Ninidrina.pdf

Amine Functionalization of Collagen Matrices withMultifunctional 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, andNetwork 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 toimprove their potential for tethering of bioactive molecules. Collagen sponges were incorporated with amine-terminated multifunctional polyethylene glycol (PEG) derivatives after N-(3-dimethylaminopropyl)-N′-ethylcar-bodiimide and N-hydroxysuccinimide (EDC/NHS) cross-linking. The extent of the incorporation of differentamounts and different numbers of active moieties of amine-terminated PEG systems into the collagen scaffoldswas evaluated using ninhydrin assay, Fourier transform infrared spectrophotometry (FTIR), collagenase degradationassay, denaturation temperature measurements, and in vitro cell studies. A 3% 8-arm amine-terminated PEG wasfound 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 improveddenaturation 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 thecells’ morphology, metabolic activity, and ability to proliferate in vitro. Overall, our results indicate that amine-terminated 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 desiredmechanical stability and proteolytic resistance on collagen fibersin connective tissues.1-3 Lysyl oxidase is secreted from fibro-genic cells as a 50KDa pro-enzyme that is proteolyticallyprocessed to the mature enzyme in the extracellular space.Inhibition of lysyl oxidase action toward collagen moleculesresults in the accumulation and ultimate proteolytic degradationof soluble collagen monomers, thus, preventing the formationof insoluble collagen fibers.4 The participation of this enzymeis therefore critical to the development and repair of connectivetissues.5 However, the lysyl oxidase mediated cross-linking doesnot occur in vitro and, consequently, reconstituted forms ofcollagen lack sufficient strength and disintegrate upon handlingor collapse under the pressure from surrounding tissue in vivo.Thus, it is necessary to introduce exogenous cross-links (chemi-cal, biological, or physical) into the molecular structure tocontrol mechanical and thermal properties, biological stability,the residence time in the body, and to some extent theimmunogenicity and antigenicity of the device.6-8 However,biomaterial design has evolved from basic constructs that matchstructural and mechanical properties to biofunctional materialsthat aim to incorporate instructive signals into scaffolds and tomodulate cellular functions such as proliferation, differentiation,and morphogenesis.9,10 At present, there is no commonlyaccepted ideal cross-linking treatment for collagen-derivedbiomaterials, and among them only transglutaminase (TGase)11,12

and polyamidoamine (PAMAM) dendrimeric systems13,14 offeropportunities of functionalization.

Indeed, tissue TGase belongs to a family of enzymes thatcatalyze several post-translational modifications of proteins byforming inter- and intramolecular bonds; the process results inthe formation of stable covalently cross-linked proteins in theextracellular matrix in a Ca2+-dependent manner.15-19 It hasbeen recently demonstrated that tissue type II and microbial(Ca2+-independent) TGase can be used to stabilize collagenscaffolds,20-24 albeit limited.11 The resultant, however, covalentγ-glutamyl-ε-lysine isopeptide bond of TGase has been usedto incorporate peptides into the molecular structure,11,12,24-26

which indicates the functionalization potential of TGase in thebiomaterials field. However, the limited stabilization potentialof TGase due to its single molecule functionality can limit itsuse in tissue engineering applications. For these reasons,multifunctional approaches based on PAMAM dendrimers havebeen developed. Such systems not only enhance the mechanicalproperties of the produced scaffolds but also offer multipleopportunities of functionalization.13,14,27-29 However, cytotox-icity complications of PAMAM dendrimers as a function ofgeneration, independent of the surface charge, have causedconcerns in regard to their use in biomaterial fields.30-34

To this end, PEG systems have been introduced as valuablealternatives to limited TGase functionalization ability and totoxicity of PAMAM dendrimers. PEG, a low toxic and lowantigenic poly(ether-diol) has been FDA approved for severalmedical and food industry applications.35 Additionally, PEG hasbeen shown to facilitate cell infiltration, tissue in-growth, andenzyme degradation with improved blood compatibility andability to resist protein adsorption.36,37 It has also beendemonstrated that linear38-41 and bifunctional42 PEGs cansignificantly increase the mechanical stability and biocompat-ibility of biomaterials. PEG-dendrimer hybrid use has beenadvocated due to the high ratio of multivalent surface moietiesto molecular volume, low toxicity, and hemolytic properties,

* To whom correspondence should be addressed. Tel.: +353-(0)-9149-3166. Fax: +353-(0)-9156-3991. E-mail: [email protected].

† University Hospital of Galway.‡ NFB, NUIG.§ Department of Mechanical and Biomedical Engineering, NUIG.

Biomacromolecules 2010, 11, 3093–3101 3093

10.1021/bm100898p 2010 American Chemical SocietyPublished on Web 10/13/2010

long blood circulation times, low organ accumulation, and highaccumulation in tumor tissue due to the enhanced permeationand retention effect.43-52 We therefore herein aim to investigatethe influence of amine-terminated PEG systems on collagenscaffolds. Figure 1 demonstrates the proposed mode of reactionbetween collagen scaffolds, EDC/NHS, and 4-arm PEG system.The influence of different quantities and different numbers ofactive moieties of amine-terminated PEGs on the propertiesof the resultant scaffolds were examined. The characteristicsof the resultant scaffold were evaluated using ninhydrin assay,FTIR, collagenase degradation assay, denaturation temperaturemeasurements, and in vitro cell studies to ascertain cell viability,proliferation, and morphology.

2. Experimental Section

2.1. Materials and Reagents. Porcine Achilles tendons wereacquired from a local slaughter house. The amine-terminated multiarmpolyethylene glycol Mw 10 KDa (PEG) derivatives were purchased fromJenKem Technology U.S.A. (Allen, TX). Alamar blue was purchasedfrom BioSource Europe (Nivelles, Belgium); Quant-iT PicoGreendsDNA reagent, rhodamine phalloidin, and 4′,6-diamidino-2-phenylin-dole, and dihydrochloride (DAPI) were purchased from Invitrogen (BioSciences Ltd., Dun Laoghaire, Ireland). All other materials and reagentswere purchased from Sigma-Aldrich (Dublin, Ireland) unless otherwisestated.

2.2. Collagen Extraction and Analysis. Typical protocols for theextraction, purification, and analysis of collagen were employed as hasbeen described in detail previously.40 Briefly, frozen porcine Achillestendons were minced, washed in a series of neutral phosphate buffers,and suspended in 0.5 M ethanoic acid in the presence of pepsin (porcinegastric 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 ElectronCorporation, Bath, U.K.) and purified by repeated salt precipitation (0.9M NaCl), centrifugation and acid solubilization (1 M ethanoic acid).The final atelocollagen solution was dialyzed (8000 Mw cut off) against

0.01 M ethanoic acid and kept refrigerated at 4 °C until used. Theatelocollagen solution purity was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, AlphaTechnologies Ltd., Co., Wicklow, Ireland) analysis (90% type I) andits concentration was determined by hydroxyproline assay (3 mg/mL).

2.3. Scaffold Stabilization and Functionalization. Collagen spongeswere obtained after pipetting 1 mL of the dialyzed atelocollagen solutioninto 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 using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)and N-hydroxysuccinimide (NHS) in 50 mM 4-morpholineethane-sulfonic acid in 40% ethanol (MES solution). A molar ratio of EDC toNHS to collagen’s carboxyl groups of 5:5:1 was used.8,53-55 The pHof the solution was adjusted to 5.5 using either 0.1 M sodium hydroxide(NaOH) or 0.1 M hydrochloric acid (HCl). Scaffolds were alsostabilized with 0.625% aqueous glutaraldehyde (GTA) solution as hasbeen described before.8,56,57

To evaluate the functionalization potential of amine-terminated PEGderivatives, 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 theEDC/NHS cross-linking solution as described above. The producedstabilized and stabilized/functionalized scaffolds were washed exten-sively in distilled water for 1 h at 37 °C, followed by overnightlyophilization.

2.4. Ninhydrin Assay. Ninhydrin assay was used to determine theamount of free amines incorporated into the collagenous structure afterfunctionalization using the amine-terminated PEG derivatives as hasbeen 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 ethyleneglycol monoethyl ether. The two solutions were mixed and the pH wasadjusted to 5.5 using 10 M NaOH (ninhydrin solution). A total of 25-30mg of each scaffold was immersed in 200 µL of distilled water, followedby 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 iceand 250 µL of 50% isopropanol was added. The samples were vortexedand the absorbance of the developed Ruhemann’s purple color wasread at 570 nm.

2.5. Fourier Transform InfraRed (FTIR) Spectroscopy. Confor-mational changes in all scaffolds due to cross-linking and cross-linking/functionalization were determined using attenuated total reflectanceFourier transform InfraRed (ATR-FTIR; Shimadzu FTIR-8600, Shi-madzu Europe Ltd., Duisburg, Germany). Spectra were recorded at RTin the mid-infrared range (4000-400 cm-1). A total of 40 scans weresignal-averaged for a single spectrum at a resolution of (8 cm-1 usinga ZnSe crystal at an incident angle of 45°. The spectra were analyzedusing the Hyper-IR software (Shimadzu Europe Ltd., Duisburg,Germany) to obtain quantitative peak information.

2.6. Evaluation of Enzymatic Stability. Dry scaffolds were ac-curately weighed and incubated for 6 h in 1 mL of 50 mM[tris(hydroxymethyl)-methyl-2-aminoethane sulfonate] (TES) buffer (pH7.4) containing 0.36 mM calcium chloride at 37 °C and 2.5 or 5 collagendigestive units (CDU) per mg of collagen collagenase type I fromClostridium histolyticum (0.25-1.0 FALGPA units/mg solid, >125CDU/mg solid). The reaction was subsequently stopped using 0.2 mLof 0.25 M ethylenediaminetetraacetic acid (EDTA) and the mixtureswere centrifuged for 10 min at 1000 rpm at 4 °C (Heraus Fresco 17,Thermo Scientific, Dublin, Ireland). The supernatants were discardedand the pellets were washed twice in distilled water followed bylyophilization. Scaffold degradation was determined from the weightof residual matrix after collagenase degradation and expressed as apercentage of the original weight.56,60

2.7. Evaluation of Thermal Stability. The denaturation temperaturewas determined using the DSC-60 (Shimadzu, Japan) differential

Figure 1. Proposed reaction mode between collagen, EDC/NHS, and4-arm amine-terminated PEG system.

3094 Biomacromolecules, Vol. 11, No. 11, 2010 Ward et al.

scanning calorimeter as has been described previously.61 Briefly, drysamples were incubated overnight in PBS at RT. The following day,the samples were blotted with filter paper to remove excess fluid andhermetically sealed in standard aluminum pans (Mettler-Toledo; MasonTechnology, Dublin, Ireland). Heating was carried out at a constanttemperature ramp of 5 °C/min in the temperature range of 15-100 °C.An empty aluminum pan was used as reference probe. The endothermictransition was recorded as a typical peak and the temperature ofmaximum power of absorption during denaturation was recorded.

2.8. Evaluation of Cell Viability, Morphology, and Activity. R3T3mouse fibroblasts (passage 3-4) were cultured to confluence in T75flasks (Sarstedt Ltd., Wexford, Ireland) containing Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and 1% (v/v) penicillin/streptomycin (PS), 25 mg/L Amphot-ericin-B, and 1% glutamine 200 mM (all were purchased fromInvitrogen, Bio Sciences Ltd., Dun Laoghaire, Ireland). All scaffoldswere disinfected in 70% aqueous ethanol solution followed by thoroughwashing in Hank’s Balanced Salt Solution (HBSS; Invitrogen, BioSciences Ltd., Dun Laoghaire, Ireland). The samples were thenincubated in media for 3 h in a 5% CO2 humidified incubator at 37 °Cprior to seeding. Each scaffold was seeded with a density of 20000cells per well and the media were changed every 24 h. Cell viabilitywas assessed at days 3 and 7 by monitoring their metabolic activityusing the Alamar Blue assay. Briefly at days 3 and 6, all cell-seededscaffolds were transferred to a 24-well plate, rinsed with HBSS and700 µL of 10% (v/v) Alamar Blue reagent in HBSS was added to eachwell. After 1 h of incubation at 37 °C, fluorescence was measured usinga 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 PicoGreenassay as per manufacturer’s guidelines. A standard curve based onknown concentration of DNA was used to determine the total cellnumber. The sample fluorescence was measured using a microplatereader (VICTOR3 V Multilabel Counter, PerkinElmer BioSignal Inc.,U.S.A.) at 480 nm excitation and 520 nm emission.

Cell morphology was evaluated through immunocytochemistry after4% aqueous paraformaldehyde solution fixation for 15 min and washingin 1% BSA in Tris-HCl buffer (pH 7.4) for 30 min to block nonspecificbinding sites. The cell cytoskeleton was stained with rhodaminephalloidin using 1:100 dilution in PBS for 1 h at RT, while the cellnuclei was stained with 1:100 DAPI in PBS for 20 min at RT. Thestained cells on the scaffolds were rinsed with HBSS and examinedunder fluorescence light microscope (Olympus IX81, Olympus Europe,Hamburg, Germany). ImagePro Plus 5.0 software (Media CybeneticsInc., 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 toevaluate cells on the surface of the scaffolds after 7 days in culture.The cell-seeded scaffolds were washed twice with HBSS; fixed in anaqueous 3% GTA solution; dehydrated first using a series of ascendingaqueous ethanol concentrations followed by hexamethyldisilazane; andfinally 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 valueof five samples ( standard deviation. Analysis was performed usingstatistical software (MINITAB version 15, Minitab, Inc., PA, U.S.A.).One-way analysis of variance (ANOVA) for multiple comparisons andtwo-sample t test for pair-wise comparisons were employed afterconfirming the following assumptions: (a) the distribution from whicheach of the samples was derived was normal (Anderson-Darlingnormality test) and (b) the variances of the population of the sampleswere equal to one another (Bartlett’s and Levene’s tests for homoge-nicity of variance). Nonparametric statistics were utilized when eitheror both of the above assumptions were violated and consequentlyKruskal-Wallis test for multiple comparisons or Mann-Whitney testfor two samples were carried out. Statistical significance was acceptedat p < 0.05.

3. Results

3.1. Evaluation of Free Amine Content. Ninhydrin assaywas used to quantitatively assess the free amines present onthe collagen scaffolds. EDC/NHS cross-linking significantlydecreased the amount of free amines when compared to the non-cross-linked collagen scaffolds (p < 0.006; Figure 2). Nosignificant difference (p > 0.05) was observed between the EDC/NHS stabilized scaffolds and the functionalized scaffolds using4- and 6-arm amine-terminated PEG system, independent ofthe 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 thecontrol scaffolds was observed for concentrations above 3% (p< 0.001), but no significant difference was observed among the3, 5, and 10% w/v amount of the 8-arm PEG (p > 0.05).

FTIR spectroscopy was used to provide information aboutchanges in the molecular structure of collagen scaffolds as afunction of cross-linking and functionalization. The representa-tive IR spectra of the various matrices in this study are shownin Figure S3. Quantitative peak information of the individualspectra was obtained using the Hyper-IR software. The absorp-tion peak area ratios of the amide I band at 1635 cm-1 to thatof amide A band were determined and mean values were plotted(Figure 3). The ratio of EDC/NHS and GTA stabilized scaffoldssignificantly increased over the non-cross-linked collagen scaf-folds (p > 0.05). The incorporation of 3, 5, and 10% 8-armamine-terminated PEG on EDC/NHS stabilized scaffolds sig-nificantly decreased the ratio of amide I to amide A over thecontrol EDC/NHS stabilized scaffolds (p < 0.001). No significantdifference (p > 0.05) was observed among the ratios of amideI to amide A of EDC/NHS stabilized and 3, 5, and 10% 8-armamine-terminated PEG-functionalized scaffolds.

3.2. Evaluation of Enzymatic Stability. Collagenase deg-radation assay was used to evaluate the resistance of theproduced scaffolds to enzymatic degradation. The percentageweight of scaffolds remained after collagenase degradation isshown in Figure 4. Noncross-linked collagen scaffolds com-pleted degraded with 5 CDU of collagenase per mg of collagenwithin 6 h. However, cross-linking with either GTA or EDCprohibited degradation by collagenase of the scaffolds for theexperimental period tested (p > 0.05). No significant losses incollagen content were observed for collagen scaffolds fixed withEDC/NHS and functionalized with variable amounts of 8-armamine-terminated PEGs when they were treated with 2.5 CDUof collagenase (p > 0.05). However, some degradation wasdetected when they were treated with 5 CDU of collagenase (p< 0.01).

3.3. Evaluation of Thermal Stability. Differential scanningcalorimetry was employed to evaluate the thermal propertiesof the different scaffolds produced in this study. Figure S4demonstrates typical DSC thermographs, while Figure 5 il-lustrates the denaturation temperatures of all scaffolds evaluatedin this study. Non-cross-linked scaffolds exhibited the lowestdenaturation temperature (44.40 ( 0.95 °C; p < 0.001). GTAand EDC/NHS cross-linked scaffolds exhibited the highestdenaturation temperatures (71.36 ( 0.44 and 70.12 ( 0.58 °C,respectively; p < 0.002). Functionalized scaffolds with 3, 5 and10% of 8-arm amine-terminated PEG exhibited denaturationtemperatures (56.87 ( 0.88, 55.98 ( 0.30 and 59.12 ( 1.92°C, respectively) significantly higher than the non-cross-linkedscaffolds (p < 0.001), but significantly lower than the EDC/NHS fixed scaffolds (p < 0.002).

Amine Functionalization of Collagen Matrices Biomacromolecules, Vol. 11, No. 11, 2010 3095

3.4. Evaluation of Cell Viability, Morphology, andActivity. The results of the Alamar Blue cell viability assay offibroblasts seeded on collagen scaffolds functionalized withvariable concentrations of amine-terminated PEG was comparedto that of fibroblasts seeded on Tissue Culture Plastic (TCP)and EDC/NHS cross-linked scaffolds (positive controls) andGTA stabilized collagen scaffolds (negative control), as mea-sured by fluorescence optical density (OD) are shown in Figure6. No statistical differences of Alamar Blue OD were observedamong 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 significantly higher forthe functionalized scaffolds when compared to the TCP valueson day 7 (p < 0.005). Collagen scaffolds functionalized with

amine-terminated PEGs, EDC/NHS stabilized scaffolds, andTCP exhibited at all tested periods significant higher AlamarBlue OD than the GTA fixed collagen scaffolds counterparts(p < 0.002).

Figure 7 shows the results of DNA content by PicoGreenDNA assay. All scaffolds showed significant increased DNAcontent from day 3 to day 7 (p < 0.003). At days 3 and 7, GTAstabilized scaffolds showed significantly lower DNA contentwhen compared to any other scaffold (p < 0.001). At days 3and 7, no significant difference was observed in the DNAcontent among the scaffolds that were stabilized with EDC/NHS and functionalized with variable amounts of 8-arm amine-terminated PEG system (p > 0.05).

The SEM and fluorescent light micrographs of scaffoldsseeded with 3T3 fibroblast cells are shown in Figure 8. Therewas no morphological difference between cells proliferating onthe different scaffolds. SEM micrographs (Figure 8a,b) illustratethe surface morphology of confluent fibroblasts at days 3 and7, respectively, on scaffolds treated with EDC/NHS and 3%8-arm amine-terminated PEG. DAPI staining shows the highnumber of cells that have been adhered on scaffolds treated withEDC/NHS and 3% 8-arm PEG on day 7 (Figure 8c). Fluorescentimages of scaffolds treated with EDC/NHS and 3% 8-arm PEGafter 7 days in culture demonstrate that the fibroblasts attach toeach other and onto the three-dimensional surface of the scaffoldby elongated dendritic projections (Figure 8d). Intact cytosk-eleton and nuclei of fibroblast cells is also apparent. Thefibroblast cell bodies were typically either round, elongated, orstar-shaped.

4. Discussion

The first generation of biomaterials was aiming to imitatestructural characteristics and the mechanical properties of native

Figure 2. Percentage of amine content of the scaffolds produced in this study, in relation to non-cross-linked collagen scaffolds. Values arepresented as mean ( SD (n ) 3). No significant difference was observed among the EDC/NHS cross-linked scaffolds and the EDC/NHS and8-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/vamount 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 inamine content of the cross-linked collagen scaffolds and an increasein amine content of the EDC/NHS stabilized scaffolds that weretreated with variable amounts of amine-terminated PEG system.


tissues.62 However, the current biomaterial concepts require theuse of biofunctional materials that will incorporate instructivesignals into the scaffolds and modulate host response.9,10 Surfacemodifications or functionalization methods based on

TGase11,12,24-26 or PAMAM dendrimers13,14,27-29 providemeans of anchoring therapeutic molecules onto the scaffoldsor targeting specific ligands. However, the limited stabilizationand functionalization potential of TGase11 and cytotoxicityconcerns of PAMAM dendrimers30-34 have encouraged re-search for alternative functionalization strategies. In this study,we evaluated the functionalization potential that multiarm amine-terminated PEG systems can bring about on collagen scaffoldsusing biochemical, biophysical, and biological assays.

Ninhydrin assay was used for the quantification of free aminesand revealed that cross-linking with EDC/NHS reduced the freeamines of collagen scaffolds in comparison to the non-cross-linked control samples. During carbodiimide cross-linking ofcollagen, carboxylic acid groups of aspartic and glutamic acidresidues in collagen react with EDC and NHS. This results inthe formation of NHS-activated carboxylic acid groups, whichupon reaction with ε-amino groups from lysine and hydroxyllysine residues form peptide-like cross-links and release ofNHS.63-65 Incorporation of 4- and 6-arm amine-terminated PEGdid not significantly contribute to the increase of free amines.However, when the 8-arm amine-terminated PEG system wasused, a significant increase in the free amines was detected,which indicates successful functionalization of the collagenscaffolds. FTIR involves the measurement of wavelength andintensity of absorption of IR light through excitation ofmolecular vibrations, which provides information about changesin molecular structure of organic materials. All spectra weretypical of that observed for proteins.66-68 FTIR analysisdemonstrated that GTA and EDC/NHS cross-linked scaffoldsexhibited 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 thenon-cross-linked scaffolds due to the effective cross-linking. Therationale behind the choice of the peaks 1635 and 1735 cm-1

was that the amount of EDC-activated -COOH groups available

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 collagencontent were observed for collagen scaffolds treated with EDC/NHS and variable amounts of 8-arm amine-terminated PEGs when they weretreated with 2.5 CDU of collagenase (p > 0.05), while some degradation was detected when they were treated with 5 CDU of collagenase for6 h (p < 0.01).

Figure 5. Denaturation temperature of collagen scaffolds evaluatedin this study. Values are presented as mean ( SD (n ) 3). Non-cross-linked scaffolds exhibited the lowest denaturation temperature(p < 0.001). GTA and EDC/NHS cross-linked scaffolds exhibited thehighest denaturation temperatures (p < 0.002). Functionalized scaf-folds with 3, 5, and 10% of 8-arm amine-terminated PEG exhibiteddenaturation temperatures significantly higher than the non-cross-linked scaffolds (p < 0.001), but significantly lower than the EDC/NHS fixed 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 stabilizedscaffolds showed significantly lower DNA content than any other scaffold (p < 0.001). No significant difference was observed in the DNA contentamong the scaffolds that were stabilized with EDC/NHS and functionalized with variable amounts of 8-arm amine-terminated PEG system indays 3 and 7 (p > 0.05).

Figure 6. Alamar Blue fluorescence optical density (OD) of fibroblast seeded collagen scaffolds as a function of time. No statistical differencesof Alamar Blue OD were observed among the TCP and the functionalized with 8-arm amine-terminated PEG systems collagen scaffolds on day3 (p > 0.05), while the Alamar Blue OD values were significant higher for the functionalized scaffolds when compared to the TCP values on day7 (p < 0.005). GTA fixed collagen scaffolds exhibited at all tested periods significant lower Alamar Blue OD than the other scaffolds (p < 0.002).


for reaction are constant because of the use of a fixed amountof EDC in the preparation for all samples. Consequently, anincrease in the 1635 to 1735 cm-1 peak ratios indicates thedecrease in the free -COOH groups and the increase in amidelinkages. 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 at1635 cm-1 to that of amide A band at 1735 cm-1 in comparisonto the EDC/NHS cross-linked scaffolds. We speculate that whenconcentrations above 3% of the 8-arm amine-terminated PEGare used, competition for the carboxyl groups of collagen takesplace that leads to less cross-linking bridges between thepolypeptide chains of the molecule. A similar observation hasbeen reported before when collagen-based scaffolds werestabilized with EDC/NHS and subsequently functionalized witheither amino acids69 or PAMAM dendrimers.13

Differential scanning calorimetry and collagenase digestionwere employed to evaluate the stability of the producedscaffolds. DSC has been used as a sensitive method to examinechanges in collagen structure due to cross-linking.61,70-72 Thedegree of cross-linking of the samples is related to the increasein shrinkage temperature after cross-linking.8,73,74 All samplesexhibited a denaturation temperature and resistance to collage-nase higher than the non-cross-linked scaffolds, which indicatesan increase in stability. The high denaturation of carbodiimidefixed scaffolds has been attributed to the addition of nucleophileNHS that increases the rate and degree of cross-linking, resultingin materials with high Ts and lower free amine groups.65,75,76

The high cross-linking stability of GTA has been attributed toits self-polymerization capabilities.77 Aldehyde groups of GTAreact with either hydroxyl groups and then condense to form aheterocyclic compound, which subsequently undergoes oxidationto a pyridinium ring or with amine groups to form Schiffbases.7,54,73,75,78 Scaffolds treated with EDC/NHS and 3, 5, and10% 8-arm amine-terminated PEG demonstrated denaturationtemperature lower than those that treated with EDC/NHS alone.We also detected some degradation when the same scaffoldswere treated with 5 CDU of collagenase (p < 0.01). These resultsconfirm our previous speculation that competition for thecarboxyl groups of collagen takes place that possibly compro-mises the cross-linking efficiency. However, given that suc-cessful incorporation of free amines was observed, usingninhydrin assay, for concentrations of 3% and above of 8-armamine-terminated PEG, we recommend this concentration asthe minimum effective concentration.

Alamar Blue and PicoGreen assays were used to assessmetabolic activity and cell proliferation on the producedscaffolds. Biological evaluation using 3T3 cells revealed thatthe EDC/NHS and the EDC/NHS and 8-arm amine-terminatedPEG system were characterized by higher biocompatibility thanthe GTA samples. It has been shown that residual EDC/NHSforms urea as a byproduct79,80 and unbound and excesschemicals can be easily washed away and, therefore, areconsidered as nontoxic cross-linking agents.81 GTA, on the other

Figure 8. SEM micrographs of scaffolds treated with EDC/NHS and 3% 8-arm amine-terminated PEG and seeded with 3T3 cells (some cellsare indicated with arrows) for 3 (a) and 7 (b) days. DAPI staining shows the high number of cells that have been adhered on scaffolds treatedwith 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).


hand, due to its self-polymerization capability is associated withcytotoxic drawbacks,77,82-92 which were also evident in thisstudy.

5. Conclusions

In this study we demonstrated that 3% 8-arm amine-terminated PEG system is the minimum effective concentrationrequired to enhance the functionality of EDC/NHS stabilizedcollagen scaffolds. The resultant scaffolds are characterized bybiological, biochemical, and biophysical properties similar orsuperior to non-cross-linked and EDC/NHS or GTA cross-linkedcollagen scaffolds. These results advocate the use of polyeth-ylene glycol systems as a strategy to tether bioactive moleculesinto scaffolds and enhance the biological activity of the producedconstructs.

Acknowledgment. The authors would like to thank M. Abu-Rub, M. Monaghan, J. Chan, C. Holladay, and E. Collin fortheir excellent technical assistance and useful discussions. A.P.would like to acknowledge the Health Research Board ProjectGrant RP/2008/188 for financial support. D.Z. would like toacknowledge the Science Foundation Ireland (SFI_09-RFP-ENM2483) for financial support.

Supporting Information Available. Supporting figures. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

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