cross-linking of extruded collagen fibers—a biomimetic three-dimensional scaffold for tissue...

Cross-linking of extruded collagen fibers—Abiomimetic three-dimensional scaffold for tissueengineering applications

Dimitrios I. Zeugolis,1,2,3 Gordon R. Paul,4 Geoffrey Attenburrow5

1Tissue Modulation Laboratory, National University of Singapore, 117510, Singapore2Division of Bioengineering, Faculty of Engineering, National University of Singapore, 117576, Singapore3Immunology Programme, Department of Microbiology, Yong Loo Lin School of Medicine, National Universityof Singapore, 117456, Singapore4Devro Plc, Glasgow, Scotland, G69 0JE, United Kingdom5School of Applied Sciences, The University of Northampton, NN2 7AL, United Kingdom

Received 1 November 2007; revised 30 November 2007; accepted 25 February 2008Published online 8 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32031

Abstract: The repair of tissue defects remains a challeng-ing clinical problem. Extruded collagen fibers comprise apromising scaffold for anterior cruciate ligament and ten-don reconstruction; however the engineering of thesefibers has still to be improved to bring this material to clin-ical practice. In this study, for the first time we investi-gated the influence of a wide range of cross-linkingapproaches (chemical, physical, and biological) on theproperties of these fibers. Ultrastructural evaluationrevealed a closely packed interfiber structure independentof the cross-linking method employed. The thermal prop-erties were dependent on the cross-linking methodemployed and closely matched native tissues. The stress–strain curves were found to depend on the water contentof the fibers, which was influenced by the cross-linking

method. An inversely proportional relationship betweenboth dry and wet fiber diameter and stress at break wasfound, which indicates that tailored-made biomaterials canbe produced. Overall, the chemical stabilizations weremore potent than both physical and biological approaches.Bifunctional agents such as hexamethylene diisocyanateand ethylene glycol diglycidyl ether or agents that pro-mote matrix formation such as glutaraldehyde producedfibers with properties similar to those of native or syn-thetic fibers to suit a wide range of tissue engineeringapplications. � 2008 Wiley Periodicals, Inc. J BiomedMater Res 89A: 895–908, 2009

Key words: collagen cross-linking; structural, thermal andmechanical properties; biomaterials; tissue engineering

INTRODUCTION

The attractiveness of collagen as a biomaterialrests largely on the view that is a natural materialand is therefore seen by the body as a normal con-stituent rather than foreign matter. Moreover,advancements in purification methods and analyticalassays have essentially assured minimal immunoge-nicity. Additionally, collagen possesses many desira-ble features making it an excellent choice as a bioma-

terial, among which are its high biodegradabilityand resistance to proteolysis; low antigenicity andinflammatory response; and its ability to promotecell attachment and growth and consequently tissuehealing and regeneration.1–4

Collagen based biomaterials in different physicalforms (fibers, films, sponges, hydrogels, powder) arewidely used for tissue engineering applications.5–9

Such collagen scaffolds or polymeric scaffolds coatedwith extracellular matrix proteins10–13 are consideredto be biologically active and consequently shouldblock wound contraction and facilitate organ regen-eration.14,15 The use of collagen fibers to fabricatethree dimensional scaffolds as substrate for nerveregeneration, soft and hard tissue replacement, wounddressing applications, suture materials and knittedmeshes for vascular applications has been advocatedbecause of their unique/advantageous properties,such as high surface area; superior biocompatibility;and easy of fabrication into different forms.16–19

Additional Supporting Information may be found in theonline version of this article.Correspondence to: D. I. Zeugolis; e-mail: dzeugolis@

gmail.comContract grant sponsor: The University of Northampton,

EPSRC

� 2008 Wiley Periodicals, Inc.

It is, however, essential that the physical proper-ties of the fibers to match those of the tissue to bereplaced. Natural cross-linking, the formation ofcovalent inter- and intra-molecular bonds betweenproteins, is utilized by nature to create new entitieswith properties completely different from the origi-nal monomeric form.20 The primary function ofnative cross-linking is to impart desired mechanicalcharacteristics and proteolytic resistance on the colla-gen fibers in connective tissue. However, the lysyloxidase mediated cross-linking would not occurin vitro and consequently reconstituted forms of col-lagen can lack sufficient strength and may disinte-grate upon handling or collapse under the pressurefrom surrounding tissue in vivo, thus a number ofcross-linking approaches (chemical, physical, and bi-ological) have been investigated through theyears3,4,21 that produce matrices with different phy-sical properties depending on the cross-linkingmethod adopted.22,23 However, at present, not onlythere is no commonly accepted ideal cross-linkingtreatment for collagen-derived bio-prostheses, butalso for extruded collagen fibers only a few cross-linking approaches have been investigated, namelyglutaraldehyde,16,24,25 cyanamide,16,24 carbodiimide,25

and dehydrothermal17,18,24 on different collagenpreparations. Since the physical properties of thesefibers depend on the collagen preparation,26,27 theamount27 and type28 of co-agent used in the fab-rication process and the extrusion tube internaldiameter,24,27,29 the influence of the already usedcross-linking approaches cannot be directly com-pared. Therefore, herein we aim to directly compare,determine and evaluate, for first time on the samecollagen preparation, the changes in structural, me-chanical, and thermal properties that different cross-linking methods could bring about.

MATERIALS AND METHODS

Materials

All chemicals, unless otherwise stated, were purchasedfrom Sigma-Aldrich (Dorset, UK). The bovine Achilles ten-dons were kindly provided by the BLC Research Centre(Northampton, UK).

Collagen preparation

Typical protocols for the extraction and purification ofcollagen were employed.26 Briefly, frozen bovine Achillestendons were minced, washed in neutral phosphate buf-fers and suspended in 0.5M ethanoic acid in the presenceof pepsin (2500 U/mg, Roche Diagnostics, Sussex, UK) for72 h at 48C. Consequently, the collagen suspension was

centrifuged (12,000g at 48C for 45 min; Gr20.22 Jouan refri-gerated centrifuge, Thermo Electron Corporation, Bath,UK) and purified by repeated salt precipitation (0.9MNaCl), centrifugation and acid solubilization (1M ethanoicacid). The final atelocollagen collagen solution was dia-lyzed (8000 Mw cut off) against 0.01M ethanoic acid andkept refrigerated at 48C until used. The atelocollagen solu-tion purity was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis (90% type I)and its concentration was determined by hydroxyprolineassay (7 mg/mL).

Micro-fiber fabrication

The fiber formation process has been described in detailpreviously26 and was based on previous publications.17,19

A 5 mL syringe (Terumo Medical Corporation UK Ltd,Merseyside, UK) containing the atelocollagen solution wasloaded on a syringe pump (KD-Scientific 200, KD-ScientificInc., MA), which was set to infuse at 0.4 mL/min andextruded through a 1.5 mm in internal diameter siliconeextrusion tube (Samco Silicone Products, Ltd., Warwick-shire, UK) into a Fiber Formation Buffer (118 mM Phos-phate Buffer and 20% of polyethylene glycol Mw 8k; pH7.50 and 378C). After a period of 15 min, the fibers weretransferred to the Fiber Incubation Buffer (6.0 mM Phos-phate Buffer and 75 mM sodium chloride; pH 7.10 and378C) for further 15 min. Thereafter, control fibers wereincubated for 10 min in distilled water (DW) or werecross-linked (see below).

Cross-linking experimentation

The conditions employed for cross-linking the extrudedcollagen fibers have commonly been reported in the litera-ture as optimum and can be classified as follows:

Chemical fixations

Tanning agents: fibers immersed in 1% basic chromiumsulphate (Elementis Chromium, Eaglescliffe, UK) overnightin distilled water (DW) at room temperature (RT).30

Aldehydes: Fibers immersed in 0.625% formaldehyde;and 0.625% glutaraldehyde (GTA) overnight in 0.01Mphosphate buffer saline (PBS) at RT31–38; and 1.3% starchdialdehyde (suspension) overnight in DW at RT.39,40

Isocyanates: Fibers incubated in a 100% 2-propanol solu-tion for 30 min and then immersed in a 5% hexamethylenediisocyanate (HMDC) solution in 100% 2-propanol over-night.41,42

Carbodiimide: Fibers transferred and remained overnightat RT in a 215 mL 0.05M 2-(N-morpholino) ethanesulfonicacid (MES) buffer containing 1.731 g 1-ethyl-3-[3-dimethy-laminopropyl]carbodiimide (EDC) and 0.415 g N-hydroxy-sulfosuccinimide (NHS); the following day 3-PBS washeswere carried out.43–45

Acyl azide: Fibers transferred and remained overnight atRT in 0.5% diphenylphosphorylazide (DPPA) in dimethyl-formamide (DMF); the following day, three-washes in

896 ZEUGOLIS, PAUL, AND ATTENBURROW

Journal of Biomedical Materials Research Part A

borate buffer (0.04M disodium tetraborate, 0.04M boricacid) were carried out.21,46–48

Epoxides: 4% ethylene glycol diglycidyl ether in 0.025Mdisodium tetraborate overnight at RT.33,34,38

Carbohydrate mediated: Fibers transferred in a 0.25Mribose in 0.2M phosphate buffer overnight at RT.49,50

Quinones: Fibers were immersed overnight at RT in 1 gnordihydroguaiaretic acid (NDGA) in 75 mL 0.1M NaOHand 125 mL of 0.01M PBS.35,51–55

Iridoid glycosides: fibers were transferred and remainedovernight at RT in 0.625% genipin (Challenge Bioproducts,Taichung, Taiwan) in 0.01M PBS.35,51,56–58

Reduction: fibers immersed in 0.05% sodium borohy-dride in DW at RT and then washed overnight in 0.01MPBS at RT.59–61

Biological approach

Enzymatic Cross-linking: Fibers incubated overnight at RTin a 0.1 mg/mL microbial transglutaminase (mTGase) (ForumBioscience Ltd., Redhill, UK) in 50mMTris-HCl.62–64

Physical methods

Ultra violet (UV) irradiation: freshly prepared fiberswere washed for 10 min in DW and dried overnight at RTunder the tension of their own weight. The following day,UV irradiation was carried out overnight at 1 mW/cm2 at1 cm distance.65–74

Dye-mediated Photo-oxidation: Freshly prepared fiberswere incubated overnight in a 0.02M phosphate buffercontaining 0.05% methylene blue and at 10 cm distancefrom the container a 150 W halogen lamp was placed.75,76

Dehydrothermal (DHT) cross-linking: Freshly preparedfibers were washed for 10 min in DW and dried overnightat RT under the tension of their own weight. The follow-ing day, DHT cross-linking was carried out at 1108C,under vacuum (50–100 mtorr) for 6h.17,25,77–79

Control and cross-linked fibers (apart from the DHT-and the UV- treated ones) were washed in DW water toremove traces of un-reacted cross-linking agents, air-driedunder the tension of their own weight at RT for 24 h andconditioned at RT at 65% relative humidity for at least 48 h.

Cross-linking efficiency

The cross-linking efficiency was evaluated through ther-mal and swelling studies.

Denaturation temperature

The hydrothermal stability of the fibers was determinedusing an 822e Mettler-Toledo differential scanning calorim-eter (Mettler-Toledo International Inc., Leicester, UK). Dryreconstituted collagen fibers were hydrated overnight atRT in 0.01M PBS at pH 7.4. The wet fibers were removedand quickly blotted with filter paper to remove excess sur-face water and hermetically sealed in aluminium pans.

Heating was carried out at a constant temperature ramp(58C/min) in the temperature range of 15–1208C, with anempty aluminium pan as the reference probe. Thermaldenaturation, the endothermic transition, was recorded asa typical peak, and two characteristic temperatures weremeasured corresponding to the peak (temperature of maxi-mum power absorption during denaturation) and onset(temperature at which the tangent to the initial power ver-sus temperature line crosses the baseline) temperatures.

Swelling studies

Collagen fibers were incubated overnight in PBS (pH7.4) at room temperature. Subsequently, the fibers wereremoved and quickly blotted using a filter paper toremove excess surface water. The swelling ratio was thencalculated as: 100 3 [(Mean Wet Fiber Diameter) 2 (MeanDry Fiber Diameter)]/(Mean Dry Fiber Diameter).

Mechanical testing and ultrastructuralmatrix analysis

Stress–strain curves of dry and wet reconstituted colla-gen fibers were determined in uniaxial tension using anInstron 1122 Universal testing machine (Instron Ltd, Buck-inghamshire, UK) operated at an extension rate of 10 mm/min. The gauge length was fixed at 3 cm and soft rubberwas used to cover the inside area of the grips to avoiddamaging the fibers at the contact points. Results obtainedwith fibers that broke at contact points with the grips wererejected. The cross sectional area of each fiber was calcu-lated by measuring the diameter at four places along itslongitudinal axis using a Nikon Eclipse E600 optical micro-scope with a calibrated eyepiece (Nikon Instruments, Sur-rey, UK). It was assumed that the fibers were circular forthe cross-sectional area determinations. Fracture surfacesof collagen fibers that had been extended to failure wereexamined using a Hitachi S3000N Variable Pressure Scan-ning Electron Microscope (Hitachi, Berkshire, UK). The fol-lowing definitions were used to calculate the mechanicaldata: stress at break was defined as the load at failure di-vided by the original cross-sectional area (engineering-stress); strain at break was defined as the increase in fiberlength required to cause failure divided by the originallength and modulus was defined as the stress at 0.02 straindivided by 0.02. Prior to wet testing, dried reconstitutedcollagen fibers were equilibrated in PBS (0.01 M; pH 7.4)at room temperature overnight.

Statistical analysis

Numerical data is expressed as mean 6 SD. Analysiswas performed using statistical software (MINITABTM

version 13.1, Minitab, Inc., State College PA). One wayanalysis of variance (ANOVA) for multiple comparisonsand 2-sample t-test for pair wise comparisons wereemployed after confirming the following assumptions: (a)the distribution from which each of the samples wasderived was normal (Anderson-Darling normality test);

CROSS-LINKING OF EXTRUDED COLLAGEN FIBERS 897


and (b) the variances of the population of the sampleswere equal to one another (Bartlett’s and Levene’s tests forhomogenicity of variance). Nonparametric statistics wereutilized when either or both of the above assumptions wereviolated and consequently Kruskal-Wallis test for multiplecomparisons or Mann-Whitney test for 2-samples were car-ried out. Statistical significance was accepted at p< 0.05.

RESULTS

Matrix morphology

Optical microscopic evaluation of the cross-linkedfibers revealed changes in colour occurred for certain

treatments. GTA, epoxide, ribose, UV and DHTcross-linked fibers turned yellow/orange; NDGAtreated fibers became brown, chromium treatedfibers were coloured green, while dye mediated pho-tooxidation and genipin fixation resulted blue fibers.Detailed scanning electron microscopy study of thenoncross-linked collagen fibers revealed a rathersmooth surface structure, with ridges and crevicesrunning roughly parallel to the longitudinal axis ofthe fibers [Fig. 1(a)]. The surface morphology of thecross-linked fibers appeared to depend on themethod employed; moderate cross-linking methods(all physical methods, sodium borohydride, mTGase,and starch dialdehyde) yielded fibers with similarsurface characteristics as the control fibers, whilst

Figure 1. Surface morphology of: (a) non-cross-linked and mild cross-linked extruded collagen fibres; and (b) extensivelycross-linked collagen fibres.

Figure 2. A scanning electron micrograph of the four different fractures modes identified that were independent of thecross-linking method employed; (a) smooth fracture (epoxide fixation); (b) rough fracture (carbodiimide fixation); (c) longi-tudinal split fracture (non-cross-linked); and (d) fibrillation fracture (ribose fixation).



more potent chemical approaches (NDGA, genipin,HMDC, aldehydes, epoxide, and chromium) resultedin fibers with more pronounced ridges and crevicesalong the longitudinal axis of the fibers [Fig. 1(b)].Microscopic evaluation of the failed ends of dryfibers that have been extended to failure revealed aclosely packed interfiber space (Fig. 2). Typical frac-tures modes were identified independent of the fixa-tion method employed and can be classified as: (a)smooth fracture [Fig. 2(a)]; (b) rough fracture withthe internal structure of the fiber appeared slightlydrawn out for the one fraction and pulled in for theother fraction [Fig. 2(b)]; (c) longitudinal splitfracture [Fig. 2(c)]; and (d) fibrillation fracture[Fig. 2(d)].

Cross-linking evaluation

Differential scanning calorimetry was utilized toevaluate the thermal properties of the fixed fibers.Mild cross-linking approaches resulted in fibers withdenaturation temperature similar to the control (p >0.05 for DHT, sodium borohydride, and mTGase),while the other cross-linking techniques significantlyincreased the denaturation temperature of the fibersproduced (p < 0.0085), with the chromium treatedones exhibiting the highest temperature (106.448C;p < 0.001) (Table I). The enthalpy of denaturationwas reduced significantly for every cross-linkingtechnique employed (p < 0.05) (Table I). A variableswelling ratio was observed depending on the cross-linking technique, with the HMDC fixed fibers toswell the least (Table I).

Biomechanical analysis

Uniaxial tensile tests revealed three distinct stress–strain curves that were dependent on the wateruptake of the fibers (Fig. 3); an s-shape curve wasobtained in low swelling/dry state [Fig. 3(a)], and aj-shape curve derived from fibers with high watercontent [Fig. 3(c)]. For the first time we report herethe specific form of the stress–strain curve seen withthe intermediate swelling and is characterized by ans-shape curve up to the knee point, followed bya j-shape curve that persists until failure [Fig. 3(b)].Curves for intermediate and low swelling consistedof a small toe region, a region of sharply increasingstress up to a knee point where the gradient of thecurve reduced, followed by a long region of constantgradient (dry/low swelling) or increasing gradient(intermediate swelling) which persisted until failure.For every treatment, the stress–strain curves showed

TABLE ICross-Linking Evaluation Through Swelling and Thermal Analysis

Dry Diameter 6SD (lm)

Wet Diameter 6SD (lm) % Swelling Peak 6 SD (8C) Energy (J g21)

DW Overnight 171 6 6 (n 5 4) 298 6 17 (n 5 5) 74.27 45.47 6 1.05 (n 5 3) 13.48 6 0.74 (n 5 3)DHT 171 6 13 (n 5 6) 264 6 23 (n 5 4) 54.39 43.23 6 1.11 (n 5 3) 8.99 6 1.62 (n 5 3)Sodium Borohydride 101 6 9 (n 5 6) 314 6 37 (n 5 5) 210.89 43.87 6 1.12 (n 5 3) 7.41 6 0.44 (n 5 3)mTGase 159 6 19 (n 5 6) 623 6 47 (n 5 3) 291.82 47.69 6 0.14 (n 5 3) 4.38 6 0.95 (n 5 3)Starch Dialdehyde 197 6 28 (n 5 7) 313 6 33 (n 5 5) 58.88 50.87 6 1.01 (n 5 3) 9.66 6 0.47 (n 5 3)UV 160 6 9 (n 5 6) 252 6 33 (n 5 5) 57.50 51.78 6 1.07 (n 5 3) 7.50 6 2.22 (n 5 3)Photo-oxidation 178 6 19 (n 5 6) 391 6 22 (n 5 4) 119.66 52.31 6 1.02 (n 5 3) 6.70 6 0.40 (n 5 3)Ribose 153 6 11 (n 5 6) 233 6 22 (n 5 4) 52.29 55.55 6 0.50 (n 5 3) 9.44 6 0.90 (n 5 3)EDC/NHS 167 6 23 (n 5 6) 373 6 47 (n 5 4) 123.35 61.25 6 2.06 (n 5 3) 9.05 6 1.25 (n 5 3)DPPA 201 6 35 (n 5 5) 310 6 44 (n 5 3) 54.23 64.53 6 0.61 (n 5 3) 8.89 6 0.99 (n 5 3)NDGA 309 6 33 (n 5 6) 446 6 64 (n 5 5) 44.34 66.20 6 0.25 (n 5 3) 7.90 6 0.67 (n 5 3)HMDC 332 6 75 (n 5 7) 336 6 26 (n 5 5) 1.20 66.67 6 0.25 (n 5 3) 2.75 6 0.21 (n 5 3)Genipin 240 6 32 (n 5 5) 340 6 47 (n 5 5) 41.67 67.43 6 0.61 (n 5 3) 6.62 6 0.43 (n 5 3)0.625% Formaldehyde 190 6 17 (n 5 6) 306 6 45 (n 5 5) 61.05 70.82 6 1.70 (n 5 3) 1.31 6 0.18 (n 5 3)0.625% GTA 260 6 26 (n 5 5) 306 6 29 (n 5 5) 17.69 75.45 6 0.61 (n 5 3) 1.35 6 0.19 (n 5 3)Epoxide 224 6 44 (n 5 5) 250 6 29 (n 5 5) 11.61 86.52 6 0.45 (n 5 3) 4.22 6 0.31 (n 5 3)Chromium 238 6 32 (n 5 5) 314 6 38 (n 5 3) 31.93 106.44 6 0.87 (n 5 3) 1.14 6 0.12 (n 5 3)

Sample number n in parentheses; SD: standard deviation.

Figure 3. The stress-strain curves observed were found tobe dependent on the water content: (a) s-shape curve forlow water content; (b) intermediate shape curve for inter-mediate water content; and (c) j-shape curve for highwater content.



a diameter dependent variation; thin fibers exhibitedhigh stress to strain ratio graphs and a short toeregion, while thick fibers yielded low stress to strainratio graphs and a long toe region (Fig. 4).

Table II summarizes the physical and mechanicalproperties of the produced fibers in both dry andwet state. The different cross-linking approachesinvestigated herein yielded fibers with variablephysical and mechanical properties in comparisonwith the control ones, in both dry and wet state (seeSupplementary Information Table SI for the statisti-cal analysis). Rehydration of the fibers resulted inreduced modulus values for every treatment (p <0.05); and increased fiber diameter and reducedstress and force at break for every treatment (p <0.05) except for the HMDC (p > 0.05) and the epox-ide (p > 0.05) fixed fibers. The strain at breakremained unaffected after rehydration for control,sodium borohydride, mTGase, UV, dye mediat-ed photo-oxidation, ribose, carbodiimide, DPPA,HMDC, chromium, and formaldehyde treated fibers(p > 0.05); increased for the DHT and NDGA treatedfibers (p < 0.05) and decreased for the starch dialde-hyde, genipin, GTA and epoxide treated fibers (p <0.05).

Fitting a linear regression model between thestress at break and fiber diameter, strong correlationswere obtained in both dry and wet state (Table II).

DISCUSSION

Structural characteristics

Certain cross-linking treatments brought aboutcolor changes on the extruded collagen fibers. Simi-lar discoloration results to ours have been reportedfor bovine, porcine and canine pericardium and der-mal sheep collagen fixed with GTA, epoxy, and gen-ipin33,34,38,51,80 as well as in nonenzymatic glycationof tendons and bones and human skin and lens.81–83

The observed discoloration was attributed to thereaction of the cross-linking agent with the aminoacid residues,33,34,38,51 while the blue color of the dyemediated cross-linking can be attributed to the meth-ylene blue that was utilized for the fixing process.Likewise, tanning using basic chromium sulfateyields green colored matrices.

All fibers exhibited an external surface character-ized by ridges and crevices running along the longi-tudinal axis of the fiber. Such unique morphologicalcharacteristics have been attributed to the substruc-ture of the fibers84 and/or to the handling of the fi-brous structure85 and have been shown to facilitatecell attachment and fibroblast migration.86,87 Thedegree of surface roughness was dependent onthe cross-linking method employed, which is inagreement with previous observations where it wasshown that different cross-linking treatments canmodify the surface properties of collagenous materi-als.88 For example, microbial transglutaminase hasno influence on the surface of the collagen matrix,89

while surface irregularities have been reported forUV-irradiated rat tail tendon fibers.90 The interfiberspace, however, was found consistently filled, whichis in agreement with previous observations for colla-gen fibers,91 composite fibers92 and elastin basedmaterials.93 The fracture modes identified are inaccord with previous publications,30,94–96 where theirrelative occurrence was attributed to the handling ofthe fibers while in the wet state; to the strain rate; oreven to flaws within the fiber structure.

Cross-linking evaluation through thermalproperties and swelling ratio

When collagen in a hydrated state is heated, thecrystalline triple helix will be transformed into amor-phous randomly coiled peptide chains that results inshrinkage of the collagen fiber.97,98 DSC is widelyused to evaluate the denaturation temperature ofbio-scaffolds by investigating the molecule and fibril

Figure 4. The influence of dry (I) and wet (II) fibre diameter on the stress-strain graph. Thin fibres (a) exhibit small toeregions and high stress/low strain graphs, whilst thick fibres (b) display long toe region and low stress/high straingraphs.



TABLEII

PhysicalandM

ech

anicalPropertiesofControlandCross-LinkedExtrudedCollagenFibers

Treatmen

tDiameter

6SD

(lm)

Stressat

Break

6SD

(MPa)

Strainat

Break

6SD

Forceat

Break

6SD

(N)

Modulusat

2.2%

Strain6

SD

(MPa)

R2Value

DW

Overnight

Dry

(n5

4)17

16

611

1.85

610

.11

0.37

60.07

2.55

60.12

154.23

622

.09

0.77

00W

et(n

55)

2986

172.97

60.87

0.33

60.07

0.20

60.04

3.78

61.10

0.90

85Chromium

Dry

(n5

5)23

86

3247

.416

13.46

0.57

60.17

1.89

60.25

23.646

6.54

0.92

82W

et(n

53)

3146

3816

.196

5.89

0.56

60.16

1.19

60.14

7.09

61.31

0.91

45Form

aldeh

yde

Dry

(n5

6)19

06

1763

.886

15.80

0.60

60.10

1.76

60.10

17.456

7.01

0.91

03W

et(n

55)

3066

4512

.006

4.18

0.57

60.08

0.82

60.15

7.92

63.94

0.98

88GTA

Dry

(n5

5)26

06

2631

.896

9.93

0.55

60.05

1.62

60.23

34.206

19.10

0.96

25W

et(n

55)

3066

2910

.856

2.85

0.43

60.04

0.77

60.04

6.90

63.29

0.94

47StarchDialdeh

yde

Dry

(n5

7)19

76

2889

.886

23.76

0.41

60.09

2.63

60.22

28.176

11.94

0.89

94W

et(n

55)

3136

332.44

60.73

0.28

60.06

0.18

60.02

1.55

60.61

0.77

28NDGA

Dry

(n5

6)30

96

3326

.136

8.83

0.22

60.10

1.87

60.28

30.616

17.09

0.82

27W

et(n

55)

4466

646.35

62.57

0.58

60.10

0.92

60.09

4.86

63.17

0.89

97Gen

ipin

Dry

(n5

5)24

06

3239

.856

12.27

0.55

60.06

1.71

60.09

102.25

622

.98

0.97

64W

et(n

55)

3406

476.89

62.52

0.40

60.03

0.59

60.09

5.54

63.95

0.84

93EDC-N

HS

Dry

(n5

6)16

76

2312

5.89

639

.21

0.53

60.08

2.63

60.23

59.186

25.20

0.94

13W

et(n

54)

3736

473.16

60.63

0.54

60.11

0.34

60.03

1.76

60.33

0.94

50DPPA

Dry

(n5

5)20

16

3570

.366

31.88

0.53

60.07

2.03

60.40

16.026

7.20

0.76

67W

et(n

53)

3106

445.11

61.32

0.44

60.13

0.37

60.02

2.95

61.33

0.89

32HMDC

Dry

(n5

7)33

26

7520

.056

8.46

0.29

60.11

1.53

60.10

14.796

5.78

0.97

16W

et(n

55)

3366

2617

.256

5.92

0.45

60.15

1.11

60.41

4.39

62.13

0.81

59Epoxide

Dry

(n5

5)22

46

4451

.466

26.26

0.37

60.05

1.76

60.37

48.906

23.78

0.93

60W

et(n

55)

2506

2930

.216

9.95

0.29

60.04

1.55

60.28

20.026

4.42

0.79

26Ribose

Dry

(n5

6)15

36

1114

5.17

623

.73

0.34

60.07

2.63

60.18

1001

.986

274.05

0.82

72W

et(n

54)

2336

225.38

61.47

0.25

60.09

0.22

60.02

5.96

62.18

0.95

25Sodium

Borohydride

Dry

(n5

6)10

16

940

2.80

645

.59

0.30

60.02

3.23

60.28

1785

.526

413.78

0.76

79W

et(n

55)

3146

372.03

60.70

0.26

60.05

0.15

60.03

4.01

62.91

0.78

05mTGase

Dry

(n5

6)15

96

1917

2.50

647

.14

0.47

60.04

3.27

60.17

351.54

612

4.32

0.98

58W

et(n

53)

6236

470.10

60.04

0.61

60.14

0.03

60.02

0.13

60.09

0.92

01DHT

Dry

(n5

6)17

16

1347

.426

8.23

0.13

60.02

1.07

60.08

35.666

17.08

0.78

85W

et(n

54)

2646

232.46

60.64

0.26

60.08

0.14

60.06

4.41

61.58

0.93

71UV

Dry

(n5

6)16

06

951

.556

9.94

0.08

60.02

1.03

60.10

582.74

629

0.27

0.81

47W

et(n

55)

2526

333.11

61.42

0.21

60.12

0.17

60.13

6.00

62.33

0.94

25Photooxidation

Dry

(n5

6)17

86

1910

5.99

627

.04

0.45

60.04

2.55

60.14

44.826

19.00

0.94

38W

et(n

54)

3916

221.71

60.49

0.47

60.11

0.20

60.04

0.84

60.14

0.87

22

Sam

ple

number

nin

paren

theses;SD:stan

darddev

iation.



integrity; melting profiles of wet tested materialslower than 39–408C (body temperature)99 wouldindicate denaturation and effectively such scaffoldswould be unsuitable for biomedical applicationssince they would decompose upon implantation.Control and cross-linked scaffolds exhibited denatu-ration temperatures ranging from 43 to 1068C,clearly indicating their suitability for tissue engineer-ing applications. Moreover, the noncross-linkedfibers exhibited denaturation temperature higherthan any other noncross-linked collagenous prepara-tion (see Supplementary Information Table SII). Ithas been shown that the intermolecular cross-linksresponsible for increased thermal stability and resist-ance to collagenase digestion, may form most easilyin collagen fibers, due to the increased energy ofcrystallization derived from the interaction betweenthe close packed molecules in the fiber form,100–104

followed by sponges, films and lastly in collagengels, where there is less formation of cross-linksbecause the molecules are more spread.21,101

After cross-linking the fibers exhibited a widerange of denaturation temperatures depending onthe cross-linking method employed which is inaccord with previous observations (see Supplemen-tary Information Table SII).21,33,101,105–107 This dif-ference could be attributed to the stabilizationchemistry of the method employed. For example,chromium, glutaraldehyde (a self-polymerized alde-hyde), and epoxide (a bi-functional agent) fixationsall brought about the highest thermal stability, sincethey could be expected to cross-link a greater num-ber of molecular subunits including those that wererelatively far apart. In accord with our results, it hasbeen shown that fixation with chromium, GTA andDenacolTM involves extensive cross-linking and poly-mer formation within the matrix, leading to a rise inmatrix complexity and thermal stability.108–110 Fur-thermore, denaturation temperature depends on thesize of the co-operating units and the nature of thebonds introduced in the shrinking process; the largerthe unit, the slower the kinetics and the higher theshrinkage temperatures,30,111 which fits with thetheory of the complex matrix formation. In contrastto these highly effective cross-linking approaches,fixation of rat tail tendon in the presence of sodiumborohydride yielded no difference in denaturationtemperature, since increasing the stability of themature aldimine bond by borohydride reductiondoes not affect the length of the bond between thetwo molecules in the native fiber.112 Similarly, it hasbeen shown that TGase cross-linking had no effecton the hydrothermal stability of bovine skin colla-gen; the authors suggested that the average numberof tanning units per collagen monomer required toelicit a change in hydrothermal stability is far greaterthan the number of cross-links that TGase could

incorporate.64,89 However, it has been shown thatmTGase itself113 or in the presence of urea114 andincreased amounts of porcine TGase [collagen toenzyme 50–1 (wt/wt)]103 made it possible to recon-struct collagen and to raise only slightly the denatu-ration temperature. Furthermore, the physical meth-ods although they are more biocompatible than thechemical approaches since they involve the modifica-tion and cross-link formation of the existing matrixcomponents and no-external chemical is used, resultin materials with little added matrix complexity andso there is no significant rise in the thermal stabilitycompared with untreated tissue.3,115–118 Photo-oxida-tion, for example, yields a material with a relativelysmall shrinkage temperature change, having ashrinkage temperature similar to the untreated mate-rial, which suggests that the tissue and/or scaffoldbehaves like the original and that the cross-links didnot influence the tissue/scaffold character.115,119,120

In all cases a significant decrease in enthalpy ofdenaturation was observed. In general, cross-linkingresults in stabilization of the triple helix structure,thus it increases the shrinkage temperature121 anddecreases the entropy of themelting transition.97,100–104

It is important to point out, however, that this transi-tion is a bulk response and does not reflect the exactnumber or location of the cross-links.122,123 Further-more, it has been reported that there is a decrease incollagen denaturation enthalpy change when specificagents have cleaved hydrogen bonds but an increase inenthalpy change in the presence of a hydrophobic exo-thermic bond breaking agent. Such results indicatethat samples with higher denaturation temperaturehave more hydrogen bonds and/or fewer hydro-phobic bonds than samples with lower denaturationtemperatures.124,125

Previous work has shown that the water holdingcapacity of collagen may be modified by cross-link-ing and as such it can be used to indirectly evaluatethe cross-linking effectiveness.88,126 As the equilib-rium water content of the fiber is modified by thecross-linking treatment, some of the water bindingsites may be occupied or removed by the formationof cross-links, which explains the reduced swellingratio.16,17,22,127 If the binding is physical, the swellingprocess is less limited, while with the chemicalcross-linking the swelling is more restricted.39,128

Moreover, it has been suggested that at a given cross-link density, the chemical structure of the cross-linksmay influence the degree of swelling .33,105-107,129. Forexample, in our study as well as in GTA cross-linkingof pericardium tissue, higher swelling ratio wasobtained in comparison to the use of epoxy counter-part. This may indicate that the surface of the epoxyfixed collagenous structure was more hydrophilic thanthe GTA one. This may be because the epoxy hastwo hydrophilic ether bonds (��O��), while GTA has



only hydrophobic carbon-carbon bonds (C��C).38 Inany case, a straight forward relationship between thedenaturation temperature and the enthalpy of denatu-ration with the swelling ratio was not observed andtherefore it can be speculated that a rise in shrinkagetemperature and/or a decrease in the swelling ratiomay not be necessary a predictor of stabilization, butrather a reflection of the particular chemical nature ofthe stabilization.115

Biomechanical evaluation

Uniaxial tensile tests of dry and partially rehy-drated extruded collagen fibers produced stress–strain curves similar to those reported for semicrys-talline polymers that yield and undergo plasticflow.130 The yielding mechanism involves some formof flow that occurs within the fiber, possibly inter-fibrillar slippage, which plays an important role inthe tensile deformation of aligned connective tissuesuch as tendon.131 In all cases, the slope of thestress-strain curve increases with strain, a character-istic of collagenous structures, such as skin, rat tailtendon, extruded collagen fibers, and leather.17,132,133

The low modulus of the toe region that gives riseto a nonlinear stress–strain curve in native tissuessuch as tendons or ligaments has been attributedto the reorientation and uncrimping of the collagenfibrils, as well as the initiation of stretching of thetriple helix, the nonhelical ends and the cross-links.134–136

Typical s-shape curves (dry fibers/low swelling)have been reported for tendon,137 ligaments,92 ex-truded collagen fibers19,29,138 and nano-fibrousmeshes10,12,139; whilst analogous j-shape stress-straincurves (wet fibers/high swelling) have been reportedfor re-hydrated reformed collagen fibers,18,140 peri-cardium tissue36 and rat tail tendon.29 As firstherein, we report the intermediate stress-straincurves (intermediate swelling). It is reasonable toassume that the effect of moisture content on stress-strain curve shapes is due to the plasticizing actionof water resulting in more freedom of movementbeing available to fibers and fibrils thus allowinggreater re-orientation of fibers and fibrils duringstretching. In hydrated collagenous tissue the occur-rence of a j-shaped curve is often associated withcollagen fibers becoming more aligned along thestrain axis during stretching.141 We would identifythe cross-linking density as an important modulatingfactor because it affects the water uptake of thefibers. Others have shown that water content playsan important role in determining the mechanicalproperties of collagen fibers95 and cross-linking den-sity influences the tensile deformation behavior70;

therefore, the higher the density of cross-links, theless water can be bound.113

The force and modulus values were reduced afterrehydration as well as the stress at break values.These large decreases in the mechanical propertiesin the hydrated state suggests that water moleculesact to break down the hydrogen and the electrostaticbonds that hold collagen fibrils together.126 The lossof the mechanical properties may be connected withbreaking up of the inter- and intra- molecular hydro-gen bonds and release of water, which controlsH��O��H��collagen bonds, that is, the number andthe length of distance between the proteinchains.69,90 It is likely that, in the absence of watermolecules, these water-binding sites are available tobond inter-molecularly to stiffen the collagen triplehelix and prevent slippage and translation to occurbetween neighboring molecules.17

In both the dry and wet state, strong inverse corre-lations between fiber diameter and stress at breakwere obtained indicating that by controlling the fiberdiameter, tailor made biomaterials can be producedto suit a wide range of biomedical applications.These strong correlations can be explained in twoways: (a) the tensile strength increases as the cross-sectional area decreases because there is less chancefor defects in thinner sections142–144; or (b) as thefiber diameter decreases, improved longitudinalalignment takes place that enhances strong interac-tions between the collagen fibrils.18,24,29,138 Thesestrong interactions are manifest in the stress–straincurves with the short toe region observed with thethin fibers, while the looser interactions would tendto give the longer toe regions observed in the thickfibers. Moreover, since (a) water acts as a plasticizerfor biopolymers145 and as a mild plasticizer for colla-gen146; (b) water molecules are not removed duringair-drying147; and (c) rigidification of fibers is accom-plished by a loss of water from the fibers,148 it is rea-sonable to assume that thicker fibers could be morehydrated than their thinner counterparts and that iswhy they exhibit longer toe regions.

The important significance of the work reportedherein is that we present for the first time a directcomparison of a wide range of different cross-linkingapproaches on one type of collagen preparation. Theresults clearly demonstrate that different cross-link-ing approaches, due to the different chemistry that isinvolved in the stabilization process, lead to diversemechanical properties. GTA, for example, can self-polymerize and consequently create a three-dimen-sional network with long-range cross-links spanninglarger gaps, which can affect the collagen fibersproperties by stiffening and strengthening thefibers.16,140 In a similar manner, epoxy and HMDCfixations (bifunctional agents) could create linearcross-linking within the collagenous matrix and con-



sequently produce scaffolds with appreciable me-chanical strength as has been observed previ-ously.38,106,108,149 On the other hand, moderate cross-linking approaches (physical methods, biologicalapproach, and sodium borohydride) did not signifi-cantly improve the mechanical properties of thefibers produced. UV irradiation cross-linking, forexample, is thought to be initiated by free radicalsformed on aromatic amino acid residues, which indi-cates a rather limited maximum degree of cross-link-ing due to the small number of tyrosine and phenyl-alanine residues in collagen.3,4,74,102 In addition, apreliminary investigation of the effect of TGasecross-linking on the physical properties of leathershowed a reduction in tensile strength as observedherein; the opposite effect to that which might beexpected from the introduction of more covalentbonds between collagen molecules. However, it wasspeculated that the TGase cross-linking may cova-lently fix the collagen fibers into a conformation thatis not necessarily the most optimal for increased ten-sile strength.64

Most important of all, the fibers produced in thisstudy are characterized by mechanical propertiesthat closely match native tissues. For example,human anterior cruciate ligament, rat tail tendon,bovine, and rabbit Achilles tendons have beenshown to have diameter ranging from 20 to 400 lm,that can withstand mechanical loads from 15 to53 MPa and exhibit strain at break from 7 to40%.18,24,54,94,138,140,150,151 Fixations using epoxide orHMDC yielded fibers with properties falling withinthis range. It has been reported that epoxy com-pounds (ethylene glycol diglycidyl ether) exhibitedlower cytotoxicity than GTA,33 a remarkable re-sistance to degradation and increased mechanicaland thermal properties of the fixed tissue/scaf-folds.38,152,153 The utilization of HMDC has beenadvocated since the reaction of isocyanates withamines does not involve any potentially toxic sideproducts and the isocyanates functional groups reactwith amines at physiological pH in aqueous solu-tions, which is essential for the health of cells. Fur-thermore, the short half-life of isocyanates in waterensures that reactive groups will not be releasedfrom the treated surface over extended time peri-ods.154,155 The bi-functional reagent HMDC has beenused extensively as an alternative for GTA cross-linking.21,33,105–107

CONCLUSIONS

Herein, we investigated for first time the influence,on the properties of the same extruded collagen fibertype, of sixteen different cross-linking approaches.

Chemical stabilization through bifunctional agents oragents that promote matrix formation brought aboutmore pronounced changes. Results obtained demon-strate that these fibers are characterized by thermal,structural, physical, and mechanical properties thatmatch those of native tissues such as tendon and an-terior cruciate ligament. This work supports the useof extruded collagen fibers to constitute an in vitrocollagen three-dimensional scaffold that imitates theextracellular matrix.

The authors thank Mrs. P. Potter, Ms. S. Lee, Mrs. T.Hayes and Mr. L. Stathopoulos for excellent technical as-sistance; and Dr. S. Jeyapalina and Dr. P. Antunes for theiruseful discussion.

References

1. Kielty CM, Grant ME. The collagen family: Structure, assem-bly and organization in the extracellular matrix. In: RoycePM, Steinmann B, editors. Connective Tissue and Its Herit-able Disorders: Molecular, Genetic and Medical Aspects.New York: Wiley; 2002. p 159–221.

2. Lynn AK, Yannas IV, Bonfield W. Antigenicity and immu-nogenicity of collagen. J Biomed Mater Res Part B: Appl Bio-mater 2004;71:343–354.

3. Friess W. Collagen—Biomaterial for drug delivery. Eur JPharm Biopharm 1998;45:113–136.

4. Paul RG, Bailey AJ. Chemical stabilisation of collagen as abiomimetic. The Sci World J 2003;3:138–155.

5. Lu JT, Lee CJ, Bent SF, Fishman HA, Sabelman EE. Thin col-lagen film scaffolds for retinal epithelial cell culture. Bioma-terials 2007;28:1486–1494.

6. Yamamoto M, Takahashi Y, Tabata Y. Controlled release bybiodegradable hydrogels enhances the ectopic bone forma-tion of bone morphogenetic protein. Biomaterials 2003;24:4375–4383.

7. Ueda H, Hong L, Yamamoto M, Shigeno K, Inoue M, TobaT, Yoshitani M, Nakamura T, Tabata Y, Shimizu Y. Use ofcollagen sponge incorporating transforming growth factor-[b]1 to promote bone repair in skull defects in rabbits. Bio-materials 2002;23:1003–1010.

8. Cote MF, Sirois E, Doillon CJ. In vitro contraction rate ofcollagen in sponge-shape matrices. J Biomater Sci Polym Ed1992;3:301–313.

9. Boccafoschi F, Habermehl J, Vesentini S, Mantovani D. Bio-logical performances of collagen-based scaffolds for vasculartissue engineering. Biomaterials 2005;26:7410–7417.

10. He W, Yong T, Ma ZW, Inai R, Teo WE, Ramakrishna S.Biodegradable polymer nanofiber mesh to maintain func-tions of endothelial cells. Tissue Eng 2006;12:2457–2466.

11. Rho KS, Jeong L, Lee G, Seo B-M, Park YJ, Hong S-D, RohS, Cho JJ, Park WH, Min B-M. Electrospinning of collagennanofibers: Effects on the behavior of normal human kerati-nocytes and early-stage wound healing. Biomaterials 2006;27:1452–1461.

12. He W, Ma Z, Yong T, Teo WE, Ramakrishna S. Fabricationof collagen-coated biodegradable polymer nanofiber meshand its potential for endothelial cells growth. Biomaterials2005;26:7606–7615.

13. Chong MSK, Lee CN, Teoh SH. Characterization of smoothmuscle cells on poly-e-caprolactone films. Mater Sci Eng C2007;27:309–312.



14. Sethi KK, Yannas IV, Mudera V, Eastwood M, McFarland C,Brown RA. Evidence for sequential utilization of fibronectin,vitronectin, and collagen during fibroblast-mediated colla-gen contraction. Wound Repair Regen 2002;10:397–408.

15. Yannas IV. Similarities and differences between inducedorgan regeneration in adults and early foetal regeneration.J Royal Soc Interface 2005;2:403–417.

16. Kato YP, Silver FH. Formation of continuous collagen fibres:Evaluation of biocompatibilily and mechanical properties.Biomaterials 1990;11:169–175.

17. Wang MC, Pins GD, Silver FH. Collagen fibres with improvedstrength for the repair of soft tissue injuries. Biomaterials1994;15:507–512.

18. Pins GD, Silver FH. A self-assembled collagen scaffold suita-ble for use in soft and hard tissue replacement. Mater SciEng C 1995;3:101–107.

19. Cavallaro JF, Kemp PD, Kraus KH. Collagen fabrics as bio-materials. Biotechnol Bioeng 1994;43:781–791.

20. Bailey AJ. The chemistry of natural enzyme-induced cross-links of proteins. Amino Acids 1991;1:293–306.

21. Rault I, Frei V, Herbage D, Abdul-Malak N, Huc A. Evalua-tion of different chemical methods for cross-linking collagengel, films and sponges. J Mater Sci Mater Med 1996;7:215–221.

22. Charulatha V, Rajaram A. Influence of different crosslinkingtreatments on the physical properties of collagen mem-branes. Biomaterials 2003;24:759–767.

23. Weadock K, Olson RM, Silver FH. Evaluation of collagencrosslinking techniques. Biomater Med Devices Artif Organs1983–84;11:293–318.

24. Dunn MG, Avasarala PN, Zawadsky JP. Optimization ofextruded collagen fibers for ACL reconstruction. J BiomedMater Res 1993;27:1545–1552.

25. Dunn MG, Tria AJ, Kato YP, Bechler JR, Ochner RS, Zawad-sky JP, Silver FH. Anterior cruciate ligament reconstructionusing a composite collagenous prosthesis. A biomechanicaland histologic study in rabbits. Am J Sports Med 1992;20:507–515.

26. Zeugolis DI, Paul RG, Attenburrow G. Factors influencingthe properties of reconstituted collagen fibers prior to selfassembly: Animal species and collagen extraction method.J Biomed Mater Res A 2008;86A:892–904.

27. Zeugolis DI, Paul RG, Attenburrow G. Extruded collagen-polyethylene glycol fibers for tissue engineering applications.J Biomed Mater Res Part B: Appl Biomater 2008;85B:343–352.

28. Zeugolis DI, Paul RG, Attenburrow G. Engineering extrudedcollagen fibers for biomedical applications. J Appl Polym Sci2008;108:2886–2894.

29. Gentleman E, Lay AN, Dickerson DA, Nauman EA, LivesayGA, Dee KC. Mechanical characterization of collagen fibersand scaffolds for tissue engineering. Biomaterials 2003;24:3805–3813.

30. Rajini KH, Usha R, Arumugam V, Sanjeevi R. Fracturebehaviour of cross-linked collagen fibres. J Mater Sci 2001;36:5589–5592.

31. Yannas IV, Burke JF, Gordon PL, Huang C, Rubenstein RH.Design of an artificial skin. II. Control of chemical composi-tion. J Biomed Mater Res 1980;14:107–132.

32. Visser CE, Voute ABE, Oosting J, Boon ME, Kok LP. Micro-wave irradiation and cross-linking of collagen. Biomaterials1992;13:34–37.

33. Sung H-W, Huang R-N, Huang LLH, Tsai C-C, Chiu C-T.Feasibility study of a natural crosslinking reagent for biolog-ical tissue fixation. J Biomed Mater Res 1998;42:560–567.

34. Huang LL, Sung HW, Tsai CC, Huang DM. Biocompatibilitystudy of a biological tissue fixed with a naturally occurr-ing crosslinking reagent. J Biomed Mater Res 1998;42:568–576.

35. Chang Y, Tsai C-C, Liang H-C, Sung H-W. In vivo evalua-tion of cellular and acellular bovine pericardia fixed with anaturally occurring crosslinking agent (genipin). Biomaterials2002;23:2447–2457.

36. Garcia Paez JM, Jorge Herrero E, Carrera Sanmartin A,Millan I, Cordon A, Martin Maestro M, Rocha A, Arenaz B,Castillo-Olivares JL. Comparison of the mechanical behav-iors of biological tissues subjected to uniaxial tensile testing:Pig, calf and ostrich pericardium sutured with Gore-Tex.Biomaterials 2003;24:1671–1679.

37. Garcia Paez JM, Jorge-Herrero E, Carrera A, Millan I, RochaA, Calero P, Cordon A, Sainz N, Castillo-Olivares JL. Os-trich pericardium, a biomaterial for the construction of valveleaflets for cardiac bioprostheses: Mechanical behaviour,selection and interaction with suture materials. Biomaterials2001;22:2731–2740.

38. Chen CN, Wu CC, Tsai CC, Sung HW, Chang Y. Assess-ment of the pericardia of four mammalian species fixedwith an epoxy compound as pericardial heterografts. J ChinInst Chem Engrs 1997;28:389–397.

39. Rehakova M, Bakos D, Vizarova K, Soldan M, Jurickova M.Properties of collagen and hyaluronic acid compositematerials and their modification by chemical crosslinking.J Biomed Mater Res 1996;30:369–372.

40. Bowes JH, Cater CW. The interaction of aldehydes with col-lagen. Biochim Biophys Acta (BBA) - Protein Struct 1968;168:341–352.

41. Koide M, Osaki K, Konishi J, Oyamada K, Katakura T, Taka-hashi A, Yoshizato K. A new type of biomaterial for artifi-cial skin: Dehydrothermally cross-linked composites offibrillar and denatured collagens. J Biomed Mater Res 1993;27:79–87.

42. Naimark WA, Pereira CA, Tsang K, Lee JM. HMDC cross-linking of bovine pericardial tissue: A potential role of thesolvent environment in the design of bioprosthetic materials.J Mater Sci: Mater Med 1995;6:235–241.

43. van Wachem PB, Plantinga JA, Wissink MJ, Beernink R,Poot AA, Engbers GH, Beugeling T, van Aken WG, Feijen J,van Luyn MJ. In vivo biocompatibility of carbodiimide-crosslinked collagen matrices: Effects of crosslink density,heparin immobilization, and bFGF loading. J Biomed MaterRes 2001;55:368–378.

44. Wissink MJB, Beernink R, Pieper JS, Poot AA, EngbersGHM, Beugeling T, van Aken WG, Feijen J. Binding andrelease of basic fibroblast growth factor from heparinizedcollagen matrices. Biomaterials 2001;22:2291–2299.

45. van Wachem PB, van Luyn MJ, Olde Damink LH, DijkstraPJ, Feijen J, Nieuwenhuis P. Biocompatibility and tissueregenerating capacity of crosslinked dermal sheep collagen.J Biomed Mater Res 1994;28:353–363.

46. Roche S, Ronziere M-C, Herbage D, Freyria A-M. Nativeand DPPA cross-linked collagen sponges seeded with fetalbovine epiphyseal chondrocytes used for cartilage tissue en-gineering. Biomaterials 2001;22:9–18.

47. Brunel G, Piantoni P, Elharar F, Benque E, Marin P, ZahediS. Regeneration of rat calvarial defects using a bioabsorbablemembrane technique: Influence of collagen cross-linking.J Periodontol 1996;67:1342–1348.

48. Petite H, Frei V, Huc A, Herbage D. Use of diphenylphos-phorylazide for cross-linking collagen-based biomaterials.J Biomed Mater Res 1994;28:159–165.

49. Hegde PS, Chandrakasan G, Chandra TS. Inhibition of colla-gen glycation and crosslinking in vitro by methanolicextracts of Finger millet (Eleusine coracana) and Kodo millet(Paspalum scrobiculatum). J Nutr Biochem 2002;13:517–521.

50. Reddy GK, Stehno-Bittel L, Enwemeka CS. Glycation-Induced Matrix Stability in the rabbit Achilles Tendon. ArchBiochem Biophys 2002;399:174–180.



51. Liang H-C, Chang Y, Hsu C-K, Lee M-H, Sung H-W. Effectsof crosslinking degree of an acellular biological tissue on itstissue regeneration pattern. Biomaterials 2004;25:3541–3552.

52. Koob TJ, Willis TA, Hernandez DJ. Biocompatibility ofNDGA-polymerized collagen fibers. I. Evaluation of cytotox-icity with tendon fibroblasts in vitro. J Biomed Mater Res2001;56:31–39.

53. Koob TJ, Willis TA, Qiu YS, Hernandez DJ. Biocompatibilityof NDGA-polymerized collagen fibers. II. Attachment,proliferation, and migration of tendon fibroblasts in vitro.J Biomed Mater Res 2001;56:40–48.

54. Koob TJ, Hernandez DJ. Material properties of polymerizedNDGA-collagen composite fibers: Development of biologi-cally based tendon constructs. Biomaterials 2002;23:203–212.

55. Koob TJ, Hernandez DJ. Mechanical and thermal propertiesof novel polymerized NDGA-gelatin hydrogels. Biomaterials2003;24:1285–1292.

56. Sung H-W, Chang Y, Chiu C-T, Chen C-N, Liang H-C. Me-chanical properties of a porcine aortic valve fixed with anaturally occurring crosslinking agent. Biomaterials 1999;20:1759–1772.

57. Sung H-W, Chen C-N, Huang R-N, Hsu J-C, Chang W-H. Invitro surface characterization of a biological patch fixedwith a naturally occurring crosslinking agent. Biomaterials2000;21:1353–1362.

58. Tsai C-C, Chang Y, Sung H-W, Hsu J-C, Chen C-N. Effectsof heparin immobilization on the surface characteristics of abiological tissue fixed with a naturally occurring crosslink-ing agent (genipin): An in vitro study. Biomaterials 2001;22:523–533.

59. Bailey AJ. Intermediate labile intermolecular crosslinks incollagen fibres. Biochim Biophys Acta (BBA) 1968;160:447–453.

60. Rousseau CF, Gagnieu CH. In vitro cytocompatibility of por-cine type I atelocollagen crosslinked by oxidized glycogen.Biomaterials 2002;23:1503–1510.

61. Tanzer ML, Mechanic G. Collagen reduction by sodium bor-ohydride: Effects of reconstitution, maturation and lathyr-ism. Biochem Biophys Res Commun 1968;32:885–892.

62. Folk JE, Cole PW. Transglutaminase: Mechanistic features ofthe active site as determined by kinetic and inhibitor stud-ies. Biochim Biophys Acta (BBA) 1966;122:244–264.

63. Jelenska MM, Fesus L, Kopec M. The comparative ability ofplasma and tissue transglutaminases to use collagen as asubstrate. Biochim Biophys Acta 1980;616:167–178.

64. Collighan RJ, Li X, Parry J, Griffin M, Clara S. Transglutami-nases as tanning agents for the leather industry. JALCA2004;99:293–302.

65. Cooper DR, Davidson RJ. The Effect of ultraviolet irradia-tion on soluble collagen. Biochem J 1965;97:139–147.

66. Cooper DR, Davidson RJ. The effect of ultraviolet irradiationon collagen-fold formation. Biochem J 1966;98:655–661.

67. Davidson RJ, Cooper DR. The effect of ultraviolet irradiationon acid-soluble collagen. Biochem J 1967;105:965–969.

68. Yunoki S, Suzuki T, Takai M. Stabilization of low denatura-tion temperature collagen from fish by physical cross-linkingmethods. J Biosci Bioeng 2003;96:575–577.

69. Sionkowska A. The influence of UV light on collagen/poly(ethylene glycol) blends. PolymDegrad Stab 2006;91:305–312.

70. Lee CR, Grodzinsky AJ, Spector M. The effects of cross-link-ing of collagen-glycosaminoglycan scaffolds on compressivestiffness, chondrocyte-mediated contraction, proliferation andbiosynthesis. Biomaterials 2001;22:3145–3154.

71. Tyan Y-C, Liao J-D, Klauser R, Wu I-D, Weng C-C. Assess-ment and characterization of degradation effect for the var-ied degrees of ultra-violet radiation onto the collagen-bonded polypropylene non-woven fabric surfaces. Biomate-rials 2002;23:65–76.

72. Ryu A, Naru E, Arakane K, Masunaga T, Shinmoto K,Nagano T, Hirobe M, Mashiko S. Cross-linking of collagenby singlet oxygen generated with UV-A. Chem Pharm Bull(Tokyo) 1997;45:1243–1247.

73. Bellincampi LD, Closkey RF, Prasad R, Zawadsky JP, DunnMG. Viability of fibroblast-seeded ligament analogs after au-togenous implantation. J Orthop Res: Official Publ OrthopRes Soc 1998;16:414–420.

74. Sionkowska A, Skopinska J, Wisniewski M. Photochemicalstability of collagen/poly (vinyl alcohol) blends. PolymDegrad Stab 2004;83:117–125.

75. Adams AK, Talman EA, Campbell L, McIlroy BK, MooreMA. Crosslink formation in porcine valves stabilized bydye-mediated photooxidation. J Biomed Mater Res 2001;57:582–587.

76. Mechanic GL (University of North Carolina, Assignee). Pro-cess for cross-linking collagenous material and resultingproduct. US Patent 5,147,514, 1992.

77. Yannas IV, Tobolsky AV. Cross-linking of gelatine by dehy-dration. Nature 1967;215:509–510.

78. Yamamoto Y, Nakamura T, Shimizu Y, Takimoto Y, Matsu-moto K, Kiyotani T, Yu L, Ueda H, Sekine T, Tamura N. Ex-perimental replacement of the thoracic esophagus with abioabsorbable collagen sponge scaffold supported by a sili-cone stent in dogs. ASAIO J 1999;45:311–316.

79. Collins RL, Christiansen D, Zazanis GA, Silver FH. Use ofcollagen film as a dural substitute: Preliminary animal stud-ies. J Biomed Mater Res 1991;25:267–276.

80. van Wachem PB, Zeeman R, Dijkstra PJ, Feijen J, HendriksM, Cahalan PT, van Luyn MJ. Characterization and biocom-patibility of epoxy-crosslinked dermal sheep collagens.J Biomed Mater Res 1999;47:270–277.

81. Reddy GK. Glucose-mediated in vitro glycation modulatesbiomechanical integrity of the soft tissues but not hard tis-sues. J Orthop Res 2003;21:738–743.

82. Nagaraj RH, Monnier VM. Protein modification by the deg-radation products of ascorbate: Formation of a novel pyrrolefrom the Maillard reaction of L-threose with proteins. Bio-chim Biophys Acta 1995;1253:75–84.

83. Hong S-B, Lee K-W, Handa JT, Joo C-K. Effect of advancedglycation end products on lens epithelial cells in vitro. Bio-chem Biophys Res Commun 2000;275:53–59.

84. Christiansen DL, Silver FH. Mineralization of an axiallyaligned collagenous matrix: A morphological study. CellsMater 1993;3:177–188.

85. Shao Y, Qiu J, Shah SP. Microstructure of extruded cement-bonded fiberboard. Cement Concr Res 2001;31:1153–1161.

86. Cornwell KG, Downing B, Pins GD Characterizing fibroblastmigration on discrete collagen threads for applications intissue regeneration. J Biomed Mater Res Part A 2004;71:55–62.

87. Cornwell KG, Lei P, Andreadis ST, Pins GD Crosslinking ofdiscrete self-assembled collagen threads: Effects on mechani-cal strength and cell-matrix interactions. J Biomed Mater ResPart A 2007;80A:362–371.

88. Cote M-F, Doillon CJ. Wettability of cross-linked collagenousbiomaterials: In vitro study. Biomaterials 1992;13:612–616.

89. Nomura Y, Toki S, Ishii Y, Shirai K. Effect of Transglutami-nase on reconstruction and Physicochemical properties ofcollagen gel from shark type I collagen. Biomacromolecules2001;2:105–110.

90. Sionkowska A, Wess T. Mechanical properties of UV irradi-ated rat tail tendon (RTT) collagen. Int J of Biol Macromol2004;34:9–12.

91. Brokaw JL, Doillon CJ, Hahn RA, Birk DE, Berg RA, SilverFH. Turbidimetric and morphological studies of type I colla-gen fibre self assembly in vitro and the influence of fibro-nectin. Int J Biol Macromol 1985;7:135–140.



92. Hepworth DG, Smith JP. The mechanical properties of com-posites manufactured from tendon fibres and pearl glue(animal glue). Compos Part A: Appl Sci Manuf 2002;33:797–803.

93. Dutoya S, Verna A, Lefebvre F, Rabaud M. Elastin-derivedprotein coating onto poly(ethylene terephthalate). Technical,microstructural and biological studies. Biomaterials 2000;21:1521–1529.

94. Pins G, Huang E, Christiansen D, Silver F. Effects of staticaxial strain on the tensile properties and failure mechanismsof self-assembled collagen fibers. J Appl Polym Sci 1997;63:1429–1440.

95. Arumugam V, Naresh MD, Somanathan N, Sanjeevi R.Effect of strain rate on the fracture behaviour of collagen.J Mater Sci 1992;27:2649–2652.

96. Arumugam V, Naresh MD, Sanjeevi R. Effect of strain rateon the fracture behaviour of skin. J Biosci 1994;19:307–313.

97. Mentink CJAL, Hendriks M, Levels AAG, WolffenbuttelBHR. Glucose-mediated cross-linking of collagen in rat ten-don and skin. Clin Chim Acta 2002;321:69–76.

98. Hormann H, Schlebusch H. Reversible and irreversibledenaturation of collagen fibers. Biochemistry 1971;10:932–937.

99. Wallace DG, Rosenblatt J, Ksander GA. Tissue compatibilityof collagen-silicone composites in a rat subcutaneous model.J Biomed Mater Res 1992;26:1517–1534.

100. Friess W, Lee G. Basic thermoanalytical studies of insolublecollagen matrices. Biomaterials 1996;17:2289–2294.

101. Chevallay B, Abdul_Malak N, Herbage D. Mouse fibroblastsin long-term culture within collagen three-dimensional scaf-folds: Influence of crosslinking with diphenylphosphoryla-zide on matrix reorganization, growth, and biosynthetic andproteolytic activities. J Biomed Mater Res 2000;49:448–459.

102. Sionkowska A. Thermal denaturation of UV-irradiated wetrat tail tendon collagen. Int J Biol Macromol 2005;35:145–149.

103. Orban JM, Wilson LB, Kofroth JA, El-Kurdi MS, Maul TM,Vorp DA. Crosslinking of collagen gels by transglutaminase.J Biomed Mater Res Part A 2004:68:756–762.

104. Bailey AJ, Light ND. Connective Tissue in Meat and MeatProducts. London: Elsevier Applied Science; 1989.

105. Damink LHHO, Dijkstra PJ, van Luyn MJA, van WachemPB, Nieuwenhuis P, Feijen J. Cross-linking of dermal sheepcollagen using a water-soluble carbodiimide. Biomaterials1996;17:765–773.

106. Damink LHHO, Dijkstra PJ, van Luyn MJA, van WachemPB, Nieuwenhuis P, Feijen J. Crosslinking of dermal sheepcollagen using hexamethylene diisocyanate. J Mater SciMater Med 1995;6:429–434.

107. Damink LHHO, Dijkstra PJ, van Luyn MJA, van WachemPB, Nieuwenhuis P, Feijen J. In vitro degradation of dermalsheep collagen cross-linked using a water-soluble carbodii-mide. Biomaterials 1996;17:679–684.

108. Chachra D, Gratzer PF, Pereira CA, Lee JM. Effect ofapplied uniaxial stress on rate and mechanical effects ofcross-linking in tissue-derived biomaterials. Biomaterials 1996;17:1865–1875.

109. Madhan B, Muralidharan C, Jayakumar R. Study on the sta-bilisation of collagen with vegetable tannins in the presenceof acrylic polymer. Biomaterials 2002;23:2841–2847.

110. Covington AD, Song L. Crosslinking—What Crosslinking?Studies on the general mechanism of tanning. IULTCS.Cancun,Mexico, 2003.

111. Usha R, Ramasami T. Structure and conformation of intra-molecularly cross-linked collagen. Colloids Surf B 2005;41:21–24.

112. Miles CA, Avery NC, Rodin VV, Bailey AJ. The Increase indenaturation temperature following cross-linking of collagen

is caused by dehydration of the fibres. J Mol Biol 2005;346:551–556.

113. Chen R-N, Ho H-O, Sheu M-T. Characterization of collagenmatrices crosslinked using microbial transglutaminase.Biomaterials 2005;26:4229–4235.

114. Nomura Y, Toki S, Ishii Y, Shirai K. Improvement of sharktype I collagen with microbial transglutaminase in urea.Biosci Biotechnol Biochem 2001;65:982–985.

115. Moore MA, Chen WM, Phillips RE, Bohachevsky IK, McIl-roy BK. Shrinkage temperature versus protein extraction asa measure of stabilization of photooxidized tissue. J BiomedMater Res 1996;32:209–214.

116. Weadock KS, Miller EJ, Keuffel EL, Dunn MG. Effect ofphysical crosslinking methods on collagen-fiber durability inproteolytic solutions. J Biomed Mater Res 1996;32:221–226.

117. Zahedi S, Bozon C, Brunel G. A 2-year clinical evaluation ofa diphenylphosphorylazide-cross-linked collagen membranefor the treatment of buccal gingival recession. J Periodontol1998;69:975–981.

118. Zahedi S, Legrand R, Brunel G, Albert A, Dewe W, Cou-mans B, Bernard JP. Evaluation of a diphenylphosphoryla-zide-crosslinked collagen membrane for guided bone regen-eration in mandibular defects in rats. J Periodontol 1998;69:1238–1246.

119. Moore MA, Bohachevsky IK, Cheung DT, Boyan BD, ChenWM, Bickers RR, McIlroy BK. Stabilization of pericardial tis-sue by dye-mediated photooxidation. J Biomed Mater Res1994;28:611–618.

120. Moore MA, Adams AK. Calcification resistance, biostability,and low immunogenic potential of porcine heart valvesmodified by dye-mediated photooxidation. J Biomed MaterRes 2001;56:24–30.

121. Wissink MJB, Beernink R, Pieper JS, Poot AA, EngbersGHM, Beugeling T, van Aken WG, Feijen J. Immobilizationof heparin to EDC/NHS-crosslinked collagen. Characteriza-tion and in vitro evaluation. Biomaterials 2001;22:151–163.

122. Anselme K, Petite H, Herbage D. Inhibition of calcificationin vivo by acyl azide cross-linking of a collagen-glycosami-noglycan sponge. Matrix (Stuttgart, Germany) 1992;12:264–273.

123. Friess W, Uludag H, Foskett S, Biron R. Bone regenerationwith recombinant human bone morphogenetic protein-2(rhBMP-2) using absorbable collagen sponges (ACS): Influ-ence of processing on ACS characteristics and formulation.Pharm Dev Technol 1999;4:387–396.

124. Kopp J, Bonnet M, Renou JP. Effect of collagen crosslinkingon collagen-water interactions (a DSC investigation). Matrix(Stuttgart, Germany) 1989;9:443–450.

125. Finch A, Ledward DA. Shrinkage of collagen fibres: A dif-ferential scanning calorimetric study. Biochim Biophys Acta(BBA) 1972;278:433–439.

126. Pieper JS, Oosterhof A, Dijkstra PJ, Veerkamp JH, van Kup-pevelt TH. Preparation and characterization of porous cross-linked collagenous matrices containing bioavailable chon-droitin sulphate. Biomaterials 1999;20:847–858.

127. Hutcheon GA, Messiou C, Wyre RM, Davies MC, DownesS. Water absorption and surface properties of novel poly(e-thylmethacrylate) polymer systems for use in bone and car-tilage repair. Biomaterials 2001;22:667–676.

128. Vizarova K, Bakos D, Rehakova M, Macho V. Modificationof layered atelocollagen by ultraviolet irradiation and chemi-cal cross-linking: Structure stability and mechanical proper-ties. Biomaterials 1994;15:1082–1086.

129. Yannas IV. Collagen and Gelatin in the Solid State. J Macro-mol Sci Part C: Polym Rev 1972;7:49–106.

130. Attenburrow GE, Bassett DC. Compliances and failuremodes of oriented chain-extended polyethylene. J Mater Sci1979;14:2679–2687.



131. Knight DP, Nash L, Hu XW, Haffegee J, Ho MW. In vitro for-mation by reverse dialysis of collagen gels containing highlyoriented arrays of fibrils. J Biomed Mater Res 1998;41:185–191.

132. Attenburrow GE. The rheology of leather - A review. J SocLeather Technol Chem 1993;77:107–114.

133. Fratzl P, Misof K, Zizak I, Rapp G, Amenitsch H, BernstorffS. Fibrillar structure and mechanical properties of collagen.J Struct Biol 1997;122:119–122.

134. Silver FH, Christiansen DL, Snowhill PB, Chen Y. Transitionfrom viscous to elastic-based dependency of mechanicalproperties of self-assembled type I collagen fibers. J ApplPolym Sci 2001;79:134–142.

135. Silver FH, Christiansen DL, Snowhill PB, Chen Y. Role ofstorage on changes in the mechanical properties of tendonand self-assembled collagen fibres. Connect Tissue Res2000;41:155–164.

136. Purslow PP, Wess TJ, Hukins DWL. Collagen orientationand molecular spacing during creep and stress-relaxation insoft connective tissues. J Exp Biol 1998;201:135–142.

137. Rigby BJ, Hirai N, Spikes JD, Eyring H. The mechanicalproperties of rat tail tendon. J Gen Physiol 1959;43:265–283.

138. Pins GD, Christiansen DL, Patel R, Silver FH. Self-assembly ofcollagen fibers. Influence of fibrillar alignment and decorin onmechanical properties. Biophys J 1997;73:2164–2172.

139. Kwon IK, Matsuda T. Co-Electrospun Nanofiber Fabrics ofPoly(L-lactide-co-e-caprolactone) with Type I Collagen orHeparin. Biomacromolecules 2005;6:2096–2105.

140. Kato YP, Christiansen D, Hahn RA, Shieh S-J, Goldstein JD,Silver FH.Mechanical properties of collagen fibres: A compari-son of reconstituted and rat tail tendon fibres. Biomaterials1989;10:38–42.

141. Viidik A. Rheology of skin with special reference to age-related parameters and their possible correlation to struc-ture. In: Robert L, editor. Frontier of Matrix Biology. Agingof Connective Tissue Skin. Basel: Karger; 1973. p 157–189.

142. Saito Y, Minami K, Kobayashi M, Nakao Y, Omiya H, Ima-mura H, Sakaida N, Okamura A. New tubular bioabsorb-able knitted airway stent: Biocompatibility and mechanicalstrength. J Thorac Cardiovasc Surg 2002;123:161–167.

143. Hull D, Clyne TW. An Introduction to Composite Materials.Cambridge Solid State Science Series. Cambridge: Cam-bridge University Press; 1996.

144. Thomason JL. The influence of fibre properties of the per-formance of glass-fibre-reinforced polyamide 6,6. ComposSci Technol 1999;59:2315–2328.

145. Taravel MN, Domard A. Collagen and its interactions withchitosan. III. Some biological and mechanical properties.Biomaterials 1996;17:451–455.

146. Menard C, Mitchell S, Spector M. Contractile behavior ofsmooth muscle actin-containing osteoblasts in collagen-GAGmatrices in vitro: Implant-related cell contraction. Biomateri-als 2000;21:1867–1877.

147. El Feninat F, Ellis T, Sacher E, Stangel I. Moisture-dependentrenaturation of collagen in phosphoric acid etched humandentin. J Biomed Mater Res 1998;42:549–553.

148. Rosenblatt J, Devereux B, Wallace DG. Injectable collagen asa pH-sensitive hydrogel. Biomaterials 1994;15:985–995.

149. Osborne CS, Barbenel JC, Smith D, Savakis M, Grant MH.Investigation into the tensile properties of collagen/chondroitin-6-sulphate gels: The effect of crosslinkingagents and diamines. Med Biol Eng Comput 1998;36:129–134.

150. Moutos FT, Freed LE, Guilak F. A biomimetic three-dimen-sional woven composite scaffold for functional tissue engi-neering of cartilage. Nat Mater 2007;6:162–167.

151. Kato YP, Dunn MG, Zawadsky JP, Tria AJ, Silver FH.Regeneration of Achilles tendon with a collagen tendonprosthesis. Results of a one-year implantation study. J BoneJoint Surg (American Volume) 1991;73:561–574.

152. Courtman DW, Errett BF, Wilson GJ. The role of crosslink-ing in modification of the immune response elicited againstxenogenic vascular acellular matrices. J Biomed Mater Res2001;55:576–586.

153. Zeeman R, Dijkstra PJ, van Wachem PB, van Luyn MJA,HendriksM, Cahalan PT, Feijen J. Successive epoxy and carbo-diimide cross-linking of dermal sheep collagen. Biomaterials1999;20:921–931.

154. Panza JL, Wagner WR, Rilo HL, Rao RH, Beckman EJ, Rus-sell AJ. Treatment of rat pancreatic islets with reactive PEG.Biomaterials 2000;21:1155–1164.

155. Deible CR, Petrosko P, Johnson PC, Beckman EJ, Russell AJ,Wagner WR. Molecular barriers to biomaterial thrombosisby modification of surface proteins with polyethylene glycol.Biomaterials 1998;19:1885–1893.



cross-linking of extruded collagen fibers—a biomimetic three-dimensional scaffold for tissue...

Documents