type I collagen and citraconylated type I collagen, (F) SEM of cells on fibres coated with type I collagen and maleylated type I collagen. Bars (AeC) ¼ 100 mm. Bars (DeF) ¼ 20 mm.
9204
Y.B. Truong et al. / Biomaterials 33 (2012) 9198e9204
4. Conclusion The present study has shown that layer-by-layer coating of an electrospun fibre network with alternating layers of triple-helical anionic and cationic collagens provides an effective method for producing homologous collagen coatings. The approach uses nondenaturing conditions, so the collagen retains its native triple helical structure and is not converted to gelatin. The electrospun base can be made from various materials, including both resorbable and non-resorbable polymers. The collagen coated fibres in this study were found to provide a good substrate for cell adhesion and spreading, while maintaining dimensional stability. The approach has the potential to be used on other surface types. This system provides an alternative approach for producing collagen-based microfibre materials for biomedical applications, such as wound dressings or adhesion barriers, and for use in tissue engineering applications. Acknowledgements K.B. was in part supported through a Research Experience for Undergraduates (REU) supplement to the NSF CAREER award CBET0748062 (S.Z.). References [1] Ramshaw JA, Werkmeister JA, Glattauer V. Collagen-based biomaterials. Biotechnol Genet Eng Rev 1996;13:335e82. [2] Ramshaw JA, Peng YY, Glattauer V, Werkmeister JA. Collagens as biomaterials. J Mater Sci Mater Med 2009;20(Suppl. 1):S3e8. [3] Brodsky B, Ramshaw JA. The collagen triple-helix structure. Matrix Biol 1997; 15:545e54. [4] Benguigui L, Busnel JP, Durand D. Study of junction zones in gelatin gels through selective enzymatic digestion. Polymer 1991;32:2680e5. [5] Reneker DH, Chun I. Nanometre diameter fibres of polymer, produced by electrospinning. Nanotechnology 1996;7:216e23. [6] Sill TJ, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 2008;29:1989e2006. [7] Holzwarth JM, Ma PX. Biomimetic nanofibrous scaffolds for bone tissue engineering. Biomaterials 2011;32:9622e9. [8] Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules 2002;3:232e8. [9] Zhong S, Teo WE, Zhu X, Beuerman RW, Ramakrishna S, Yung LY. An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture. J Biomed Mater Res A 2006;79:456e63. [10] Yang L, Fitié CF, van der Werf KO, Bennink ML, Dijkstra PJ, Feijen J. Mechanical properties of single electrospun collagen type I fibers. Biomaterials 2008;29: 955e62. [11] Zeugolis DI, Khew ST, Yew ES, Ekaputra AK, Tong YW, Yung LY, et al. Biomaterials 2008;29:2293e305. [12] Tillman BW, Yazdani SK, Lee SJ, Geary RL, Atala A, Yoo JJ. The in vivo stability of electrospun polycaprolactoneecollagen scaffolds in vascular reconstruction. Biomaterials 2009;30:583e8. [13] Buttafoco L, Kolkman NG, Engbers-Buijtenhuijs P, Poot AA, Dijkstra PJ, Vermes I, et al. Electrospinning of collagen and elastin for tissue engineering applications. Biomaterials 2006;27:724e34. [14] Dong B, Arnoult O, Smith ME, Wnek GE. Electrospinning of collagen nanofiber scaffolds from benign solvents. Macromol Rapid Commun 2009;30:539e42. [15] Feng ZQ, Lu HJ, Leach MK, Huang NP, Wang YC, Liu CJ, et al. The influence of type-I collagen-coated PLLA aligned nanofibers on growth of blood outgrowth endothelial cells. Biomed Mater 2010;5:065011. [16] Chen JP, Li SF, Chiang YP. Bioactive collagen-grafted poly-L-lactic acid nanofibrous membrane for cartilage tissue engineering. J Nanosci Nanotechnol 2010;10:5393e8. [17] Duan Y, Wang Z, Yan W, Wang S, Zhang S, Jia J. Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties. J Biomater Sci Polym Ed 2007;18:1153e64. [18] Peng YY, Glattauer V, Werkmeister JA, Ramshaw JAM. Evaluation of collagen products for cosmetic application. J Cosmet Sci 2004;55:327e41.
[19] Grant GGS, Koktysh DS, Yun B, Matts RL, Kotov RL. Layer-by-layer assembly of collagen thin films: controlled thickness and biocompatibility. Biomed Microdevices 2001;3:301e6. [20] Zhang J, Senger B, Vautier D, Picart C, Schaaf P, Voegel JC, et al. Natural polyelectrolyte films based on layer-by layer deposition of collagen and hyaluronic acid. Biomaterials 2005;26:3353e61. [21] Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol 1982;82(Pt A):33e64. [22] Trelstad RL, Catanese VM, Rubin DF. Collagen fractionation: separation of native types I, II and III by differential precipitation. Anal Biochem 1976;71: 114e8. [23] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680e5. [24] Sykes B, Puddle B, Francis M, Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun 1976;72:1472e80. [25] Rauterberg J, Kühn K. The renaturation behaviour of modified collagen molecules. Hoppe Seylers Z Physiol Chem 1968;349:611e22. [26] Butler PJ, Harris JI, Hartley BS, Leberman R. Reversible blocking of peptide amino groups by maleic anhydride. Biochem J 1967;103:78Pe9P. [27] Dixon HB, Perham RN. Reversible blocking of amino groups with citraconic anhydride. Biochem J 1968;109:312e4. [28] Truong YB, Glattauer V, Lang G, Hands K, Kyratzis IL, Werkmeister JA, et al. A comparison of the effects of fibre alignment of smooth and textured fibres in electrospun membranes on fibroblast cell adhesion. Biomed Mater 2010;5: 25005. [29] Gupta ML, Gupta B, Oppermann W, Hardtmann G. Surface modification of polyacrylonitrile staple fibers via alkaline hydrolysis for superabsorbent applications. J Appl Polym Sci 2004;91:3127e33. [30] Werkmeister JA, Ramshaw JA, Ellender G. Characterisation of a monoclonal antibody against native human type I collagen. Eur J Biochem 1990;187: 439e43. [31] ChandraRajan J. Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation. Biochem Biophys Res Commun 1978;83:180e6. [32] Zhang Z, Liu W, Li D, Li G. Physicochemical properties of succinylated calfskin pepsin-solubilized collagen. Biosci Biotechnol Biochem 2007;71: 2057e60. [33] Werkmeister JA, Ramshaw JA. Recombinant protein scaffolds for tissue engineering. Biomed Mater 2012;7:012002. [34] Rasmussen M, Jacobsson M, Björck L. Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins. J Biol Chem 2003;278:32313e6. [35] Xu C, Yu Z, Inouye M, Brodsky B, Mirochnitchenko O. Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability. Biomacromolecules 2010;11: 348e56. [36] Moroni L, Licht R, de Boer J, de Wijin JR, van Blitterwijk CA. Fiber diameter and texture of electrospun PEOT/PBT scaffolds influence human mesenchymal stem cell proliferation and morphology, and the release of incorporated compounds. Biomaterials 2006;27:4911e22. [37] Jin HJ, Fridrikh SV, Rutledge GC, Kaplan DL. Electrospinning Bombyx mori silk with poly(ethylene oxide). Biomacromolecules 2002;3:1233e9. [38] Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang ZM. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res B Appl Biomater 2005;72:156e65. [39] Rnjak-Kovacina J, Wise SG, Li Z, Maitz PK, Young CJ, Wang Y, et al. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. Biomaterials 2011;32:6729e36. [40] Ramshaw JAM, Glattauer V, Werkmeister JA. Stabilisation of collagen in clinical applications. In: Wise DL, editor. Handbook of biomaterials engineering. New York: Marcel Dekker Inc; 2000. p. 717e38. [41] Hartley BS. Strategy and tactics in protein chemistry. Biochem J 1970;119: 805e22. [42] Abraham LC, Zuena E, Perez-Ramirez B, Kaplan DL. Guide to collagen characterization for biomaterial studies. J Biomed Mater Res B Appl Biomater 2008;87:264e85. [43] Rocha LB, Goissis G, Rossi MA. Biocompatibility of anionic collagen matrix as scaffold for bone healing. Biomaterials 2002;23:449e56. [44] Ji W, Sun Y, Yang F, van den Beucken JJ, Fan M, Chen Z, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm Res 2011;28:1259e72. [45] Meinel AJ, Germershaus O, Luhmann T, Merkle HP, Meinel L. Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications. Eur J Pharm Biopharm 2012;81:1e13.