Amine Functionalization of Collagen Matrices with Multifunctional

Biomacromolecules 2010, 11, 3093–3101

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Amine Functionalization of Collagen Matrices with Multifunctional Polyethylene Glycol Systems John Ward,†,‡ Jack Kelly,† Wenxin Wang,‡,§ Dimitrios I. Zeugolis,*,‡,§ and Abhay Pandit‡,§ Department of Plastic and Reconstructive Surgery, University Hospital of Galway, Galway, Ireland, and Network of Excellence for Functional Biomaterials (NFB), National University of Ireland, Galway (NUI Galway), Galway, Ireland Received August 3, 2010; Revised Manuscript Received September 7, 2010

A method to functionalize collagen-based biomaterials with free amine groups was established in an attempt to improve their potential for tethering of bioactive molecules. Collagen sponges were incorporated with amineterminated multifunctional polyethylene glycol (PEG) derivatives after N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide (EDC/NHS) cross-linking. The extent of the incorporation of different amounts and different numbers of active moieties of amine-terminated PEG systems into the collagen scaffolds was evaluated using ninhydrin assay, Fourier transform infrared spectrophotometry (FTIR), collagenase degradation assay, denaturation temperature measurements, and in vitro cell studies. A 3% 8-arm amine-terminated PEG was found to be the minimum required effective concentration to functionalize EDC/NHS stabilized collagen scaffolds. EDC/NHS stabilized scaffolds treated with 3% 8-arm amine-terminated PEG exhibited significantly improved denaturation temperature and resistance to collagenase degradation over non-cross-linked scaffolds (p < 0.002). Biological evaluation using 3T3 cells demonstrated that the produced scaffolds facilitated maintenance of the cells’ morphology, metabolic activity, and ability to proliferate in vitro. Overall, our results indicate that amineterminated PEG systems can be used as means to enhance the functionality of collagenous structures.

1. Introduction In vivo, native cross-linking takes place to impart desired mechanical stability and proteolytic resistance on collagen fibers in connective tissues.1-3 Lysyl oxidase is secreted from fibrogenic cells as a 50KDa pro-enzyme that is proteolytically processed to the mature enzyme in the extracellular space. Inhibition of lysyl oxidase action toward collagen molecules results in the accumulation and ultimate proteolytic degradation of soluble collagen monomers, thus, preventing the formation of insoluble collagen fibers.4 The participation of this enzyme is therefore critical to the development and repair of connective tissues.5 However, the lysyl oxidase mediated cross-linking does not occur in vitro and, consequently, reconstituted forms of collagen lack sufficient strength and disintegrate upon handling or collapse under the pressure from surrounding tissue in vivo. Thus, it is necessary to introduce exogenous cross-links (chemical, biological, or physical) into the molecular structure to control mechanical and thermal properties, biological stability, the residence time in the body, and to some extent the immunogenicity and antigenicity of the device.6-8 However, biomaterial design has evolved from basic constructs that match structural and mechanical properties to biofunctional materials that aim to incorporate instructive signals into scaffolds and to modulate cellular functions such as proliferation, differentiation, and morphogenesis.9,10 At present, there is no commonly accepted ideal cross-linking treatment for collagen-derived biomaterials, and among them only transglutaminase (TGase)11,12 and polyamidoamine (PAMAM) dendrimeric systems13,14 offer opportunities of functionalization. * To whom correspondence should be addressed. Tel.: +353-(0)-91493166. Fax: +353-(0)-9156-3991. E-mail: [email protected]. † University Hospital of Galway. ‡ NFB, NUIG. § Department of Mechanical and Biomedical Engineering, NUIG.

Indeed, tissue TGase belongs to a family of enzymes that catalyze several post-translational modifications of proteins by forming inter- and intramolecular bonds; the process results in the formation of stable covalently cross-linked proteins in the extracellular matrix in a Ca2+-dependent manner.15-19 It has been recently demonstrated that tissue type II and microbial (Ca2+-independent) TGase can be used to stabilize collagen scaffolds,20-24 albeit limited.11 The resultant, however, covalent γ-glutamyl-ε-lysine isopeptide bond of TGase has been used to incorporate peptides into the molecular structure,11,12,24-26 which indicates the functionalization potential of TGase in the biomaterials field. However, the limited stabilization potential of TGase due to its single molecule functionality can limit its use in tissue engineering applications. For these reasons, multifunctional approaches based on PAMAM dendrimers have been developed. Such systems not only enhance the mechanical properties of the produced scaffolds but also offer multiple opportunities of functionalization.13,14,27-29 However, cytotoxicity complications of PAMAM dendrimers as a function of generation, independent of the surface charge, have caused concerns in regard to their use in biomaterial fields.30-34 To this end, PEG systems have been introduced as valuable alternatives to limited TGase functionalization ability and to toxicity of PAMAM dendrimers. PEG, a low toxic and low antigenic poly(ether-diol) has been FDA approved for several medical and food industry applications.35 Additionally, PEG has been shown to facilitate cell infiltration, tissue in-growth, and enzyme degradation with improved blood compatibility and ability to resist protein adsorption.36,37 It has also been demonstrated that linear38-41 and bifunctional42 PEGs can significantly increase the mechanical stability and biocompatibility of biomaterials. PEG-dendrimer hybrid use has been advocated due to the high ratio of multivalent surface moieties to molecular volume, low toxicity, and hemolytic properties,

10.1021/bm100898p  2010 American Chemical Society Published on Web 10/13/2010

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Figure 1. Proposed reaction mode between collagen, EDC/NHS, and 4-arm amine-terminated PEG system.

long blood circulation times, low organ accumulation, and high accumulation in tumor tissue due to the enhanced permeation and retention effect.43-52 We therefore herein aim to investigate the influence of amine-terminated PEG systems on collagen scaffolds. Figure 1 demonstrates the proposed mode of reaction between collagen scaffolds, EDC/NHS, and 4-arm PEG system. The influence of different quantities and different numbers of active moieties of amine-terminated PEGs on the properties of the resultant scaffolds were examined. The characteristics of the resultant scaffold were evaluated using ninhydrin assay, FTIR, collagenase degradation assay, denaturation temperature measurements, and in vitro cell studies to ascertain cell viability, proliferation, and morphology.

2. Experimental Section 2.1. Materials and Reagents. Porcine Achilles tendons were acquired from a local slaughter house. The amine-terminated multiarm polyethylene glycol Mw 10 KDa (PEG) derivatives were purchased from JenKem Technology U.S.A. (Allen, TX). Alamar blue was purchased from BioSource Europe (Nivelles, Belgium); Quant-iT PicoGreen dsDNA reagent, rhodamine phalloidin, and 4′,6-diamidino-2-phenylindole, and dihydrochloride (DAPI) were purchased from Invitrogen (Bio Sciences Ltd., Dun Laoghaire, Ireland). All other materials and reagents were purchased from Sigma-Aldrich (Dublin, Ireland) unless otherwise stated. 2.2. Collagen Extraction and Analysis. Typical protocols for the extraction, purification, and analysis of collagen were employed as has been described in detail previously.40 Briefly, frozen porcine Achilles tendons were minced, washed in a series of neutral phosphate buffers, and suspended in 0.5 M ethanoic acid in the presence of pepsin (porcine gastric mucosa; 3200-4500 units/mg protein) for 72 h at 4 °C. Following that, the collagen suspension was centrifuged (12000 g at 4 °C for 45 min; Gr20.22 Jouan refrigerated centrifuge, Thermo Electron Corporation, Bath, U.K.) and purified by repeated salt precipitation (0.9 M NaCl), centrifugation and acid solubilization (1 M ethanoic acid). The final atelocollagen solution was dialyzed (8000 Mw cut off) against

Ward et al. 0.01 M ethanoic acid and kept refrigerated at 4 °C until used. The atelocollagen solution purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Alpha Technologies Ltd., Co., Wicklow, Ireland) analysis (90% type I) and its concentration was determined by hydroxyproline assay (3 mg/mL). 2.3. Scaffold Stabilization and Functionalization. Collagen sponges were obtained after pipetting 1 mL of the dialyzed atelocollagen solution into 24-well tissue culture plates (Sarstedt Ltd., Wexford, Ireland) followed by lyophilization using a VirTis freeze-dryer (Suffolk, U.K.) overnight. The lyophilized collagen scaffolds were stabilized using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in 50 mM 4-morpholineethanesulfonic acid in 40% ethanol (MES solution). A molar ratio of EDC to NHS to collagen’s carboxyl groups of 5:5:1 was used.8,53-55 The pH of the solution was adjusted to 5.5 using either 0.1 M sodium hydroxide (NaOH) or 0.1 M hydrochloric acid (HCl). Scaffolds were also stabilized with 0.625% aqueous glutaraldehyde (GTA) solution as has been described before.8,56,57 To evaluate the functionalization potential of amine-terminated PEG derivatives, lyophilized collagen scaffolds were incubated with 4-, 6-, and 8-arm amine-terminated PEG derivatives of different concentrations (0.001, 0.01, 0.1, 1, 3, 5, 10% w/v) in 0.1 M phosphate buffered saline (PBS) for 1 h at 37 °C, followed by a 5 h incubation at 37 °C in the EDC/NHS cross-linking solution as described above. The produced stabilized and stabilized/functionalized scaffolds were washed extensively in distilled water for 1 h at 37 °C, followed by overnight lyophilization. 2.4. Ninhydrin Assay. Ninhydrin assay was used to determine the amount of free amines incorporated into the collagenous structure after functionalization using the amine-terminated PEG derivatives as has been described before.13,14,58,59 Briefly, 200 mM citric acid and 0.16% (w/v) stannous chloride were dissolved in 100 mL of distilled water. A total of 4% (w/v) of ninhydrin was dissolved in 100 mL of ethylene glycol monoethyl ether. The two solutions were mixed and the pH was adjusted to 5.5 using 10 M NaOH (ninhydrin solution). A total of 25-30 mg of each scaffold was immersed in 200 µL of distilled water, followed by 1 mL of ninhydrin solution. The samples were then incubated at 95 °C for 30 min. To stop the reaction, the samples were cooled in ice and 250 µL of 50% isopropanol was added. The samples were vortexed and the absorbance of the developed Ruhemann’s purple color was read at 570 nm. 2.5. Fourier Transform InfraRed (FTIR) Spectroscopy. Conformational changes in all scaffolds due to cross-linking and cross-linking/ functionalization were determined using attenuated total reflectance Fourier transform InfraRed (ATR-FTIR; Shimadzu FTIR-8600, Shimadzu Europe Ltd., Duisburg, Germany). Spectra were recorded at RT in the mid-infrared range (4000-400 cm-1). A total of 40 scans were signal-averaged for a single spectrum at a resolution of (8 cm-1 using a ZnSe crystal at an incident angle of 45°. The spectra were analyzed using the Hyper-IR software (Shimadzu Europe Ltd., Duisburg, Germany) to obtain quantitative peak information. 2.6. Evaluation of Enzymatic Stability. Dry scaffolds were accurately weighed and incubated for 6 h in 1 mL of 50 mM [tris(hydroxymethyl)-methyl-2-aminoethane sulfonate] (TES) buffer (pH 7.4) containing 0.36 mM calcium chloride at 37 °C and 2.5 or 5 collagen digestive units (CDU) per mg of collagen collagenase type I from Clostridium histolyticum (0.25-1.0 FALGPA units/mg solid, >125 CDU/mg solid). The reaction was subsequently stopped using 0.2 mL of 0.25 M ethylenediaminetetraacetic acid (EDTA) and the mixtures were centrifuged for 10 min at 1000 rpm at 4 °C (Heraus Fresco 17, Thermo Scientific, Dublin, Ireland). The supernatants were discarded and the pellets were washed twice in distilled water followed by lyophilization. Scaffold degradation was determined from the weight of residual matrix after collagenase degradation and expressed as a percentage of the original weight.56,60 2.7. Evaluation of Thermal Stability. The denaturation temperature was determined using the DSC-60 (Shimadzu, Japan) differential

Amine Functionalization of Collagen Matrices scanning calorimeter as has been described previously.61 Briefly, dry samples were incubated overnight in PBS at RT. The following day, the samples were blotted with filter paper to remove excess fluid and hermetically sealed in standard aluminum pans (Mettler-Toledo; Mason Technology, Dublin, Ireland). Heating was carried out at a constant temperature ramp of 5 °C/min in the temperature range of 15-100 °C. An empty aluminum pan was used as reference probe. The endothermic transition was recorded as a typical peak and the temperature of maximum power of absorption during denaturation was recorded. 2.8. Evaluation of Cell Viability, Morphology, and Activity. R3T3 mouse fibroblasts (passage 3-4) were cultured to confluence in T75 flasks (Sarstedt Ltd., Wexford, Ireland) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (PS), 25 mg/L Amphotericin-B, and 1% glutamine 200 mM (all were purchased from Invitrogen, Bio Sciences Ltd., Dun Laoghaire, Ireland). All scaffolds were disinfected in 70% aqueous ethanol solution followed by thorough washing in Hank’s Balanced Salt Solution (HBSS; Invitrogen, Bio Sciences Ltd., Dun Laoghaire, Ireland). The samples were then incubated in media for 3 h in a 5% CO2 humidified incubator at 37 °C prior to seeding. Each scaffold was seeded with a density of 20000 cells per well and the media were changed every 24 h. Cell viability was assessed at days 3 and 7 by monitoring their metabolic activity using the Alamar Blue assay. Briefly at days 3 and 6, all cell-seeded scaffolds were transferred to a 24-well plate, rinsed with HBSS and 700 µL of 10% (v/v) Alamar Blue reagent in HBSS was added to each well. After 1 h of incubation at 37 °C, fluorescence was measured using a microplate fluorescence reader (FLx800, Bio-Tek Instruments, Inc., Vermont) at excitation and emission wavelengths of 528 and 590 nm, respectively. DNA was quantified at days 3 and 7 of the PicoGreen assay as per manufacturer’s guidelines. A standard curve based on known concentration of DNA was used to determine the total cell number. The sample fluorescence was measured using a microplate reader (VICTOR3 V Multilabel Counter, PerkinElmer BioSignal Inc., U.S.A.) at 480 nm excitation and 520 nm emission. Cell morphology was evaluated through immunocytochemistry after 4% aqueous paraformaldehyde solution fixation for 15 min and washing in 1% BSA in Tris-HCl buffer (pH 7.4) for 30 min to block nonspecific binding sites. The cell cytoskeleton was stained with rhodamine phalloidin using 1:100 dilution in PBS for 1 h at RT, while the cell nuclei was stained with 1:100 DAPI in PBS for 20 min at RT. The stained cells on the scaffolds were rinsed with HBSS and examined under fluorescence light microscope (Olympus IX81, Olympus Europe, Hamburg, Germany). ImagePro Plus 5.0 software (Media Cybenetics Inc., MD) was used to acquire digital images from the microscope. A low-voltage, high-resolution Scanning Electron Microscope (SEM; S-4700 Hitachi Scientific Instruments, Berkshire, U.K.) was used to evaluate cells on the surface of the scaffolds after 7 days in culture. The cell-seeded scaffolds were washed twice with HBSS; fixed in an aqueous 3% GTA solution; dehydrated first using a series of ascending aqueous ethanol concentrations followed by hexamethyldisilazane; and finally gold-coated (Emitech K-550X Sputter Coater, Emitech Ltd., Ashford, Kent, U.K.) prior to SEM observation. 2.9. Statistical Analysis. Numerical data is expressed as mean value of five samples ( standard deviation. Analysis was performed using statistical software (MINITAB version 15, Minitab, Inc., PA, U.S.A.). One-way analysis of variance (ANOVA) for multiple comparisons and two-sample t test for pair-wise comparisons were employed after confirming the following assumptions: (a) the distribution from which each of the samples was derived was normal (Anderson-Darling normality test) and (b) the variances of the population of the samples were equal to one another (Bartlett’s and Levene’s tests for homogenicity of variance). Nonparametric statistics were utilized when either or both of the above assumptions were violated and consequently Kruskal-Wallis test for multiple comparisons or Mann-Whitney test for two samples were carried out. Statistical significance was accepted at p < 0.05.


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3. Results 3.1. Evaluation of Free Amine Content. Ninhydrin assay was used to quantitatively assess the free amines present on the collagen scaffolds. EDC/NHS cross-linking significantly decreased the amount of free amines when compared to the noncross-linked collagen scaffolds (p < 0.006; Figure 2). No significant difference (p > 0.05) was observed between the EDC/ NHS stabilized scaffolds and the functionalized scaffolds using 4- and 6-arm amine-terminated PEG system, independent of the concentration used (0.001 to 10% w/v; Figures S1 and S2, respectively). When the 8-arm PEG system was evaluated (Figure 2), a significant increase in amine content over the control scaffolds was observed for concentrations above 3% (p < 0.001), but no significant difference was observed among the 3, 5, and 10% w/v amount of the 8-arm PEG (p > 0.05). FTIR spectroscopy was used to provide information about changes in the molecular structure of collagen scaffolds as a function of cross-linking and functionalization. The representative IR spectra of the various matrices in this study are shown in Figure S3. Quantitative peak information of the individual spectra was obtained using the Hyper-IR software. The absorption peak area ratios of the amide I band at 1635 cm-1 to that of amide A band were determined and mean values were plotted (Figure 3). The ratio of EDC/NHS and GTA stabilized scaffolds significantly increased over the non-cross-linked collagen scaffolds (p > 0.05). The incorporation of 3, 5, and 10% 8-arm amine-terminated PEG on EDC/NHS stabilized scaffolds significantly decreased the ratio of amide I to amide A over the control EDC/NHS stabilized scaffolds (p < 0.001). No significant difference (p > 0.05) was observed among the ratios of amide I to amide A of EDC/NHS stabilized and 3, 5, and 10% 8-arm amine-terminated PEG-functionalized scaffolds. 3.2. Evaluation of Enzymatic Stability. Collagenase degradation assay was used to evaluate the resistance of the produced scaffolds to enzymatic degradation. The percentage weight of scaffolds remained after collagenase degradation is shown in Figure 4. Noncross-linked collagen scaffolds completed degraded with 5 CDU of collagenase per mg of collagen within 6 h. However, cross-linking with either GTA or EDC prohibited degradation by collagenase of the scaffolds for the experimental period tested (p > 0.05). No significant losses in collagen content were observed for collagen scaffolds fixed with EDC/NHS and functionalized with variable amounts of 8-arm amine-terminated PEGs when they were treated with 2.5 CDU of collagenase (p > 0.05). However, some degradation was detected when they were treated with 5 CDU of collagenase (p < 0.01). 3.3. Evaluation of Thermal Stability. Differential scanning calorimetry was employed to evaluate the thermal properties of the different scaffolds produced in this study. Figure S4 demonstrates typical DSC thermographs, while Figure 5 illustrates the denaturation temperatures of all scaffolds evaluated in this study. Non-cross-linked scaffolds exhibited the lowest denaturation temperature (44.40 ( 0.95 °C; p < 0.001). GTA and EDC/NHS cross-linked scaffolds exhibited the highest denaturation temperatures (71.36 ( 0.44 and 70.12 ( 0.58 °C, respectively; p < 0.002). Functionalized scaffolds with 3, 5 and 10% of 8-arm amine-terminated PEG exhibited denaturation temperatures (56.87 ( 0.88, 55.98 ( 0.30 and 59.12 ( 1.92 °C, respectively) significantly higher than the non-cross-linked scaffolds (p < 0.001), but significantly lower than the EDC/ NHS fixed scaffolds (p < 0.002).

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Figure 2. Percentage of amine content of the scaffolds produced in this study, in relation to non-cross-linked collagen scaffolds. Values are presented as mean ( SD (n ) 3). No significant difference was observed among the EDC/NHS cross-linked scaffolds and the EDC/NHS and 8-arm amine-terminated PEG of 0.001, 0.01, 0.1 and 1% concentration. No significant difference was observed among the 3, 5 and 10% w/v amount of the 8-arm amine-terminated PEG (p > 0.05) and all of them exhibited higher amine content than the EDC/NHS stabilized scaffolds (p < 0.001).

Figure 3. FTIR spectra of collagen matrices indicate a decrease in amine content of the cross-linked collagen scaffolds and an increase in amine content of the EDC/NHS stabilized scaffolds that were treated with variable amounts of amine-terminated PEG system.

3.4. Evaluation of Cell Viability, Morphology, and Activity. The results of the Alamar Blue cell viability assay of fibroblasts seeded on collagen scaffolds functionalized with variable concentrations of amine-terminated PEG was compared to that of fibroblasts seeded on Tissue Culture Plastic (TCP) and EDC/NHS cross-linked scaffolds (positive controls) and GTA stabilized collagen scaffolds (negative control), as measured by fluorescence optical density (OD) are shown in Figure 6. No statistical differences of Alamar Blue OD were observed among the TCP and the functionalized with 8-arm amineterminated PEG systems collagen scaffolds on day 3 (p > 0.05), while the Alamar Blue OD values were significantly higher for the functionalized scaffolds when compared to the TCP values on day 7 (p < 0.005). Collagen scaffolds functionalized with

amine-terminated PEGs, EDC/NHS stabilized scaffolds, and TCP exhibited at all tested periods significant higher Alamar Blue OD than the GTA fixed collagen scaffolds counterparts (p < 0.002). Figure 7 shows the results of DNA content by PicoGreen DNA assay. All scaffolds showed significant increased DNA content from day 3 to day 7 (p < 0.003). At days 3 and 7, GTA stabilized scaffolds showed significantly lower DNA content when compared to any other scaffold (p < 0.001). At days 3 and 7, no significant difference was observed in the DNA content among the scaffolds that were stabilized with EDC/ NHS and functionalized with variable amounts of 8-arm amineterminated PEG system (p > 0.05). The SEM and fluorescent light micrographs of scaffolds seeded with 3T3 fibroblast cells are shown in Figure 8. There was no morphological difference between cells proliferating on the different scaffolds. SEM micrographs (Figure 8a,b) illustrate the surface morphology of confluent fibroblasts at days 3 and 7, respectively, on scaffolds treated with EDC/NHS and 3% 8-arm amine-terminated PEG. DAPI staining shows the high number of cells that have been adhered on scaffolds treated with EDC/NHS and 3% 8-arm PEG on day 7 (Figure 8c). Fluorescent images of scaffolds treated with EDC/NHS and 3% 8-arm PEG after 7 days in culture demonstrate that the fibroblasts attach to each other and onto the three-dimensional surface of the scaffold by elongated dendritic projections (Figure 8d). Intact cytoskeleton and nuclei of fibroblast cells is also apparent. The fibroblast cell bodies were typically either round, elongated, or star-shaped.

4. Discussion The first generation of biomaterials was aiming to imitate structural characteristics and the mechanical properties of native

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Figure 4. Percentage degradation of collagen matrices after 6 h exposure to collagenase solution at 37 °C. Values are presented as mean ( SD (n ) 3). Non-cross-linked collagen scaffolds completed degraded with 5 CDU of collagenase within 6 h. No significant losses in collagen content were observed for collagen scaffolds treated with EDC/NHS and variable amounts of 8-arm amine-terminated PEGs when they were treated with 2.5 CDU of collagenase (p > 0.05), while some degradation was detected when they were treated with 5 CDU of collagenase for 6 h (p < 0.01).

Figure 5. Denaturation temperature of collagen scaffolds evaluated in this study. Values are presented as mean ( SD (n ) 3). Noncross-linked scaffolds exhibited the lowest denaturation temperature (p < 0.001). GTA and EDC/NHS cross-linked scaffolds exhibited the highest denaturation temperatures (p < 0.002). Functionalized scaffolds with 3, 5, and 10% of 8-arm amine-terminated PEG exhibited denaturation temperatures significantly higher than the non-crosslinked scaffolds (p < 0.001), but significantly lower than the EDC/ NHS fixed scaffolds (p < 0.002).

tissues.62 However, the current biomaterial concepts require the use of biofunctional materials that will incorporate instructive signals into the scaffolds and modulate host response.9,10 Surface modifications or functionalization methods based on

TGase11,12,24-26 or PAMAM dendrimers13,14,27-29 provide means of anchoring therapeutic molecules onto the scaffolds or targeting specific ligands. However, the limited stabilization and functionalization potential of TGase11 and cytotoxicity concerns of PAMAM dendrimers30-34 have encouraged research for alternative functionalization strategies. In this study, we evaluated the functionalization potential that multiarm amineterminated PEG systems can bring about on collagen scaffolds using biochemical, biophysical, and biological assays. Ninhydrin assay was used for the quantification of free amines and revealed that cross-linking with EDC/NHS reduced the free amines of collagen scaffolds in comparison to the non-crosslinked control samples. During carbodiimide cross-linking of collagen, carboxylic acid groups of aspartic and glutamic acid residues in collagen react with EDC and NHS. This results in the formation of NHS-activated carboxylic acid groups, which upon reaction with ε-amino groups from lysine and hydroxyl lysine residues form peptide-like cross-links and release of NHS.63-65 Incorporation of 4- and 6-arm amine-terminated PEG did not significantly contribute to the increase of free amines. However, when the 8-arm amine-terminated PEG system was used, a significant increase in the free amines was detected, which indicates successful functionalization of the collagen scaffolds. FTIR involves the measurement of wavelength and intensity of absorption of IR light through excitation of molecular vibrations, which provides information about changes in molecular structure of organic materials. All spectra were typical of that observed for proteins.66-68 FTIR analysis demonstrated that GTA and EDC/NHS cross-linked scaffolds exhibited an increased ratio of the amide I band at 1635 cm-1 to that of the amide A band at 1735 cm-1 in comparison to the non-cross-linked scaffolds due to the effective cross-linking. The rationale behind the choice of the peaks 1635 and 1735 cm-1 was that the amount of EDC-activated -COOH groups available

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Figure 6. Alamar Blue fluorescence optical density (OD) of fibroblast seeded collagen scaffolds as a function of time. No statistical differences of Alamar Blue OD were observed among the TCP and the functionalized with 8-arm amine-terminated PEG systems collagen scaffolds on day 3 (p > 0.05), while the Alamar Blue OD values were significant higher for the functionalized scaffolds when compared to the TCP values on day 7 (p < 0.005). GTA fixed collagen scaffolds exhibited at all tested periods significant lower Alamar Blue OD than the other scaffolds (p < 0.002).

Figure 7. PicoGreen DNA assay shows an increase in DNA content from day 3 to day 7 for all scaffolds. At days 3 and 7, GTA stabilized scaffolds showed significantly lower DNA content than any other scaffold (p < 0.001). No significant difference was observed in the DNA content among the scaffolds that were stabilized with EDC/NHS and functionalized with variable amounts of 8-arm amine-terminated PEG system in days 3 and 7 (p > 0.05).

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Figure 8. SEM micrographs of scaffolds treated with EDC/NHS and 3% 8-arm amine-terminated PEG and seeded with 3T3 cells (some cells are indicated with arrows) for 3 (a) and 7 (b) days. DAPI staining shows the high number of cells that have been adhered on scaffolds treated with EDC/NHS and 3% 8-arm PEG on day 7 (c). Intact cytoskeleton and nuclei of fibroblast cells and round cell bodies are apparent (d).

for reaction are constant because of the use of a fixed amount of EDC in the preparation for all samples. Consequently, an increase in the 1635 to 1735 cm-1 peak ratios indicates the decrease in the free -COOH groups and the increase in amide linkages. On the other hand, scaffolds functionalized with 3, 5, and 10% 8-arm amine-terminated PEG and stabilized with EDC/ NHS exhibited significantly lower ratios of the amide I band at 1635 cm-1 to that of amide A band at 1735 cm-1 in comparison to the EDC/NHS cross-linked scaffolds. We speculate that when concentrations above 3% of the 8-arm amine-terminated PEG are used, competition for the carboxyl groups of collagen takes place that leads to less cross-linking bridges between the polypeptide chains of the molecule. A similar observation has been reported before when collagen-based scaffolds were stabilized with EDC/NHS and subsequently functionalized with either amino acids69 or PAMAM dendrimers.13 Differential scanning calorimetry and collagenase digestion were employed to evaluate the stability of the produced scaffolds. DSC has been used as a sensitive method to examine changes in collagen structure due to cross-linking.61,70-72 The degree of cross-linking of the samples is related to the increase in shrinkage temperature after cross-linking.8,73,74 All samples exhibited a denaturation temperature and resistance to collagenase higher than the non-cross-linked scaffolds, which indicates an increase in stability. The high denaturation of carbodiimide fixed scaffolds has been attributed to the addition of nucleophile NHS that increases the rate and degree of cross-linking, resulting in materials with high Ts and lower free amine groups.65,75,76

The high cross-linking stability of GTA has been attributed to its self-polymerization capabilities.77 Aldehyde groups of GTA react with either hydroxyl groups and then condense to form a heterocyclic compound, which subsequently undergoes oxidation to a pyridinium ring or with amine groups to form Schiff bases.7,54,73,75,78 Scaffolds treated with EDC/NHS and 3, 5, and 10% 8-arm amine-terminated PEG demonstrated denaturation temperature lower than those that treated with EDC/NHS alone. We also detected some degradation when the same scaffolds were treated with 5 CDU of collagenase (p < 0.01). These results confirm our previous speculation that competition for the carboxyl groups of collagen takes place that possibly compromises the cross-linking efficiency. However, given that successful incorporation of free amines was observed, using ninhydrin assay, for concentrations of 3% and above of 8-arm amine-terminated PEG, we recommend this concentration as the minimum effective concentration. Alamar Blue and PicoGreen assays were used to assess metabolic activity and cell proliferation on the produced scaffolds. Biological evaluation using 3T3 cells revealed that the EDC/NHS and the EDC/NHS and 8-arm amine-terminated PEG system were characterized by higher biocompatibility than the GTA samples. It has been shown that residual EDC/NHS forms urea as a byproduct79,80 and unbound and excess chemicals can be easily washed away and, therefore, are considered as nontoxic cross-linking agents.81 GTA, on the other

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hand, due to its self-polymerization capability is associated with cytotoxic drawbacks,77,82-92 which were also evident in this study.

5. Conclusions In this study we demonstrated that 3% 8-arm amineterminated PEG system is the minimum effective concentration required to enhance the functionality of EDC/NHS stabilized collagen scaffolds. The resultant scaffolds are characterized by biological, biochemical, and biophysical properties similar or superior to non-cross-linked and EDC/NHS or GTA cross-linked collagen scaffolds. These results advocate the use of polyethylene glycol systems as a strategy to tether bioactive molecules into scaffolds and enhance the biological activity of the produced constructs. Acknowledgment. The authors would like to thank M. AbuRub, M. Monaghan, J. Chan, C. Holladay, and E. Collin for their excellent technical assistance and useful discussions. A.P. would like to acknowledge the Health Research Board Project Grant RP/2008/188 for financial support. D.Z. would like to acknowledge the Science Foundation Ireland (SFI_09-RFPENM2483) for financial support. Supporting Information Available. Supporting figures. This material is available free of charge via the Internet at http:// pubs.acs.org.

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