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Biofunctionalization of collagen for improved biological response: Scaffolds for corneal tissue engineering
ARTICLE IN PRESS
Biomaterials 28 (2007) 78–88 www.elsevier.com/locate/biomaterials
Biofunctionalization of collagen for improved biological response: Scaffolds for corneal tissue engineering Xiaodong Duana, Christopher McLaughlinb, May Griffithb, Heather Sheardowna, a
Department of Chemical Engineering, McMaster University, 1280 Main St. W., Hamilton, Ont., Canada L8S 4L7 b Department of Ophthalmology, University of Ottawa, 501 Smyth Rd., Ottawa, Ont., Canada Received 5 June 2006; accepted 19 August 2006 Available online 7 September 2006
Abstract Residual dendrimer amine groups were modified with incorporate COOH group containing biomolecules such as cell adhesion peptides into collagen scaffolds. YIGSR, as a model cell adhesion peptide, was incorporated into both the bulk structure of the gels and onto the gel surface. The effects of the peptide modified collagen gels on corneal epithelial cell behavior were examined with an aim of improving the potential of these materials as tissue-engineering scaffolds. YIGSR was first chemically attached to dendrimers and the YIGSR attached dendrimers were then used as collagen crosslinkers, incorporating the peptide into the bulk structure of the collagen gels. YIGSR was also attached to the surface of dendrimer crosslinked collagen gels through reaction with excess amine groups. The YIGSR modified dendrimers were characterized by H-NMR and MALDI mass spectra. The amount of YIGSR incorporated into collagen gels was determined by 125I radiolabelling at maximum to be 3.1–3.4 102 mg/mg collagen when reacted with the bulk and 88.9–95.6 mg/cm2 when attached to the surface. The amount of YIGSR could be tuned by varying the amount of peptide reacted with the dendrimer or the amount of modified dendrimer used in the crosslinking reaction. It was found that YIGSR incorporation into the bulk and YIGSR modification of surface promoted the adhesion and proliferation of human corneal epithelial cells as well as neurite extension from dorsal root ganglia. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cornea; Collagen; Dendrimers; YIGSR; Nerve outgrowth; Epithelialization
1. Introduction The extracellular matrix (ECM) is the natural scaffold for the cells, acting as a mechanical support and creating a microenvironment to which the cells can respond. Constructing a matrix or scaffold that simulates the ECM environment is therefore desirable and a widely used strategy in tissue engineering. Such a scaffold has the potential to promote cell growth and to restore key functions to damaged tissues and organs. To mimic the high proportion of collagen present in most native tissues, collagen scaffolds are widely used in tissue engineering. However, the biological function of these tissues is in large part due to the presence of other extracellular components. Corresponding author. Tel.: +1 905 525 9140x24794; fax: +1 905 521 1350. E-mail address: [email protected] (H. Sheardown).
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.08.034
Among the macromolecules present in the corneal ECM, laminin, a complex trimer glycoprotein and a major component of the basement membrane, is important for corneal epithelial adhesion and has a neurite-promoting activity, stimulating Schwann cell mitosis [1]. The presence of a confluent corneal epithelial layer and its integration with functional nerves in a tissue-engineered cornea are essential for success of the device and for vision restoration, by promoting tear production and corneal sensitivity, while maintaining a barrier to tear and airborne pathogens [2]. The specific amino acid sequences involved in receptor interactions with laminin include the RGD and IKVAV sequences of the a-chain, YIGSR of the b1-chain, and RNIAEIIKDI of the g-chain [3]. In tissue-engineering applications, laminin itself has been previously used to promote neurite growth [4]. The YIGSR sequence of laminin has been incorporated into tissue-engineering scaffold materials to promote peripheral [1], and central
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[5] nerve regeneration. In corneal applications, YIGSR grafted to a collagen–acrylic copolymer scaffold has been shown to promote human corneal epithelial stratification and neurite ingrowth [2]. Previously we have reported on crosslinking of collagen solutions with multifunctional dendrimers, obtaining transparent, mechanically stronger collagen hydrogels with high crosslinking densities that were able to support corneal epithelial cell adhesion [6,7]. While the dynamic interactions of collagen scaffolds with the surrounding biological environment in vivo make it desirable to incorporate additional biological functionality into a crosslinked collagen matrix in the form of cell adhesion molecules like peptides and growth factors, most currently available crosslinking technologies will not permit functionalization of the matrix without potentially altering the biological properties of the collagen itself. However, presence of excess of amine groups on the dendrimers used for collagen crosslinking can be further exploited to graft biomolecules without significantly altering the crosslinking density or the biological properties of the collagen matrices. Grafting of biomolecules into the hydrogels will further enhance the potential of these materials as tissueengineering scaffolds. Our objective in the current work was therefore to incorporate the laminin-based peptide YIGSR, as a model peptide, into collagen scaffolds by way of the dendrimer crosslinkers, and to examine the effects of the incorporated peptide on corneal epithelial cell behavior and nerve regeneration. 2. Materials and methods All the reagents used were purchased from Sigma Aldrich (Oakville, Ont.) except when otherwise specified.
2.1. Covalent attachment of YIGSR to dendrimers YIGSR was added to aqueous solutions of generation 2 polypropyleneimine octamine dendrimers (Fig. 1) containing 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and the mixture reacted overnight at room temperature with stirring. The molar ratio of YIGSR to dendrimer was 1:1, meaning that the number of NH2 groups for covalent attachment of the peptide was in significant excess leaving significant amounts of residual amine groups that could be used for collagen crosslinking. A ratio of 5:5:1 EDC:NHS:COOH of YIGSR was used. The YIGSR modified dendrimer product was purified by dialysis using Spectro/Por membranes (MWCO 500, Spectrum Medical Industries Inc., Houston TX, USA) against water for 2 days. The purified product was freeze dried for characterization or further reaction.
2.2. Characterization of YIGSR attached dendrimers The purified YIGSR modified dendrimer (YIGSR-m-dendrimer) was reconstituted into deuterated water for H-NMR analysis. Spectra for the dendrimer, YIGSR and YIGSR-m-dendrimer were recorded and the peaks compared. MALDI-TOF (Matrix-Assist Laser Desorption Ionization time-of-flight) mass spectrometry was also used to characterize the dendrimer, YIGSR and YIGSR-m-dendrimer. The Micromass TofSpec 2E MALDI-TOF mass spectrometer was operated in reflectron mode using alpha-cyano-4-hydroxycinnamic acid as the matrix. In reflectron
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NH2 NH2 NH2 N N N N H2N
N
NH2
N
H2N
H2N NH2
Fig. 1. Schematic of a generation 2 polypropyleneimine dendrimer. The number of amine groups increase with increasing generations. mode, an electrostatic mirror bounces the ions back and focuses them at a second detector allowing for better resolution and mass accuracy.
2.3. Collagen gel preparation Concentrated collagen suspensions (6%, in phosphate buffered saline, pH 7.0–7.6), the generous gift of Inamed Corporation (USA), contained predominantly pepsin digested bovine cornium purified type I collagen with less than 20% type III collagen. The 6% suspension was in phosphate buffered saline, pH 7.0–7.6. All of the suspensions were acidified with 1 N HCl and diluted to make clear collagen solutions prior to further treatment. Dendrimer crosslinked collagens were prepared by mixing the collagen solution with an aqueous solution containing EDC, generation 2 polypropyleneimine octaamine dendrimer and NHS (molar ratio of EDC:NHS:COOH ¼ 5:5:1) in pre-cooled syringes on ice. The pH of the solution was adjusted to 5.5, the optimal reaction condition for carbodiimide crosslinking [8] and the solution was injected into glass molds in a 37 1C oven overnight for crosslinking and gelation. Three percent collagen gels and a collagen to dendrimer weight ratio of 10:1 were used throughout this study based on previous results [6]. In all cases, due to the high viscosity of the collagen solutions used for gel preparation, it was necessary to avoid the introduction of air into the mixture as this altered the appearance and mechanical properties of the gels formed. This was achieved by carefully removing air bubbles from the collagen suspensions before they became viscous solutions. YIGSR bulk modified collagen gels were prepared following the same procedure using a combination of dendrimers with chemically attached YIGSR and unmodified dendrimers as crosslinkers. A series of YIGSR modified collagen gels with different amounts of YIGSR were prepared by using various YIGSR-m-dendrimer percentages (100%, 10%, 1%) in the crosslinking solution. Once crosslinked, the gels were removed from the molds and immersed in glycine solution (0.5% in PBS) at room temperature to react with any residual activated carboxylic acid groups and to extract out the NHS reaction product. The final gels were rinsed with PBS at least three times over a period of 12 h to remove any residual reaction products and stored in 4 1C refrigerator until use. Gels for in vitro cell culture studies were prepared under sterile conditions in a class II biosafety cabinet to maintain sterility. All reagents were either autoclaved or sterilized by filtering with 0.2 mm filters.
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2.4. Quantification of YIGSR in collagen gels
2.8. In vitro corneal epithelial cell culture
In order to directly quantify the YIGSR content in the modified collagen gels, YIGSR was radiolabeled with 125I using Iodogen method [9]. The labeled YIGSR was purified by dialysis against water using Spectra/Por dialysis membranes (MWCO 500). The radioactivity of the dialysate was monitored until no further free iodide was detected. YIGSR solutions containing 10% 125I labeled was used to attach to dendrimer and then collagen gel preparation. These collagen gels were counted in a gamma counter to determine the amount of YIGSR in the gels.
The response of human corneal epithelial cells to the modified surfaces was examined to assess the biological effect of the YIGSR modification. For cell culture, 0.5 cm disks of the sterile gels containing various amounts of YIGSR were pretreated with keratinocyte serum-free medium (KSFM, Invitrogen Life Technologies, Burlington Ont.) containing antibiotics (penicillin/streptomycin 1:100, gentamycin 1:1000). Immortalized human corneal epithelial cells [10], were seeded on the gels at a density of 104 cells per well. The cells, in a small volume of medium (50 ml), were incubated on the surfaces for approximately 15 min. This permitted the cells to adhere to the surfaces and ensured that the cells were not washed off the surface of the disks. Following this, epidermal growth factor-containing keratinocyte serum-free medium was added to cover the surfaces. Medium was replaced every 2 days and the surfaces were examined and photographed daily. To quantify cell adhesion and proliferation, a CYQUANT (Molecular Probes, Invitrogen Life Technolgies, Burlington, Ont.) assay was performed at times of 1,2,3 and 4 days. Adhesion studies were performed at 30, 60, and 120 min post seeding.
2.5. YIGSR surface modification of the collagen gels Dendrimer only crosslinked collagen gels were immersed in an aqueous solution containing EDC, NHS and the YIGSR. Surfaces with varying peptide coverage were prepared by applying solutions with different amounts of peptide. The molar ratio of EDC:NHS:YIGSR remained constant at 5:5:1. The pH of the reaction solution was maintained at 5.5 and the reaction was carried out at room temperature overnight with slight agitation. The modified surfaces were thoroughly rinsed with Milli-Q water to remove unreacted peptides and excess EDC and NHS. Surface density of YIGSR was determined using 125I radiolabeled peptide.
2.6. Surface characterization of modified gels X-ray photoelectron spectroscopy (XPS) analysis was performed with a Leybold MAX 200 XPS System (Cologne, Germany), using a nonmonochromatised Mg Ka X-ray source operating at 15 kV and 20 mA. The spot size used was 2 4 mm. The energy range was calibrated by placing the Au 4f peak at 84 eV or the main C1s peak at 284.5 eV. Survey scans were performed from 0 to 1000 eV, and low resolution and highresolution C1s spectra were obtained at 901 and 201 takeoff angles of the collagen gels before and after YIGSR modification.
2.7. Bulk characterization of dendrimer modified collagen gels Denaturation temperatures of collagen gels were measured by using a TA differential scanning calorimeter (DSC) as previously described [6]. Denaturation temperature may provide information about the crosslinking density of collagen samples before and after YIGSR modification. A heating rate of 5 1C/min was applied over a temperature range from 15 to 100 1C, and the endothermic peak(s) of the thermogram were monitored and recorded. Mechanical properties of collagen gels were examined to test the effects of YIGSR modification. In order to prepare collagen gel samples for Instron testing, a custom designed mold was prepared. A polymer mesh was incorporated in the gel sample in the area where the gels would be gripped in the test in order to make the handling and gripping of the samples in the testing machine easier as well as to provide an accurate measure of the strength of the gel that was unaffected by the grips. The area between the grips was free of mesh so that only the gel was tested. Gel forming solution was poured into the mold, and the mold was placed under two flat glass plates in order to make the samples. A weight was placed on top of the glass plate to ensure solution contact with the mold and the plates; otherwise the gel preparation procedure was as with samples for other tests. Prior to testing, the gels were blotted dry gently with filter paper and mounted on the grips of an Instron Series IX Automated Materials Testing System. A crosshead speed of 5 mm/min and full-scale load range of 500 N were used for the test that was conducted at 23 1C and a humidity of 50%. Young’s modulus, maximum load and displacement at maximum load were recorded as indications of the mechanical properties of the various collagen samples.
2.9. In vitro early nerve in-growth Early nerve in-growth studies were performed using dorsal root ganglia (DRG) from chicken embryo. Collagen gel samples with varying amounts of incorporated YIGSR were sterilized by incubating in 1% chloroform in PBS for 4 days at 4 1C and subsequently washed in PBS followed by PBS containing antibiotics. Low concentration collagen gels were prepared from diluted collagen solutions for initial adhesion of the DRG. The DRG were isolated from chick embryos and separated from fibroblasts as previously described [4]. Selected DRG’s were then dipped into low concentration collagen gels on ice and placed on sample surfaces. Cells were cultured in KSFM (Invitrogen Life Technologies, Burlington, Ont.) supplemented with dexamethasone, dibutryl cyclic adenosine monophosphate (dB cAMP; Sigma), dimethylsulfoxide (DMSO; BDH chemicals). Media was changed every other day. DRG’s were allowed to extend for 5 days. After 5 days of culture, samples were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, Oakville Ont.) in PBS. Fluorescent immunostaining for neurofilament-200 was performed using mouse monoclonal antineurofilament-200 (diluted 1:200) as the primary antibody and fluorescently labeled goat anti-mouse (diluted 1:400) (Amersham Biosciences, Piscataway, NJ) as the secondary antibody. Fluorescent microscopy images were taken of the gels at a magnification of 10 times and a montage was created to show the extension. The numbers of nerves extending 150, 300, 450 and 600 mm were counted as a quantitative measure of neurite extension.
3. Results 3.1. Collagen gel preparation All the dendrimer crosslinked collagen gels before and after YIGSR peptide modification were transparent and strong enough to manipulate. The bulk properties of the gels appeared unaltered when they were stored in PBS/water at 4 1C for at least 8 months. 3.2. Covalent attachment of peptides to dendrimers and characterization YIGSR was attached to dendrimers using the same reaction as was used for dendrimer-mediated collagen crosslinking. The carboxylic acid groups in the peptides were activated by EDC and NHS to form reactive NHS
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esters, which reacted with amine groups in dendrimers to form chemical bonds. The reaction between the dendrimers and the peptides was confirmed by H-NMR and MALDI TOF. H-NMR spectra of dendrimer, YIGSR and YIGSR modified dendrimer are shown in Fig. 2. Specifically characteristic peaks from both the dendrimers (2.29–2.47 ppm) and YIGSR (6.62–6.91 ppm) were found in the purified YIGSR modified dendrimer spectra, which are indicative of successful attachment of YIGSR to dendrimers. The estimated molar ratio of YIGSR:dendrimer was found to be 1:5.4 based on these peaks. Therefore, compared with the initial molar ratio of YIGSR:dendrimer (1:1), it is estimated that only 18.5% of the initial YIGSR present was attached to the dendrimers after reaction and purification. Assuming all the YIGSR modified dendrimers are involved in the crosslinking reaction of collagens, the maximum YIGSR content in the collagen gels would be expected to be 1.6 102 mg/mg collagen. MALDI mass spectra of dendrimer, YIGSR and YIGSR modified dendrimer further confirmed the formation of YIGSR modified dendrimer (Fig. 3). Peaks for the dendrimer (773.7) and YIGSR (595.3) as well as for the YIGSR modified dendrimer (1354.1) were found in the spectra as expected. Additional peaks present (1186, 1086, 1029, 955) are thought to result from deposition of the chemically attached YIGSR due to its thermally labile nature [11]. 3.3. Incorporation of peptides in collagen gels and characterization of peptide modified gels 125
I labeled YIGSR was used to quantify the amount of YIGSR incorporated to collagen gels. It was found that 3.1–3.4 102 mg of YIGSR/mg collagen could be incorporated, suggesting that 24–26% of initial the YIGSR present was attached to collagen, a result consistent with the estimate from the H-NMR analysis of the peptide modified dendrimers. 3.4. Gel characterization Surfaces of collagen gels before and after YIGSR modification were examined by XPS. Not unexpectedly, there were no significant differences (data not shown), which demonstrates that the reaction with the peptide modified dendrimers did not adversely affect the surface properties of the gels. Denaturation temperatures of the collagen gels were determined by DSC to determine whether changes in the crosslinking density occurred with YIGSR modification. Multiple denaturation temperature peaks were found in YIGSR modified collagen samples similar to the previously examined dendrimer crosslinked collagens [6]. As shown in Table 1, overall the number of peaks in the YIGSR modified gels was similar to the unmodified gels, although the highest denaturation temperature was slightly lower with the presence of YIGSR.
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This may potentially indicate possible interference of YIGSR with the dendrimer-mediated crosslinking reaction resulting in a lower crosslinking density. Possibly due to this, slightly poorer mechanical properties in terms of modulus were observed in the YIGSR modified collagens as shown in Fig. 4a. However, they had a similar maximum load to the unmodified dendrimer crosslinked collagens (Fig. 4b). 3.5. Surface modification of collagen gels with YIGSR The surface coverage of YIGSR on the collagen surfaces was found to be in the range of 88.9–95.6 mg/cm2. While this accounts for only 5–6% of the maximal YIGSR coverage calculated theoretically from the availability of amine groups, it is much higher than the densities 2.5 105 mg/cm2 reported previously [12]. 3.6. In vitro corneal epithelial cell culture 3.6.1. YIGSR bulk modified collagens Representative photographs of human corneal epithelial cells on YIGSR bulk modified (1.6 104–1.6 102 mg YIGSR/mg collagen)/unmodified collagen gel surfaces at 120 min are shown in Fig. 5. It was found, not surprisingly, that the cells adhered to all of the collagen surfaces within 30–60 min. Furthermore, morphology changes were observed in all cases. In comparison, the cells did not adhere to the control tissue culture plates in this time frame and remained round and non-adherent after 2 h of culture. The presence of YIGSR resulted in the formation of clusters and visibly greater levels of cell attachment. Over longer periods of time, there was a trend showing that the cells proliferated faster on collagen gels with higher YIGSR content as shown in Figs. 6 and 7. This trend was confirmed by Cyquant assay as shown in Fig. 8. 3.6.2. YIGSR surface modified collagens Similar to the YIGSR bulk modified collagens, the cells adhered to peptide modified collagen surfaces within 60 min and changed morphology. However, over a period of 1 week of culture, there was no significant improvement in the adhesion and growth of the cells on these surfaces relative to the unmodified collagen gels (data not shown). 3.7. DRG neurite extension Neurite extension from DRG cells was found to be significantly enhanced by the presence of the YIGSR in the collagen gels. The length of the neurites and number of neurites, summarized in Fig. 9 was significantly (po0:05) enhanced by the presence of the cell adhesion peptide. Surprisingly, there was little or no effect of peptide concentration in the gel, although it is possible that the surface density of the peptide on these surfaces
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Fig. 2. H-NMR spectra of YIGSR (a), dendrimer G2 (b), and YIGSR modified dendrimer (c). Characteristic peaks from dendrimers (2.29–2.47 ppm) and YIGSR (6.62–6.91 ppm) were found in the purified YIGSR modified dendrimer spectra, which indicated the successful attachment of YIGSR to dendrimers.
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Fig. 3. MALDI-TOF spectra of (a) dendrimer and (b) YIGSR modified dendrimer. Peaks characteristic of the dendrimer and the YIGSR as well as a peak demonstrating reaction between the two species were observed in the modified dendrimer.
Table 1 Collagen sample
YIGSR modified Control
Denaturation temperature (1C) Peak 1
Peak 2
Peak 3
56.0 40.0
59.8 82.0
78.2 89.0
was relatively similar as this was a bulk modification. Visually, it is clear that the nerve density on these materials was also enhanced by the presence of the peptide. Additional studies to assess nerve in-growth into the gels may provide more insight into the effect of peptide concentration in the gel on the neurites (Fig. 10).
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4. Discussion
2.0 Young's Modulus (MPa)
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Displacement (mm)
6
Glutaraldehyde Dendrimer
YIGSR 3.0
Displacement Maximum Load
5
2.5
4
2.0
3
1.5
2
1.0
1
0.5 0.0
0
(b)
Maximum Load (N)
EDC
(a)
EDC
Glutaraldehyde Dendrimer
YIGSR
Fig. 4. Comparison of mechanical properties (a, Young’s modulus; b, Maximum load and displacement at maximum load) of YIGSR modified (6.4 mg/mg collagen) and unmodified collagen samples. Slightly lower modulus was observed in the YIGSR modified collagens. However, they had a similar maximum load to the unmodified dendrimer crosslinked collagens.
The YIGSR sequence of laminin has been studied to improve neurite growth and corneal epithelial cell stratification [2]. In the current work, YIGSR was chemically attached to multifunctional dendrimer and the peptide modified dendrimers were then used as novel crosslinkers for collagens using a combination of unmodified and YIGSR modified dendrimers as crosslinkers. The modification of the dendrimers with the peptide was confirmed by H-NMR and MALDI mass spectra. Although the extent of the overall reaction was not high, with only 18.5–26% of the initial YIGSR attached to the collagens based on H-NMR estimation and 125I radiolabelling studies, the amount of YIGSR in the gels (1.6–3.2 102 mg/mg collagen) was comparatively significantly higher than reported previously (0.0013 mg/mg collagen) [2] and could be easily tuned by crosslinking with fewer peptide modified dendrimers. This demonstrates that the multifunctional dendrimers allowed for the modification of collagen gels with high levels of biological molecules. While this is possibly higher than necessary for significant cell growth improvement, the use of dendrimers in this capacity allows for a tunable amount of biological molecules to be incorporated into collagen gels by simply altering the fraction of peptide modified dendrimers in the crosslinking mixture. Furthermore, combinations of biological molecules can potentially be incorporated in the gels. When the surfaces of the collagen gels were modified with the peptides, the measured surface density was found to be 88.9–95.6 mg/cm2, several orders of magnitude higher than
Fig. 5. HCEC adhesion on YIGSR modified collagen gels after 2 h of culture. The presence of YIGSR resulted in the formation of clusters and visibly greater levels of cell attachment.
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Fig. 6. HCEC proliferation on YIGSR modified collagen gels after 2 days of culture. The cell became confluent on YIGSR modified collagen gels within 2 days.
Fig. 7. HCEC proliferation on YIGSR modified collagen gels after 4 days of culture. No difference was found on all surfaces after the cells became confluent.
the peptide densities reported earlier [12] possibly due to the large amount of amine groups introduced by dendrimers being available for peptide attachment. While there were no differences in the surface chemistry of the modified
gels relative to the unmodified gels as determined by XPS, this is not surprising given the similarity in the structures of the collagen and the peptides. The denaturation temperatures measured for the YIGSR modified gels were slightly
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30000
Cell Number
25000 20000 15000 10000 5000 0
1
2
4
3
Day of Culture 0.0160 mg YIGSR/mg collagen 0.0016 mg YIGSR/mg collagen 0.00016 mg YIGSR/mg collagen unmodified gels
Fig. 8. HCEC proliferation on YIGSR modified collagen gels determined by Cyquant assay. The cells proliferated faster on collagen gels with higher YIGSR content.
lower than those observed on the unmodified gels, indicating that there were lower levels of crosslinking in these gels. However, the differences were relatively small. The slightly lower denaturation temperatures found in YIGSR bulk modified collagens are thought to be due to the interference of the peptide to the crosslinking of collagens with dendrimers. This might also account for the slightly poorer mechanical properties observed in YIGSR bulk modified collagens. Interactions of the modified gels with human corneal epithelial cells were used to characterize the potential of the materials as tissue-engineering scaffolds. Cell adhesion peptides including RGD, YIGSR, IKVAV have been covalently coupled to various biomaterial surfaces/bulk structures, including corneal materials, to improve cellular interactions [1,5,13–15]. The advantage of modification with the cell adhesion peptides rather than immobilization of the entire protein include minimizing potential for immunologic reactions and protein denaturation as well as maximizing surface uniformity and density since smaller
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Number of Neurons
30 25
150 µm 300 µm 450 µm 600 µm
20 15 10 5 0 Dendrimer
0.064 YIGSR 0.64 YIGSR Surface
6.4 YIGSR
Fig. 9. DRG neurite extension on YIGSR modified collagen gels compared with unmodified control. The length of the neurites and number of neuritis was significantly (po0:05) enhanced by the presence of the cell adhesion peptide.
Fig. 10. DRG nerve cell in-growth on unmodified control (left) and YIGSR modified (right, 6.4 mg/mg collagen) collagen gels. Neurites extended longer on YIGSR modified collagens.
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molecular peptides are more accessible to the receptors (such as integrins) on the cell surfaces [16–18]. Collagen itself was found to be a compatible scaffold for corneal epithelial cells growth when properly strengthened by dendrimer crosslinking. With the help of incorporated YIGSR either in bulk or on surface, the epithelial cells tended to form more clusters and attach preferably to the collagen surfaces in the short term. Over longer periods of in vitro culture, the cells grew more rapidly on YIGSR bulk modified collagen gel surfaces and became confluent earlier than on unmodified collagens. However, no difference was noted on different collagen gels after the cells became confluent. Incorporation of the laminin-derived peptide, YIGSR, was also found to significantly promote neurite extension from DRG. Both the nerve density and the length of the extended neurites were increased. However, there was no obvious trend with increasing peptide concentration, potentially due to the fact that the peptide concentration in the gels was higher than needed to promote neurite extension or that the differences on peptide concentration on the gel surfaces were relatively small. While it would be preferable to assess nerve ingrowth into these materials, these preliminary studies, similar to those performed by other groups [19,20], clearly demonstrate the potential of these materials for the support of nerves. Ongoing nerve ingrowth studies may provide additional insight into the effect of peptide concentration on innervation of tissueengineering scaffolds. Ongoing work is also examining the effect of such combination peptides as YIGSRIKVAV and IKVAVYIGSR, which have been reported [5] to further enhance nerve in-growth. 5. Conclusions The YIGSR peptide sequence of laminin was either chemically incorporated into the bulk structure of collagen gels or attached on collagen gel surfaces by way of dendrimers. The structure of YIGSR modified dendrimer was confirmed by H-NMR, MALDI mass spectra and the amount of YIGSR incorporated in collagen was determined by 125I radiolabelling. The incorporation reaction was carried out under mild aqueous conditions at room temperature and the amount of peptide incorporated can be tuned by varying reaction conditions such as the percentage of peptide modified dendrimers in crosslinker solutions for the collagens. The crosslinking density of the collagen gels was slightly affected by the incorporated YIGSR, resulting in small decreases of the modulus of the gels. However, overall the mechanical properties of the gels did not deteriorate significantly. The incorporated YIGSR peptide promoted the growth of the corneal epithelial cells on collagen gel surfaces in both terms of adhesion and proliferation. As well, neurite extension and nerve cell density was enhanced on these materials relative to unmodified or control peptide modified controls, although no effect of peptide concentration was observed. Materials
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with additional biological function have significant potential as tissue-engineering scaffolds. The ability to tune the biological function through the dendrimer crosslinker can be used to develop a variety of collagen-based tissueengineering scaffolds. Acknowledgments The authors acknowledge funding support from the Natural Sciences and Engineering Research Council of Canada. Inamed Corporation is acknowledged for providing the concentrated collagen suspensions. References [1] Itoh S, Takakuda K, Samejima H, Ohta T, Shinomiya K, Ichinose S. Synthetic collagen fibers coated with a synthetic peptide containing the YIGSR sequence of laminin to promote peripheral nerve regeneration in vivo. J Mater Sci: Mater Med 1999;10:129–34. [2] Li F, Carlsson D, Lohmann C, Suuronen E, Vascotto S, Kobuch K, et al. Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation. Proc Natl Acad Sci USA 2003;100:15346–51. [3] Meiners S, Mercado M. Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors. Mol Neurol 2002;27:177–95. [4] Suuronen EJ, Nakamura M, Watsky MA, Stys PK, Muller L, Munger R, et al. Innervated human corneal equivalents as in vitro models for nerve–target cell interatctions. FASEB J 2004;18: 170–2. [5] Tong Y, Shoichet M. Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers. Biomaterials 2001;22:1029–34. [6] Duan X, Sheardown H. Crosslinking of collagen with dendrimers. J Biomed Mater Res A 2005;75:510–8. [7] Duan X, Sheardown H. Dendrimer crosslinked collagen as a corneal tissue engineering scaffold: mechanical properties and corneal epithelial cell interactions. Biomaterials 2006;27:4608–17. [8] Olde Damink LHH, Dijkstra PJ, van Luyn MJA, van Wachem PB, Nieuwnehuis P, Feijen J. Cross-linking of dermal sheep collagen using a water-soluble carbodiimide. Biomaterials 1996;17: 765–73. [9] Behr TM, Gotthardt M, Becker W, Behe M. Radioiodination of monoclonal antibodies, proteins and peptides for diagnosis and therapy. Neuk Med 2002;41:71–9. [10] Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NLC, et al. Functional corneal equivalents constructed from cell lines. Science 1999;286:2169–72. [11] Covey TR, Huang EC, Henion JD. Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions. Anal Chem 1991;63:1193–200. [12] Shaw D, Shoichet MS. Toward spinal cord injury repair strategies: peptide surface modification of expanded poly(tetrafluoroethylene) fibers for guided neurite outgrowth in vitro. J Cranio Surg 2003;14: 308–16. [13] Schense JC, Bloch J, Aebischer P, Hubbell JA. Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension. Nat Biotech 2000;18:415–9. [14] Merrett K, Griffith CM, Deslandes Y, Pleizier G, Sheardown H. Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces. J Biomater Sci Polym Edn 2001;12:647–71. [15] Aucoin L, Griffith CM, Pleizier G, Deslandes Y, Sheardown H. Interactions of corneal epithelial cells and surfaces modified with cell
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adhesion peptide combinations. J Biomater Sci Polym Edn 2002; 13:447–62. [16] Hubbell JA. Bioactive biomaterials. Curr Opin Biotech 1999;10:123–9. [17] Lin HB, Lewis KB, Leach-Scampavia D, Ratner BD, Cooper SL. Surface properties of RGD-peptide grafted polyurethane block copolymers: variable take-off angle and cold-stage ESCA studies. J Biomater Sci Polym Edn 1993;4:183–98. [18] Massia SP, Rao SS, Hubbell JA. Covalently immobilized laminin peptide Tyr–Ile–Gly–Ser–Arg (YIGSR) supports cell spreading
and co-localization of the 67-kilodalton laminin receptor with alpha-actinin and vinculin. J Biol Chem 1993;268:8053–9. [19] Liu Y, Gan L, Carlsson DJ, Fagerholm P, Lagali N, Watsky MA, et al. A simple, cross-linked collagen tissue substitute for corneal implantation. Invest Ophthalmol Vis Sci 2006;47:1869–75. [20] Liu Y, Griffith M, Watsky MA, Forrester JV, Kuffova L, Grant D, et al. Properties of porcine and recombinant human collagen matrices for optically clear tissue engineering applications. Biomacromolecules 2006;7:1819–28.
Biomaterials 28 (2007) 78–88 www.elsevier.com/locate/biomaterials
Biofunctionalization of collagen for improved biological response: Scaffolds for corneal tissue engineering Xiaodong Duana, Christopher McLaughlinb, May Griffithb, Heather Sheardowna, a
Department of Chemical Engineering, McMaster University, 1280 Main St. W., Hamilton, Ont., Canada L8S 4L7 b Department of Ophthalmology, University of Ottawa, 501 Smyth Rd., Ottawa, Ont., Canada Received 5 June 2006; accepted 19 August 2006 Available online 7 September 2006
Abstract Residual dendrimer amine groups were modified with incorporate COOH group containing biomolecules such as cell adhesion peptides into collagen scaffolds. YIGSR, as a model cell adhesion peptide, was incorporated into both the bulk structure of the gels and onto the gel surface. The effects of the peptide modified collagen gels on corneal epithelial cell behavior were examined with an aim of improving the potential of these materials as tissue-engineering scaffolds. YIGSR was first chemically attached to dendrimers and the YIGSR attached dendrimers were then used as collagen crosslinkers, incorporating the peptide into the bulk structure of the collagen gels. YIGSR was also attached to the surface of dendrimer crosslinked collagen gels through reaction with excess amine groups. The YIGSR modified dendrimers were characterized by H-NMR and MALDI mass spectra. The amount of YIGSR incorporated into collagen gels was determined by 125I radiolabelling at maximum to be 3.1–3.4 102 mg/mg collagen when reacted with the bulk and 88.9–95.6 mg/cm2 when attached to the surface. The amount of YIGSR could be tuned by varying the amount of peptide reacted with the dendrimer or the amount of modified dendrimer used in the crosslinking reaction. It was found that YIGSR incorporation into the bulk and YIGSR modification of surface promoted the adhesion and proliferation of human corneal epithelial cells as well as neurite extension from dorsal root ganglia. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cornea; Collagen; Dendrimers; YIGSR; Nerve outgrowth; Epithelialization
1. Introduction The extracellular matrix (ECM) is the natural scaffold for the cells, acting as a mechanical support and creating a microenvironment to which the cells can respond. Constructing a matrix or scaffold that simulates the ECM environment is therefore desirable and a widely used strategy in tissue engineering. Such a scaffold has the potential to promote cell growth and to restore key functions to damaged tissues and organs. To mimic the high proportion of collagen present in most native tissues, collagen scaffolds are widely used in tissue engineering. However, the biological function of these tissues is in large part due to the presence of other extracellular components. Corresponding author. Tel.: +1 905 525 9140x24794; fax: +1 905 521 1350. E-mail address: [email protected] (H. Sheardown).
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.08.034
Among the macromolecules present in the corneal ECM, laminin, a complex trimer glycoprotein and a major component of the basement membrane, is important for corneal epithelial adhesion and has a neurite-promoting activity, stimulating Schwann cell mitosis [1]. The presence of a confluent corneal epithelial layer and its integration with functional nerves in a tissue-engineered cornea are essential for success of the device and for vision restoration, by promoting tear production and corneal sensitivity, while maintaining a barrier to tear and airborne pathogens [2]. The specific amino acid sequences involved in receptor interactions with laminin include the RGD and IKVAV sequences of the a-chain, YIGSR of the b1-chain, and RNIAEIIKDI of the g-chain [3]. In tissue-engineering applications, laminin itself has been previously used to promote neurite growth [4]. The YIGSR sequence of laminin has been incorporated into tissue-engineering scaffold materials to promote peripheral [1], and central
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[5] nerve regeneration. In corneal applications, YIGSR grafted to a collagen–acrylic copolymer scaffold has been shown to promote human corneal epithelial stratification and neurite ingrowth [2]. Previously we have reported on crosslinking of collagen solutions with multifunctional dendrimers, obtaining transparent, mechanically stronger collagen hydrogels with high crosslinking densities that were able to support corneal epithelial cell adhesion [6,7]. While the dynamic interactions of collagen scaffolds with the surrounding biological environment in vivo make it desirable to incorporate additional biological functionality into a crosslinked collagen matrix in the form of cell adhesion molecules like peptides and growth factors, most currently available crosslinking technologies will not permit functionalization of the matrix without potentially altering the biological properties of the collagen itself. However, presence of excess of amine groups on the dendrimers used for collagen crosslinking can be further exploited to graft biomolecules without significantly altering the crosslinking density or the biological properties of the collagen matrices. Grafting of biomolecules into the hydrogels will further enhance the potential of these materials as tissueengineering scaffolds. Our objective in the current work was therefore to incorporate the laminin-based peptide YIGSR, as a model peptide, into collagen scaffolds by way of the dendrimer crosslinkers, and to examine the effects of the incorporated peptide on corneal epithelial cell behavior and nerve regeneration. 2. Materials and methods All the reagents used were purchased from Sigma Aldrich (Oakville, Ont.) except when otherwise specified.
2.1. Covalent attachment of YIGSR to dendrimers YIGSR was added to aqueous solutions of generation 2 polypropyleneimine octamine dendrimers (Fig. 1) containing 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and the mixture reacted overnight at room temperature with stirring. The molar ratio of YIGSR to dendrimer was 1:1, meaning that the number of NH2 groups for covalent attachment of the peptide was in significant excess leaving significant amounts of residual amine groups that could be used for collagen crosslinking. A ratio of 5:5:1 EDC:NHS:COOH of YIGSR was used. The YIGSR modified dendrimer product was purified by dialysis using Spectro/Por membranes (MWCO 500, Spectrum Medical Industries Inc., Houston TX, USA) against water for 2 days. The purified product was freeze dried for characterization or further reaction.
2.2. Characterization of YIGSR attached dendrimers The purified YIGSR modified dendrimer (YIGSR-m-dendrimer) was reconstituted into deuterated water for H-NMR analysis. Spectra for the dendrimer, YIGSR and YIGSR-m-dendrimer were recorded and the peaks compared. MALDI-TOF (Matrix-Assist Laser Desorption Ionization time-of-flight) mass spectrometry was also used to characterize the dendrimer, YIGSR and YIGSR-m-dendrimer. The Micromass TofSpec 2E MALDI-TOF mass spectrometer was operated in reflectron mode using alpha-cyano-4-hydroxycinnamic acid as the matrix. In reflectron
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NH2 NH2 NH2 N N N N H2N
N
NH2
N
H2N
H2N NH2
Fig. 1. Schematic of a generation 2 polypropyleneimine dendrimer. The number of amine groups increase with increasing generations. mode, an electrostatic mirror bounces the ions back and focuses them at a second detector allowing for better resolution and mass accuracy.
2.3. Collagen gel preparation Concentrated collagen suspensions (6%, in phosphate buffered saline, pH 7.0–7.6), the generous gift of Inamed Corporation (USA), contained predominantly pepsin digested bovine cornium purified type I collagen with less than 20% type III collagen. The 6% suspension was in phosphate buffered saline, pH 7.0–7.6. All of the suspensions were acidified with 1 N HCl and diluted to make clear collagen solutions prior to further treatment. Dendrimer crosslinked collagens were prepared by mixing the collagen solution with an aqueous solution containing EDC, generation 2 polypropyleneimine octaamine dendrimer and NHS (molar ratio of EDC:NHS:COOH ¼ 5:5:1) in pre-cooled syringes on ice. The pH of the solution was adjusted to 5.5, the optimal reaction condition for carbodiimide crosslinking [8] and the solution was injected into glass molds in a 37 1C oven overnight for crosslinking and gelation. Three percent collagen gels and a collagen to dendrimer weight ratio of 10:1 were used throughout this study based on previous results [6]. In all cases, due to the high viscosity of the collagen solutions used for gel preparation, it was necessary to avoid the introduction of air into the mixture as this altered the appearance and mechanical properties of the gels formed. This was achieved by carefully removing air bubbles from the collagen suspensions before they became viscous solutions. YIGSR bulk modified collagen gels were prepared following the same procedure using a combination of dendrimers with chemically attached YIGSR and unmodified dendrimers as crosslinkers. A series of YIGSR modified collagen gels with different amounts of YIGSR were prepared by using various YIGSR-m-dendrimer percentages (100%, 10%, 1%) in the crosslinking solution. Once crosslinked, the gels were removed from the molds and immersed in glycine solution (0.5% in PBS) at room temperature to react with any residual activated carboxylic acid groups and to extract out the NHS reaction product. The final gels were rinsed with PBS at least three times over a period of 12 h to remove any residual reaction products and stored in 4 1C refrigerator until use. Gels for in vitro cell culture studies were prepared under sterile conditions in a class II biosafety cabinet to maintain sterility. All reagents were either autoclaved or sterilized by filtering with 0.2 mm filters.
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2.4. Quantification of YIGSR in collagen gels
2.8. In vitro corneal epithelial cell culture
In order to directly quantify the YIGSR content in the modified collagen gels, YIGSR was radiolabeled with 125I using Iodogen method [9]. The labeled YIGSR was purified by dialysis against water using Spectra/Por dialysis membranes (MWCO 500). The radioactivity of the dialysate was monitored until no further free iodide was detected. YIGSR solutions containing 10% 125I labeled was used to attach to dendrimer and then collagen gel preparation. These collagen gels were counted in a gamma counter to determine the amount of YIGSR in the gels.
The response of human corneal epithelial cells to the modified surfaces was examined to assess the biological effect of the YIGSR modification. For cell culture, 0.5 cm disks of the sterile gels containing various amounts of YIGSR were pretreated with keratinocyte serum-free medium (KSFM, Invitrogen Life Technologies, Burlington Ont.) containing antibiotics (penicillin/streptomycin 1:100, gentamycin 1:1000). Immortalized human corneal epithelial cells [10], were seeded on the gels at a density of 104 cells per well. The cells, in a small volume of medium (50 ml), were incubated on the surfaces for approximately 15 min. This permitted the cells to adhere to the surfaces and ensured that the cells were not washed off the surface of the disks. Following this, epidermal growth factor-containing keratinocyte serum-free medium was added to cover the surfaces. Medium was replaced every 2 days and the surfaces were examined and photographed daily. To quantify cell adhesion and proliferation, a CYQUANT (Molecular Probes, Invitrogen Life Technolgies, Burlington, Ont.) assay was performed at times of 1,2,3 and 4 days. Adhesion studies were performed at 30, 60, and 120 min post seeding.
2.5. YIGSR surface modification of the collagen gels Dendrimer only crosslinked collagen gels were immersed in an aqueous solution containing EDC, NHS and the YIGSR. Surfaces with varying peptide coverage were prepared by applying solutions with different amounts of peptide. The molar ratio of EDC:NHS:YIGSR remained constant at 5:5:1. The pH of the reaction solution was maintained at 5.5 and the reaction was carried out at room temperature overnight with slight agitation. The modified surfaces were thoroughly rinsed with Milli-Q water to remove unreacted peptides and excess EDC and NHS. Surface density of YIGSR was determined using 125I radiolabeled peptide.
2.6. Surface characterization of modified gels X-ray photoelectron spectroscopy (XPS) analysis was performed with a Leybold MAX 200 XPS System (Cologne, Germany), using a nonmonochromatised Mg Ka X-ray source operating at 15 kV and 20 mA. The spot size used was 2 4 mm. The energy range was calibrated by placing the Au 4f peak at 84 eV or the main C1s peak at 284.5 eV. Survey scans were performed from 0 to 1000 eV, and low resolution and highresolution C1s spectra were obtained at 901 and 201 takeoff angles of the collagen gels before and after YIGSR modification.
2.7. Bulk characterization of dendrimer modified collagen gels Denaturation temperatures of collagen gels were measured by using a TA differential scanning calorimeter (DSC) as previously described [6]. Denaturation temperature may provide information about the crosslinking density of collagen samples before and after YIGSR modification. A heating rate of 5 1C/min was applied over a temperature range from 15 to 100 1C, and the endothermic peak(s) of the thermogram were monitored and recorded. Mechanical properties of collagen gels were examined to test the effects of YIGSR modification. In order to prepare collagen gel samples for Instron testing, a custom designed mold was prepared. A polymer mesh was incorporated in the gel sample in the area where the gels would be gripped in the test in order to make the handling and gripping of the samples in the testing machine easier as well as to provide an accurate measure of the strength of the gel that was unaffected by the grips. The area between the grips was free of mesh so that only the gel was tested. Gel forming solution was poured into the mold, and the mold was placed under two flat glass plates in order to make the samples. A weight was placed on top of the glass plate to ensure solution contact with the mold and the plates; otherwise the gel preparation procedure was as with samples for other tests. Prior to testing, the gels were blotted dry gently with filter paper and mounted on the grips of an Instron Series IX Automated Materials Testing System. A crosshead speed of 5 mm/min and full-scale load range of 500 N were used for the test that was conducted at 23 1C and a humidity of 50%. Young’s modulus, maximum load and displacement at maximum load were recorded as indications of the mechanical properties of the various collagen samples.
2.9. In vitro early nerve in-growth Early nerve in-growth studies were performed using dorsal root ganglia (DRG) from chicken embryo. Collagen gel samples with varying amounts of incorporated YIGSR were sterilized by incubating in 1% chloroform in PBS for 4 days at 4 1C and subsequently washed in PBS followed by PBS containing antibiotics. Low concentration collagen gels were prepared from diluted collagen solutions for initial adhesion of the DRG. The DRG were isolated from chick embryos and separated from fibroblasts as previously described [4]. Selected DRG’s were then dipped into low concentration collagen gels on ice and placed on sample surfaces. Cells were cultured in KSFM (Invitrogen Life Technologies, Burlington, Ont.) supplemented with dexamethasone, dibutryl cyclic adenosine monophosphate (dB cAMP; Sigma), dimethylsulfoxide (DMSO; BDH chemicals). Media was changed every other day. DRG’s were allowed to extend for 5 days. After 5 days of culture, samples were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, Oakville Ont.) in PBS. Fluorescent immunostaining for neurofilament-200 was performed using mouse monoclonal antineurofilament-200 (diluted 1:200) as the primary antibody and fluorescently labeled goat anti-mouse (diluted 1:400) (Amersham Biosciences, Piscataway, NJ) as the secondary antibody. Fluorescent microscopy images were taken of the gels at a magnification of 10 times and a montage was created to show the extension. The numbers of nerves extending 150, 300, 450 and 600 mm were counted as a quantitative measure of neurite extension.
3. Results 3.1. Collagen gel preparation All the dendrimer crosslinked collagen gels before and after YIGSR peptide modification were transparent and strong enough to manipulate. The bulk properties of the gels appeared unaltered when they were stored in PBS/water at 4 1C for at least 8 months. 3.2. Covalent attachment of peptides to dendrimers and characterization YIGSR was attached to dendrimers using the same reaction as was used for dendrimer-mediated collagen crosslinking. The carboxylic acid groups in the peptides were activated by EDC and NHS to form reactive NHS
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esters, which reacted with amine groups in dendrimers to form chemical bonds. The reaction between the dendrimers and the peptides was confirmed by H-NMR and MALDI TOF. H-NMR spectra of dendrimer, YIGSR and YIGSR modified dendrimer are shown in Fig. 2. Specifically characteristic peaks from both the dendrimers (2.29–2.47 ppm) and YIGSR (6.62–6.91 ppm) were found in the purified YIGSR modified dendrimer spectra, which are indicative of successful attachment of YIGSR to dendrimers. The estimated molar ratio of YIGSR:dendrimer was found to be 1:5.4 based on these peaks. Therefore, compared with the initial molar ratio of YIGSR:dendrimer (1:1), it is estimated that only 18.5% of the initial YIGSR present was attached to the dendrimers after reaction and purification. Assuming all the YIGSR modified dendrimers are involved in the crosslinking reaction of collagens, the maximum YIGSR content in the collagen gels would be expected to be 1.6 102 mg/mg collagen. MALDI mass spectra of dendrimer, YIGSR and YIGSR modified dendrimer further confirmed the formation of YIGSR modified dendrimer (Fig. 3). Peaks for the dendrimer (773.7) and YIGSR (595.3) as well as for the YIGSR modified dendrimer (1354.1) were found in the spectra as expected. Additional peaks present (1186, 1086, 1029, 955) are thought to result from deposition of the chemically attached YIGSR due to its thermally labile nature [11]. 3.3. Incorporation of peptides in collagen gels and characterization of peptide modified gels 125
I labeled YIGSR was used to quantify the amount of YIGSR incorporated to collagen gels. It was found that 3.1–3.4 102 mg of YIGSR/mg collagen could be incorporated, suggesting that 24–26% of initial the YIGSR present was attached to collagen, a result consistent with the estimate from the H-NMR analysis of the peptide modified dendrimers. 3.4. Gel characterization Surfaces of collagen gels before and after YIGSR modification were examined by XPS. Not unexpectedly, there were no significant differences (data not shown), which demonstrates that the reaction with the peptide modified dendrimers did not adversely affect the surface properties of the gels. Denaturation temperatures of the collagen gels were determined by DSC to determine whether changes in the crosslinking density occurred with YIGSR modification. Multiple denaturation temperature peaks were found in YIGSR modified collagen samples similar to the previously examined dendrimer crosslinked collagens [6]. As shown in Table 1, overall the number of peaks in the YIGSR modified gels was similar to the unmodified gels, although the highest denaturation temperature was slightly lower with the presence of YIGSR.
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This may potentially indicate possible interference of YIGSR with the dendrimer-mediated crosslinking reaction resulting in a lower crosslinking density. Possibly due to this, slightly poorer mechanical properties in terms of modulus were observed in the YIGSR modified collagens as shown in Fig. 4a. However, they had a similar maximum load to the unmodified dendrimer crosslinked collagens (Fig. 4b). 3.5. Surface modification of collagen gels with YIGSR The surface coverage of YIGSR on the collagen surfaces was found to be in the range of 88.9–95.6 mg/cm2. While this accounts for only 5–6% of the maximal YIGSR coverage calculated theoretically from the availability of amine groups, it is much higher than the densities 2.5 105 mg/cm2 reported previously [12]. 3.6. In vitro corneal epithelial cell culture 3.6.1. YIGSR bulk modified collagens Representative photographs of human corneal epithelial cells on YIGSR bulk modified (1.6 104–1.6 102 mg YIGSR/mg collagen)/unmodified collagen gel surfaces at 120 min are shown in Fig. 5. It was found, not surprisingly, that the cells adhered to all of the collagen surfaces within 30–60 min. Furthermore, morphology changes were observed in all cases. In comparison, the cells did not adhere to the control tissue culture plates in this time frame and remained round and non-adherent after 2 h of culture. The presence of YIGSR resulted in the formation of clusters and visibly greater levels of cell attachment. Over longer periods of time, there was a trend showing that the cells proliferated faster on collagen gels with higher YIGSR content as shown in Figs. 6 and 7. This trend was confirmed by Cyquant assay as shown in Fig. 8. 3.6.2. YIGSR surface modified collagens Similar to the YIGSR bulk modified collagens, the cells adhered to peptide modified collagen surfaces within 60 min and changed morphology. However, over a period of 1 week of culture, there was no significant improvement in the adhesion and growth of the cells on these surfaces relative to the unmodified collagen gels (data not shown). 3.7. DRG neurite extension Neurite extension from DRG cells was found to be significantly enhanced by the presence of the YIGSR in the collagen gels. The length of the neurites and number of neurites, summarized in Fig. 9 was significantly (po0:05) enhanced by the presence of the cell adhesion peptide. Surprisingly, there was little or no effect of peptide concentration in the gel, although it is possible that the surface density of the peptide on these surfaces
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Fig. 2. H-NMR spectra of YIGSR (a), dendrimer G2 (b), and YIGSR modified dendrimer (c). Characteristic peaks from dendrimers (2.29–2.47 ppm) and YIGSR (6.62–6.91 ppm) were found in the purified YIGSR modified dendrimer spectra, which indicated the successful attachment of YIGSR to dendrimers.
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Fig. 3. MALDI-TOF spectra of (a) dendrimer and (b) YIGSR modified dendrimer. Peaks characteristic of the dendrimer and the YIGSR as well as a peak demonstrating reaction between the two species were observed in the modified dendrimer.
Table 1 Collagen sample
YIGSR modified Control
Denaturation temperature (1C) Peak 1
Peak 2
Peak 3
56.0 40.0
59.8 82.0
78.2 89.0
was relatively similar as this was a bulk modification. Visually, it is clear that the nerve density on these materials was also enhanced by the presence of the peptide. Additional studies to assess nerve in-growth into the gels may provide more insight into the effect of peptide concentration in the gel on the neurites (Fig. 10).
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4. Discussion
2.0 Young's Modulus (MPa)
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Displacement (mm)
6
Glutaraldehyde Dendrimer
YIGSR 3.0
Displacement Maximum Load
5
2.5
4
2.0
3
1.5
2
1.0
1
0.5 0.0
0
(b)
Maximum Load (N)
EDC
(a)
EDC
Glutaraldehyde Dendrimer
YIGSR
Fig. 4. Comparison of mechanical properties (a, Young’s modulus; b, Maximum load and displacement at maximum load) of YIGSR modified (6.4 mg/mg collagen) and unmodified collagen samples. Slightly lower modulus was observed in the YIGSR modified collagens. However, they had a similar maximum load to the unmodified dendrimer crosslinked collagens.
The YIGSR sequence of laminin has been studied to improve neurite growth and corneal epithelial cell stratification [2]. In the current work, YIGSR was chemically attached to multifunctional dendrimer and the peptide modified dendrimers were then used as novel crosslinkers for collagens using a combination of unmodified and YIGSR modified dendrimers as crosslinkers. The modification of the dendrimers with the peptide was confirmed by H-NMR and MALDI mass spectra. Although the extent of the overall reaction was not high, with only 18.5–26% of the initial YIGSR attached to the collagens based on H-NMR estimation and 125I radiolabelling studies, the amount of YIGSR in the gels (1.6–3.2 102 mg/mg collagen) was comparatively significantly higher than reported previously (0.0013 mg/mg collagen) [2] and could be easily tuned by crosslinking with fewer peptide modified dendrimers. This demonstrates that the multifunctional dendrimers allowed for the modification of collagen gels with high levels of biological molecules. While this is possibly higher than necessary for significant cell growth improvement, the use of dendrimers in this capacity allows for a tunable amount of biological molecules to be incorporated into collagen gels by simply altering the fraction of peptide modified dendrimers in the crosslinking mixture. Furthermore, combinations of biological molecules can potentially be incorporated in the gels. When the surfaces of the collagen gels were modified with the peptides, the measured surface density was found to be 88.9–95.6 mg/cm2, several orders of magnitude higher than
Fig. 5. HCEC adhesion on YIGSR modified collagen gels after 2 h of culture. The presence of YIGSR resulted in the formation of clusters and visibly greater levels of cell attachment.
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Fig. 6. HCEC proliferation on YIGSR modified collagen gels after 2 days of culture. The cell became confluent on YIGSR modified collagen gels within 2 days.
Fig. 7. HCEC proliferation on YIGSR modified collagen gels after 4 days of culture. No difference was found on all surfaces after the cells became confluent.
the peptide densities reported earlier [12] possibly due to the large amount of amine groups introduced by dendrimers being available for peptide attachment. While there were no differences in the surface chemistry of the modified
gels relative to the unmodified gels as determined by XPS, this is not surprising given the similarity in the structures of the collagen and the peptides. The denaturation temperatures measured for the YIGSR modified gels were slightly
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30000
Cell Number
25000 20000 15000 10000 5000 0
1
2
4
3
Day of Culture 0.0160 mg YIGSR/mg collagen 0.0016 mg YIGSR/mg collagen 0.00016 mg YIGSR/mg collagen unmodified gels
Fig. 8. HCEC proliferation on YIGSR modified collagen gels determined by Cyquant assay. The cells proliferated faster on collagen gels with higher YIGSR content.
lower than those observed on the unmodified gels, indicating that there were lower levels of crosslinking in these gels. However, the differences were relatively small. The slightly lower denaturation temperatures found in YIGSR bulk modified collagens are thought to be due to the interference of the peptide to the crosslinking of collagens with dendrimers. This might also account for the slightly poorer mechanical properties observed in YIGSR bulk modified collagens. Interactions of the modified gels with human corneal epithelial cells were used to characterize the potential of the materials as tissue-engineering scaffolds. Cell adhesion peptides including RGD, YIGSR, IKVAV have been covalently coupled to various biomaterial surfaces/bulk structures, including corneal materials, to improve cellular interactions [1,5,13–15]. The advantage of modification with the cell adhesion peptides rather than immobilization of the entire protein include minimizing potential for immunologic reactions and protein denaturation as well as maximizing surface uniformity and density since smaller
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Number of Neurons
30 25
150 µm 300 µm 450 µm 600 µm
20 15 10 5 0 Dendrimer
0.064 YIGSR 0.64 YIGSR Surface
6.4 YIGSR
Fig. 9. DRG neurite extension on YIGSR modified collagen gels compared with unmodified control. The length of the neurites and number of neuritis was significantly (po0:05) enhanced by the presence of the cell adhesion peptide.
Fig. 10. DRG nerve cell in-growth on unmodified control (left) and YIGSR modified (right, 6.4 mg/mg collagen) collagen gels. Neurites extended longer on YIGSR modified collagens.
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molecular peptides are more accessible to the receptors (such as integrins) on the cell surfaces [16–18]. Collagen itself was found to be a compatible scaffold for corneal epithelial cells growth when properly strengthened by dendrimer crosslinking. With the help of incorporated YIGSR either in bulk or on surface, the epithelial cells tended to form more clusters and attach preferably to the collagen surfaces in the short term. Over longer periods of in vitro culture, the cells grew more rapidly on YIGSR bulk modified collagen gel surfaces and became confluent earlier than on unmodified collagens. However, no difference was noted on different collagen gels after the cells became confluent. Incorporation of the laminin-derived peptide, YIGSR, was also found to significantly promote neurite extension from DRG. Both the nerve density and the length of the extended neurites were increased. However, there was no obvious trend with increasing peptide concentration, potentially due to the fact that the peptide concentration in the gels was higher than needed to promote neurite extension or that the differences on peptide concentration on the gel surfaces were relatively small. While it would be preferable to assess nerve ingrowth into these materials, these preliminary studies, similar to those performed by other groups [19,20], clearly demonstrate the potential of these materials for the support of nerves. Ongoing nerve ingrowth studies may provide additional insight into the effect of peptide concentration on innervation of tissueengineering scaffolds. Ongoing work is also examining the effect of such combination peptides as YIGSRIKVAV and IKVAVYIGSR, which have been reported [5] to further enhance nerve in-growth. 5. Conclusions The YIGSR peptide sequence of laminin was either chemically incorporated into the bulk structure of collagen gels or attached on collagen gel surfaces by way of dendrimers. The structure of YIGSR modified dendrimer was confirmed by H-NMR, MALDI mass spectra and the amount of YIGSR incorporated in collagen was determined by 125I radiolabelling. The incorporation reaction was carried out under mild aqueous conditions at room temperature and the amount of peptide incorporated can be tuned by varying reaction conditions such as the percentage of peptide modified dendrimers in crosslinker solutions for the collagens. The crosslinking density of the collagen gels was slightly affected by the incorporated YIGSR, resulting in small decreases of the modulus of the gels. However, overall the mechanical properties of the gels did not deteriorate significantly. The incorporated YIGSR peptide promoted the growth of the corneal epithelial cells on collagen gel surfaces in both terms of adhesion and proliferation. As well, neurite extension and nerve cell density was enhanced on these materials relative to unmodified or control peptide modified controls, although no effect of peptide concentration was observed. Materials
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with additional biological function have significant potential as tissue-engineering scaffolds. The ability to tune the biological function through the dendrimer crosslinker can be used to develop a variety of collagen-based tissueengineering scaffolds. Acknowledgments The authors acknowledge funding support from the Natural Sciences and Engineering Research Council of Canada. Inamed Corporation is acknowledged for providing the concentrated collagen suspensions. References [1] Itoh S, Takakuda K, Samejima H, Ohta T, Shinomiya K, Ichinose S. Synthetic collagen fibers coated with a synthetic peptide containing the YIGSR sequence of laminin to promote peripheral nerve regeneration in vivo. J Mater Sci: Mater Med 1999;10:129–34. [2] Li F, Carlsson D, Lohmann C, Suuronen E, Vascotto S, Kobuch K, et al. Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation. Proc Natl Acad Sci USA 2003;100:15346–51. [3] Meiners S, Mercado M. Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors. Mol Neurol 2002;27:177–95. [4] Suuronen EJ, Nakamura M, Watsky MA, Stys PK, Muller L, Munger R, et al. Innervated human corneal equivalents as in vitro models for nerve–target cell interatctions. FASEB J 2004;18: 170–2. [5] Tong Y, Shoichet M. Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers. Biomaterials 2001;22:1029–34. [6] Duan X, Sheardown H. Crosslinking of collagen with dendrimers. J Biomed Mater Res A 2005;75:510–8. [7] Duan X, Sheardown H. Dendrimer crosslinked collagen as a corneal tissue engineering scaffold: mechanical properties and corneal epithelial cell interactions. Biomaterials 2006;27:4608–17. [8] Olde Damink LHH, Dijkstra PJ, van Luyn MJA, van Wachem PB, Nieuwnehuis P, Feijen J. Cross-linking of dermal sheep collagen using a water-soluble carbodiimide. Biomaterials 1996;17: 765–73. [9] Behr TM, Gotthardt M, Becker W, Behe M. Radioiodination of monoclonal antibodies, proteins and peptides for diagnosis and therapy. Neuk Med 2002;41:71–9. [10] Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NLC, et al. Functional corneal equivalents constructed from cell lines. Science 1999;286:2169–72. [11] Covey TR, Huang EC, Henion JD. Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions. Anal Chem 1991;63:1193–200. [12] Shaw D, Shoichet MS. Toward spinal cord injury repair strategies: peptide surface modification of expanded poly(tetrafluoroethylene) fibers for guided neurite outgrowth in vitro. J Cranio Surg 2003;14: 308–16. [13] Schense JC, Bloch J, Aebischer P, Hubbell JA. Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension. Nat Biotech 2000;18:415–9. [14] Merrett K, Griffith CM, Deslandes Y, Pleizier G, Sheardown H. Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces. J Biomater Sci Polym Edn 2001;12:647–71. [15] Aucoin L, Griffith CM, Pleizier G, Deslandes Y, Sheardown H. Interactions of corneal epithelial cells and surfaces modified with cell
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