Biomaterials 25 (2004) 2831–2841

ARTICLE IN PRESS

*Correspondin

6806.

E-mail addres

0142-9612/$ - see

doi:10.1016/j.bio

Influence of different collagen species on physico-chemical propertiesof crosslinked collagen matrices

Peter Angelea,*, Jochen Abkeb, Richard Kujata, Hubert Faltermeiera,Detlef Schumanna, Michael Nerlicha, Bernd Kinnera, Carsten Englerta,

Zbigniew Ruszczakc, Robert Mehrlc, Rainer Muellerb

a Department of Trauma Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93051, Germanyb Laboratory of Interface Chemistry, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany

c Innocoll– Innovative Collagen Products, DonaustraX e 24, 93342 Saal/Donau, Germany

Received 25 July 2003; accepted 17 September 2003

Abstract

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present

study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl)

carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino

acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress–strain characteristics were

determined to evaluate the physico-chemical properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA

analysis revealed a highly significant effect of collagen type (p ¼ 0:0001), crosslinking (p ¼ 0:0001) and time (p ¼ 0:0001) on

degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced

swelling ratio compared to bovine collagen samples (po0:0001). The amino acid composition of equine collagen revealed a higher

amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between

equine (60�C) and bovine collagen (57�C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type

(p ¼ 0:0001), crosslinking (p ¼ 0:0001) and matrix condition (p ¼ 0:0001) on rupture strength measured by stress–strain analysis.

The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant

differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of

transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for

scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-

chemical properties.

r 2003 Elsevier Ltd. All rights reserved.

Keywords: Crosslinking; Collagen; Tissue engineering; DSC; Degradation; Mechanical properties

1. Introduction

The repair of musculoskeletal tissue defects, e.g.cartilage, bone, tendon, muscle, nerve, skin, is still achallenging clinical problem. With the development oftissue engineering and its promising repair strategies,there is an increasing interest in developing biocompa-tible and biodegradable materials for tissue regenera-tion. As well as absorbable and non-absorbable

g author. Tel.: +49-941-944-6805; fax: +49-941944-

s: angelepeter@aol.com (P. Angele).

front matter r 2003 Elsevier Ltd. All rights reserved.

materials.2003.09.066

synthetics, biomaterials with chondroconductive proper-ties based on natural polymers, particularly collagenhave been developed [1–14]. Collagen substrates areknown to influence the growth characteristics of cellsand also to modulate various aspects of cell behaviorlike cell-adhesion, proliferation and differentiation[3,9,10,15–21].

The disadvantages of using collagen as a biomaterialfor tissue repair are its low biomechanical stiffness andrapid biodegradation [22]. The high enzymatic turnoverrate of natural collagen in vivo makes stabilization ofcollagen-based biomaterials necessary. This can beachieved by chemical crosslinking methods, which

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–28412832

provide biomaterials with desired mechanical propertiesfor implantation and defect repair [23–25]. Severalchemical agents have been used to achieve this goal.Glutaraldehyde (GA), a bifunctional reagent for brid-ging amino groups, is the most widely used reagent forcrosslinking collagen. However, GA is associated withcytotoxicity in vitro and in vivo, caused by the presenceof unreacted functional groups or by the release of thosegroups during enzymatic degradation of the crosslinkedbiomaterials [26,27].

Methods have been developed that allow the cross-linking of collagen materials directly without incorpora-tion of the crosslinking reagent. For example, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) oracylazide were used to generate peptide-like bonds inbiomaterials [28,29]. Use of EDC and N-hydroxysucci-nimide (NHS) to crosslink collagen seems to yieldbiomaterials with good biocompatibility, higher cellulardifferentiation potential [20,30,31] and with increasedresistance against enzymatic degradation [28,32]. Thephysicochemical and biological properties after cross-linking have been described extensively [29,33–35]. ForEDC/NHS-crosslinking, collagen of bovine [20,25,31,32,36–38], ovine [24,28,39–41] or porcine [30,42] origin hasbeen used.

Potential risks with the use of bovine collagen, such asthe possibility of transmitting mad cow disease, hasproven a significant drawback for manufacturingbiomaterials based on this material. The use of equinecollagen would serve as an alternative, free of thepotential risk of this infection. However systematiccomparative studies on the species-related properties ofbovine and equine collagen are missing. The principalgoal of this study was to assess the differences betweenthe properties of bovine and equine-based collagen astissue engineering scaffolds. The physico-chemical andultrastructural differences were evaluated with andwithout crosslinking with EDC and NHS.

2. Material and methods

2.1. Substrate materials

Non-soluble, native equine and bovine fibrillar type Icollagen was separated from Achilles tendons andpurified by acidic and basic hydrolysis as well ascontrolled enzymatic treatment with pepsin, accordingto a standardized industrial protocol, which is also usedfor the production of the commercially availablecollagen-based hemostyptika Collatampt, Colla-tampEt, and for commercial collagen-based drugdelivery systems by Innocoll GmbH, Saal/Donau,Germany (Medical Grade Collagen, ISO 9001). Thenewly developed scaffolds were especially designed fortissue engineering purposes with a higher collagen

concentration (25 mg/ml) for improved mechanicalstiffness and cell friendly environment. The scaffold’sstructure and porosity were engineered to fulfill specificcell and tissue growth requirements.

Due to the preparation and purification processes(enzymatic treatment, acid and base treatment, oxida-tion, multiple washing processes) no non-collagencomponents were left in the highly purified collagenmaterial, which was then used to prepare appropriatecollagen dispersions for further freeze- or air-drying.During this process the native collagen reconstitutedinto final fibril structures creating a porous net similar tothose found in natura. For collagen films, 100 ml ofbovine or equine collagen in 10 mm acetic acid weredispensed into 24-well-plates (Sarstedt, N .umbrecht,Germany) and dried. Collagen matrices were cut in sizeand swollen in morpholinoethanesulfonic acid (MES)buffer (pH 5.5) with low vacuum evaporation for 60 minprior to crosslinking.

2.2. Crosslinking of collagen

Crosslinking of bovine or equine collagen testmaterials was carried out in MES buffer (pH 5.5) withvariable concentrations of EDC. NHS was added in anEDC:NHS ratio of 4:1. After crosslinking with areaction time of 4 h the samples were washed twice with0.1m Na2HPO4 for 1 h and 4 times with deionized waterfor 30min. Collagen films were air dried. Collagensponges were dehydrated by continual extraction inacetone for 6 h and dried in vacuum.

2.3. Amino acid analysis

For amino acid analysis, uncrosslinked equine andbovine collagen samples (5 mg) were hydrolyzed with 6nhydrochloric acid for 24 h at 120�C. The resultingmixture was analyzed by a Biotronic Amino AcidAnalyser LC 5001 using the column resin BTC 2710 intriplicate [44].

2.4. Characterization of collagen crosslinking

Amino group content: To assess the degree of collagen-crosslinking, the free amino group content was deter-mined. This was done by reaction of the non-crosslinkedamino groups of the samples with 2,4,6-trinitrobenze-nesulfonic acid (TNBS), according to an assay describedby Bubnis et al. [43]. Five replicates were used for eachdetermination and the number of amino groups per1000 residues calculated.

Shrinkage temperature: The shrinkage temperaturewas determined by differential scanning calorimetry(DSC) using a modified protocol by Pieper et al. [44].Calorimetric measurements were performed using aDSC-7 (Perkin-Elmer Corporation, Norwalk, USA)

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–2841 2833

equipped with a RC 6 cryostat (Lauda, Germany).Temperature and enthalpy calibration were performedby using high purity indium (Mp ¼ 156:61�C) asstandard. Collagen matrices, 4� 4� 4mm3 in size, wereimmersed in deionized water (Millipore, Eschborn,Germany) at 4�C for 1 h. The wet samples were wipedwith filter paper to remove excess water and hermeticallysealed in aluminum pans. Heating was carried out at arate of 5�C/min in the temperature range 15–95�C withan empty aluminum pan as the reference probe.Shrinkage temperature (TS) was determined as the onsetvalue of the occurring endothermic peak. The value ofdenaturation enthalpy (DHS) was calculated with respectto the mass of vacuum dried collagen matrices. Themean value and standard deviation of three independentmeasurement runs per matrix type and degree ofcrosslinking were determined.

2.5. Macroscopic matrix morphology

Swelling ratios of natural and crosslinked collagenmatrices of equine and bovine origin (n ¼ 12/group)were assessed as follows [29]. After hydrating inwater for 1 h, the samples were equilibrated for 1 h inPBS (pH 7.4) at room temperature, blotted with filterpaper to remove excess surface water and then weighed.The collagen matrices were then placed in deionizedwater to remove the buffer salts and air-dried toconstant weight. The swelling ratio was calculated as aratio of the weight of the hydrated to that of driedmatrices.

In addition to assessment of weight and calculation ofthe swelling ratio, the volume changes of the differentcylindrical formed collagen matrices (n ¼ 12/group)before contact with fluid (dry samples) and afterremoval of fluid (dried samples) were determined.

2.6. Ultrastructural matrix morphology

Bovine and equine collagen matrices with differentdegrees of EDC/NHS-crosslinking (0, 5, 30 mg/ml)(n ¼ 2/group) were prepared for scanning electronmicroscopy as described previously [14]. Briefly, alde-hyde-fixed samples were rinsed in phosphate buffer(pH 7.4), osmicated, washed again in buffer, dehydratedin increasing concentrations of ethanol and subse-quently in acetone. Dehydrated specimens were sub-jected to critical-point drying using liquid carbon-dioxide substitution, mounted on aluminum stubs andgold-coated in a sputter coater (Polaron). Samples wereexamined and photographed with a Zeiss DSM 940microscope.

Replicates of bovine and equine collagen-basedmatrices with different degrees of EDC/NHS-cross-linking (0, 5, 30 mg/ml) were pretreated for 2 h with 2 mlof DMEM culture medium containing 1mg (2.1 Units/

ml) of collagenase P from clostridium histolyticum(EC3.4.24.3, Boehringer, Mannheim, Germany) andthen analyzed as described above. The range of poresize of each sample was assessed. The diameter ofcollagen fibers (10 fibers/group) was measured photo-morphometrically and compared statistically.

2.7. Enzymatic stability of collagen matrices

Enzymatic stability of collagen samples was testedwith in vitro collagenase digestion experiments accord-ing to a modified protocol described in the literature[29,31,40]. Films with an accurately weighed amount ofcollagen (the weights were a mean of five measurementswithin an error of 72%) were incubated at 37�C with1ml of DMEM culture medium containing 1mg(2.1 Units) of collagenase P from clostridium histolyti-cum (EC3.4.24.3, Boehringer, Mannheim, Germany),for 1.5, 3, 6, 24 h and for 6 days. In the last group theenzyme solution was changed daily. Thereafter, theresidues of non-digested collagen were rinsed with PBSand stained with picro-syrius-red according to a colori-metric method of Junqueira et al. [45]. After removal ofnon-bound dye by washing the collagen films with 0.01nhydrochloric acid (HCl) and dissolving the stainedmaterial in 0.1n NaOH, the optical density of thesamples was measured with a micro-plate reader (Emaxand Softmax, Molecular Devices, USA).

2.8. Stress–strain characteristics of collagen matrices

Stress–strain curves of matrix samples were deter-mined by uniaxial measurements using a Zwick-1445mechanical tester. Cylindrical testing bars (4 mmlength� 4mm diameter) were cut from the matrixsheets using circular punches. Each sample was gluedwith epoxy resin between two standardized aluminumdiscs with a diameter of 6mm and a thickness of 2mm.The metal discs allowed a convenient fixation of thesamples between two vertically positioned grips. One ofthem was mounted under the upper fixed frame ofthe testing device, and the second on the movablecrosshead below. The tension–compression load cell wasconnected to the crosshead. For testing the tensilestrength, an initial gauge length of 4mm was used andeach sample was initially pre-stretched with a load of0.1 g. Thereafter, the movement of the crosshead wascontinued with a speed of 1mm/min until rupture of thetest specimen occurred. Elongation and stress weredigitalized and recorded by a chart recorder. Curveregistration and analysis was performed with Lab View5.0-software. The tensile strength, the elongation atbreak and the elastic modulus were calculated for eachsample. Specimens for wet tests were prepared bysoaking the matrix bars in PBS at room temperaturefor 10 min.

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15

20

25

30

35

grou

pco

nten

t [n

/100

0]

Bovine collagenEquine collagen

P. Angele et al. / Biomaterials 25 (2004) 2831–28412834

2.9. Statistical analysis

Data are reported as the mean7SD. One-factor, two-factor and three-factor analyses of variance (ANOVAs)with Bonnferoni-Dunn and Fisher’s post-hoc tests formultiple comparisons as well as Student’s t-test wereperformed using Sigma Statt Software for WindowsVersion 2.03, SPSS Inc. Significance was accepted at alevel of po0:05:

0 5 10 15 20 25 30

5

10

Am

ino

EDC-concentration [mg/ml]

Fig. 1. Concentration of free amino groups in the sample using an

assay reported by Bubnis et al. [43]. With increasing EDC-concentra-

tion the amount of free amino groups decreased. No significant

difference (two-factor ANOVA, p ¼ 0:95) between the equine and

bovine collagen samples in respect to the concentration of free amino

groups was detected.

31

32

33

34

35

36

∆H

Onsettemperature

Hea

t flo

w[m

W]

Bovine coll., not crosslinkedEquine coll., not crosslinkedEquine coll., 5 mg/ml EDCEquine coll., 30 mg/ml EDC

3. Results

3.1. Amino acid composition

Differences between bovine and equine collagen couldbe detected in the amino acid composition. Equinecollagen contained a higher amount of hydroxylysineand lysine, whereas bovine collagen had a slightly higheramount of proline and hydroxyproline residues. Cy-steine was absent in both samples and both containedsmall amounts of tyrosine (Table 1).

3.2. Characterization of collagen crosslinking

Crosslinking of the collagen matrices could becontrolled by variation of EDC/NHS concentration.The degree of crosslinking is inversely proportional tothe amount of free amino groups. With increasing EDC-concentration, the amount of free amino groupsdecreased and the degree of crosslinking increased(Fig. 1). At concentrations of EDC higher than 2 mg/

50 60 70 80 9030

Temperature [°C ]

Fig. 2. DSC uncrosslinked and crosslinked collagen matrices. This

graph shows a representative helix-to-coil-transition of uncrosslinked

bovine and equine collagen and of equine collagen in different degrees

of EDC/NHS-crosslinking. Shrinkage temperature (Ts) is determined

as the onset value of the occurring endothermic peak.

Table 1

Amino acid composition of equine and bovine collagen

Equine collagen

(Residues/1000

residues)

Bovine collagen

(Residues/1000

residues)

Hydroxyproline 10477 103712

Aspartic acid/asparagine 5071 5371

Threonine 2271 2072

Serine 3975 3575

Glutamic acid/glutamine 9772 9773

Proline 14276 14777

Glycine 219715 22275

Alanine 12072 12479

Cysteine 070 070

Valine 2872 2771

Methionine 473 273

Isoleucine 1371 1572

Leucine 3272 3072

Tyrosine 571 371

Phenylalanine 1972 1871

Histidine 1171 772

Hydroxylysine 1371 1071

Lysine 2674 2171

Arginine 5671 6174

The values indicated represent mean 7SD, where n ¼ 3:

ml no further increase in crosslinking was obtained.There was no significant difference (two-factorANOVA, p ¼ 0:95) between equine and bovine collagenwith respect to the concentration of free amino groups(Fig. 1).

With DSC the helix-to-coil-transition of collagenfibers can be measured as a temperature dependentendothermic signal, which indicates the extent ofintermolecular crosslinking (Fig. 2; Table 2). The resultsindicated differences in the hydrothermal stability ofscaffolds depending on the collagen species and thedegree of crosslinking. Non-crosslinked equinecollagen exhibited a higher stability, expressed by a5% higher shrinkage temperature, when compared with

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Table 2

Shrinkage temperatures Ts (�C) and enthalpies DHs (J/g) of control and EDC/NHS crosslinked collagen samples

Bovine collagen Equine collagen

Ts (�C) DHs (J/g) Ts (�C) DHs (J/g)

Non-crosslinked 54.270.1 31.970.9 57.870.1 27.071.3

5mg/ml EDC/NHS 78.470.4 11.870.5 76.570.2 6.270.4

30mg/ml EDC/NHS 86.370.1 9.370.1 85.770.2 7.271.0

The values indicated represent mean7SD, where n ¼ 3: Statistical analysis was performed using a two-factor ANOVA and values of po0:05 were

considered to be significant.

bovine equine0

1

2

3

4

5

6

Sw

ellin

gra

tio

Collagen species

not crosslinkedcrossl. 5 mg/ml EDCcrossl. 30 mg/ml EDC

Fig. 3. Swelling ratio of scaffolds based on collagen of different species

and with different degrees of EDC/NHS-crosslinking. No relevant

differences can be detected between the swelling ratios of non-

crosslinked bovine- and equine-collagen samples. EDC/NHS-cross-

linking results in a significant decrease of the swelling ratio compared

to the non-crosslinked collagen (two-factor ANOVA, po0:0001).

EDC/NHS-crosslinked equine-collagen matrices showed significantly

higher reduction of swelling ratios compared with the crosslinked

bovine-collagen samples (two-factor ANOVA, po0:0001). The values

indicated represent mean7SD, where n ¼ 12:

P. Angele et al. / Biomaterials 25 (2004) 2831–2841 2835

the non-crosslinked bovine samples (two-factor ANO-VA, p ¼ 0:0001). With increasing EDC/NHS-crosslink-ing, an increase in shrinkage temperature was detected(p ¼ 0:0001). No species differences were found with thecrosslinked collagen matrices. Shrinking enthalpiesdecreased with increasing degree of crosslinking forboth collagen species. For the non-crosslinked bovinescaffold a higher shrinkage enthalpy was found com-pared with the non-crosslinked equine collagen scaffold.

3.3. Macroscopic form stability of collagen matrices

The different collagen matrices were comparedmorphologically with respect to their form stabilityusing their swelling ratios and their volume changesafter being soaked with fluid. No relevant differencescould be detected between the swelling ratios of non-crosslinked bovine and equine collagen samples (Fig. 3).However, EDC/NHS-crosslinking resulted in a signifi-cant decrease in swelling ratio compared to non-cross-linked collagen (two-factor ANOVA, po0:0001; Fig. 3).EDC/NHS-crosslinking of equine collagen samplesshowed a significantly higher reduction of swellingratios compared with the crosslinked bovine collagen(two-factor ANOVA, po0:0001; Fig. 3). After fluidremoval the non-crosslinked bovine and equine collagensamples collapsed to approximately 30% of their dryvolume (volume change of the samples (mean7SD):Bovine: 29%76; equine: 32%75). No significantdifferences could be found for volume changes betweensamples of the different collagens (Student’s t-test,p ¼ 0:3). Crosslinking, as well as collagen species, hada significant effect on volume changes (two-factorANOVA, p ¼ 0:001). After 5mg/ml EDC/NHS-cross-linking (bovine: 35%78; equine: 51%79) and 30 mg/ml EDC/NHS-crosslinking (bovine: 49%79; equine:87%710), equine collagen samples had significantlyhigher form stability (before and after fluid treatment)than bovine collagen samples (Student’s t-test,po0:0001).

3.4. Ultrastructure of collagen matrices

The ultrastructure of bovine and equine collagenmatrices with and without EDC/NHS-crosslinking (0, 5,

30 mg/ml) was analyzed by scanning electron micro-scopy (Fig. 4). No differences between the two collagenswere noted in uncrosslinked or EDC/NHS-crosslinkedcollagen matrices. The matrices showed highly inter-connective pores with pore sizes between 50 and 350 mm.The walls of the pores contained collagen fibers, whichwere covered by a non-fibrillar ground substance. Therewas no significant difference in the diameter of thecollagen fibers between uncrosslinked (0 mg/ml-EDC/NHS: bovine: 0.2170.04 mm; equine: 0.2270.06 mm) orcrosslinked (30 mg/ml-EDC/NHS: bovine: 0.2270.07 mm; equine: 0.2070.08 mm) bovine and equinecollagen. After collagenase treatment for 2 h, the non-crosslinked collagen matrices revealed a degradation ofthe thinner pore walls. At higher magnification it couldbe seen that collagen fibers were still present, whereasthe non-fibrillar collagen was degraded. EDC/NHS-crosslinking (5, 30 mg/ml) protected the matrix materialagainst collagenase degradation, as seen by the pre-served non-fibrillar collagen portion (Fig. 4). No

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Fig. 4. Scanning electron microscopy of uncrosslinked and EDC/NHS-crosslinked collagen scaffolds. Equine-collagen-based matrix without EDC/

NHS-crosslinking (A,B). Representative cross section of replicates without (C,D) and with 5 mg/ml EDC/NHS-crosslinking (E,F) after 2 h of

collagenase treatment. Bars represent 400mm for lower (A,C,D) and 5 mm for higher magnification. Note: No obvious differences between equine and

bovine (not shown) based collagen matrices could be detected.

P. Angele et al. / Biomaterials 25 (2004) 2831–28412836

significant differences could be detected between theresistance of bovine and equine collagen matrices inrespect to this short term collagenase treatment.

3.5. Enzymatic stability

Bovine and equine collagen samples were evaluatedfor their enzymatic stability against collagenase diges-tion (Fig. 5). Without EDC/NHS-crosslinking, bovineand equine collagen samples were almost completely

degraded after 1.5 h. No significant difference betweenbovine and equine collagen samples could be detected.Three-factor ANOVA with Fisher’s PLSD post-hoc testrevealed a highly significant effect of collagen type(p ¼ 0:0001), crosslinking (p ¼ 0:0001) and time(p ¼ 0:0001) on degradation of the collagen films bycollagenase treatment. With low concentration of thecrosslinking reagents (1 mg/ml EDC/NHS) an increasein enzymatic stability could, however, only be detectedin equine collagen films (significant different in compar-

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–2841 2837

ison with bovine collagen samples with Student’s t-test,p ¼ 0:0001). With high concentrations of crosslinkingreagents (30 mg/ml EDC/NHS) a complete resistance of

0 1 5 30

0

20

40

60

80

100

120

140

EDC-concentration [mg/ml]

Rem

aini

ngeq

uine

colla

gen

[%]

0 1 5 30

0

20

40

60

80

100

120

Rem

aini

ngbo

vine

colla

gen

[%]

0 h 1.5 h 3 h6 h 24 h 6 d

Fig. 5. Relative collagen concentration [%] after different time points

of collagenase digestion. Different EDC/NHS-crosslinking using

bovine (upper half) and equine (lower half) collagen samples were

analyzed. The values indicated represent mean7SD, where n ¼ 8:

Table 3

Biomechanical properties of uncrosslinked and crosslinked collagen scaffold

Collagen scaffold crosslinked

with EDC/NHS (mg/ml)

Bovine collagen

Elastic modulus

(kPa)

Strength at

break (kPa)

0 Dry 3127127 119736

Wet 1473 3375

1 Dry 2167110 156730

Wet 2474 4974

5 Dry 2097153 128731

Wet 2876 5076

30 Dry 105729 113731

Wet 1975 3074

The tests were obtained in dry and wet material condition. The values indicate

using the student t-test and values of po0:05 were considered to be significa

equine collagen samples against collagenase induceddegradation could be achieved for at least six days. Incontrast to equine collagen, 30 mg/ml EDC/NHS-cross-linked bovine collagen was degraded to more than 50%after collagenase digestion for six days (Student’s t-test,p ¼ 0:0001; Fig. 5). After each time period of collage-nase treatment, the collagen concentration were sig-nificantly higher in the equine compared with the bovinesamples (unpaired Student’s t-test, po0:0001).

3.6. Biomechanical analysis

Tensile stress–strain analysis was performed onbovine and equine collagen matrices with 0, 1, 5 and30 mg/ml EDC/NHS-crosslinking reagent. All sampleswere analyzed under dry and wet condition (Table 3).

Three-factor ANOVA revealed a highly significanteffect of collagen type (p ¼ 0:0001), crosslinking(p ¼ 0:0001) and matrix condition (p ¼ 0:0001) on therupture strength of matrices measured by stress–strainanalysis. The rupture strength of non-crosslinked andEDC/NHS-crosslinked matrices decreased significantlyunder wet compared to dry matrix condition (two-factorANOVA; po0:0001). For dry matrix condition decreas-ing levels of rupture strength under increasing EDC/NHS-crosslinking could be detected for equine collagenmatrices (two-factor ANOVA; po0:0001). In contrast,bovine collagen-based matrices showed no significantdifference (two-factor ANOVA with Fisher’s PLSD;p ¼ 0:13). Under wet condition, 1 and 5mg/ml EDC/NHS-crosslinking of both collagen types resulted in asignificant increase in rupture strength compared touncrosslinked controls (one-factor ANOVA with Fish-er’s PLSD; po0:0001). 30 mg/ml EDC/NHS-crosslink-ing showed a significant decrease in rupture strengthcompared to 1 and 5 mg/ml EDC/NHS-crosslinking(one-factor ANOVA with Fisher’s PLSD; po0:0001).

s

Equine collagen

Elongation

at break (%)

Elastic modulus

(kPa)

Strength at

break (kPa)

Elongation

at break (%)

974 97744 209722 1575

2675 1474 2875 3079

973 88722 173711 2078

2775 3075 5876 3179

973 224751 157716 971

2074 3477 5778 2274

1273 1697110 123723 1074

1672 2777 1876 1876

d represent mean7SD, where n ¼ 8: Statistical analysis was performed

nt.

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–28412838

The rupture strength of equine collagen matrices with30 mg/ml EDC/NHS-crosslinking (wet condition) wasfound to be significantly higher compared to compar-able matrices of bovine origin (one-factor ANOVA withFisher’s PLSD; po0:005).

The corresponding strain at rupture of non-cross-linked and EDC/NHS-crosslinked matrices decreasedsignificantly under wet compared to dry condition (two-factor ANOVA with Fisher’s PLSD; po0:0001). Fordry matrix condition no significant differences could bedetected between the collagen origin or the degree ofEDC/NHS-crosslinking. However, under wet condition,the strain at rupture of 0 and 1 mg/ml EDC/NHS-crosslinking was found to be significantly highercompared with matrices crosslinked with 30 mg/mlEDC/NHS (two-factor ANOVA with Fisher’s PLSD;po0:001). The equine collagen-based matrices revealeda tendency to higher strain at rupture without reachingsignificance compared to bovine collagen matrices.

4. Discussion

Collagen-based biomaterials are widely used for tissueengineering. Because of the high degradation rate ofnatural collagen in vivo, crosslinking is necessary toreduce biodegradation of the biomaterial for sufficienttissue repair. EDC/NHS-collagen crosslinking results inbiomaterials with good biocompatibility, with highcellular differentiation potential [20,30,31] and goodresistance against enzymatic degradation [28,32]. In thisstudy, matrices of different collagen origin and differentdegrees of EDC/NHS-crosslinking were compared interms of their physicochemical properties.

The amino acid composition analysis revealed com-parable results to other collagen-based matrices in theliterature [31,37]. The two collagens showed small butdistinct differences in amino acid composition. Impor-tantly, equine collagen has a higher number of lysineand hydroxylysine residues compared with collagen ofbovine origin. These amino acids are necessary fornatural intra- and intermolecular crosslinking [46].According to Notbohm et al. [47], an increase of lysinehydroxylation parallels the increase in thermostability ofcollagen. A direct relationship exists between thecontent of the amino acids proline and hydroxyprolineand thermal stability [48]. This becomes evident inanimals of different body temperature, with warm-blooded animals having the highest content of theseamino acids. However, in our series, the differencesbetween the two mammalian collagens examined weretoo small to draw definitive conclusions. The absence ofcysteine indicated that there were no traces of othercollagen types or procollagen that contains disulfidelinkages in the collagen samples. Tyrosine was detectedin both equine and bovine samples, indicating that

telopeptides, crucial for natural collagen crosslinks, arestill present, since it is not present in the collagen triplehelix [49]. The higher amount of lysine and hydro-xylysine was not found in the analyses of free aminogroups. Taken together with the slightly higher hydro-thermal stability determined by DSC, this may indicatethat there is a higher degree of natural crosslinking inequine collagen.

Carbodiimides react with carboxyl groups of theasparaginic and glutaminic acid residues to form anactivated, but unstable form of O-urea. The use of NHSto improve the crosslinking yield of carbodiimides byforming a more stable ester is well-documented [32,40].Consistent with this, the results of the present studydemonstrate that EDC/NHS-crosslinked collagen ma-trices increased in shrinkage temperature to 85.7�C forequine and to 86.3�C for bovine collagen compared with57.8�C and 54.2�C, for the non-crosslinked equine andbovine samples, respectively. However, no significantdifferences could be detected between equine- andbovine-based collagen samples after crosslinking. Thedegree of collagen crosslinking could be monitored bythe reduced number of free amino groups with increas-ing concentration of EDC/NHS-crosslinking. There wasno significant difference in the crosslinking behaviorbetween equine and bovine collagen samples in thepresent study, consistent with results reported in theliterature [28,31,37,38,40].

Collagen crosslinking was further assessed by mea-suring swelling ratio and matrix volume changes duringcontact with fluid. With increasing degree of EDC/NHS-crosslinking, a highly significant reduction inswelling ratio and reduced volume changes duringcontact with fluid could be detected. A reduced swellingratio after crosslinking was also described by others[28,38]. The reduction of swelling ratio was significantlyhigher for the equine compared with the bovine collagenmatrices after crosslinking. Ultrastructural analysisrevealed no significant differences in pore size andmatrix morphology with respect to the collagen originand to the degree of EDC/NHS-crosslinking. This isconsistent with the findings of others; a retainment ofultrastructural morphology for similar collagen-based matrices was described after EDC/NHS-cross-linking [38].

One effect of crosslinking is increased resistance toenzymatic degradation by bacterial collagenase. In thepresent study, enzymatic degradation of non-crosslinkedmatrices resulted in a removal of a non-fibrillarcollagenous ground substance as detected by ultrastruc-tural analysis. The amorphous substance found betweenthe collagen fibrils can be interpreted as low molecularcollagen fragments which were not yet reconstituted tothe fully developed macrofibrils. Even a low degree ofEDC/NHS-collagen crosslinking (0.5 mg/ml) was ableto dramatically change the rate of enzymatic degrada-

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–2841 2839

tion by preserving the matrix ultrastructure under short-term-collagenase treatment. No obvious differences inthe ultrastructure could be seen between bovine andequine collagen samples. In vitro degradation wasfurthermore evaluated using a biochemical, colorimetricmethod described by Junqueira et al. [45]. A significantincrease in resistance to enzymatic degradation could beshown after crosslinking. This data is consistent withthat of others [20,30–32,40]. In contrast to the ultra-structural analysis with only short-term collagenaseincubation, as described above, long-term treatmentwith collagenase revealed significant differences betweenbovine and equine collagen samples. EDC/NHS-cross-linked matrices of equine collagen were significantlymore resistant against collagenase treatment comparedwith matrices of bovine collagen. With 5 mg/ml EDC/NHS-crosslinking equine collagen samples revealed onlya partial degradation (up to 20%) after six days ofcollagenase treatment, whereas bovine-based collagensamples showed complete degradation. Equine collagenmatrices with 30 mg/ml EDC/NHS-crosslinking werecompletely resistant to collagenase in contrast to bovinecollagen-based matrices, which still had 60% degrada-tion after six days of collagenase treatment. Onepossible explanation is that collagenase cleavage sitesare more effectively masked by the crosslinking inequine-based collagen samples. Bacterial collagenasecatalyses hydrolytic cleavage of collagen in non-polarregions, either in a single alpha-chain or simultaneouslyacross three chains of the triple helix in lateral fashion[50]. EDC/NHS-crosslinking of equine collagen originmay have been more effective in blocking collagenase’sspecific ability to cleave alpha-chain linkages. Theselinkages would not be expected to affect the shrinkagetemperature or the free amino group content incollagens of different origin, but perhaps their enzymaticsusceptibility.

Tensile testing was performed to analyze the biome-chanical properties of the collagen matrices. In thepresent study, 1 and 5mg/ml EDC/NHS-crosslinkingincreased significantly the rupture strength of wetmatrices compared with non-crosslinked controls. Withhigh degrees of crosslinking (30 mg/ml EDC/NHS) adecrease in rupture strength to strength levels of non-crosslinked controls could be detected. Furthermore, adecrease of corresponding strain at rupture could befound with increasing degrees of EDC/NHS-crosslink-ing. These results are consistent with the literatureindicating a decrease in rupture strength and a reductionin corresponding strain at rupture for highly EDC/NHS-crosslinked scaffolds [28,40]. It indicates a changein biomechanical properties and could be explained bythe production of stress concentrations due to failure ofbrittle collagen fibers [28,51]. The low rupture strength,corresponding strain at rupture and tensile modulus ofnon-crosslinked and EDC/NHS-crosslinked collagen

matrices in the present study may be due to differentmatrix porosities and geometries compared with thematrices described in the literature [28,32].

The present study revealed a highly significant effectof collagen type (p ¼ 0:0001) on the rupture strength ofmatrices. Furthermore, the equine collagen-based ma-trices revealed a tendency to higher strain at rupturewithout reaching significance compared to bovinecollagen matrices. These differences between ovine[28,40], bovine and equine collagen-based matricesindicate species differences in biomechanical propertiesbefore and after EDC/NHS-crosslinking.

There are several possible explanations for thedifferent physico-chemical properties of equine andbovine collagen matrices. EDC/NHS-crosslinking mayoccur within an alpha chain, between alpha chains, or asintermolecular or interfibrillar linkages as described byOlde Damink et al. [28]. This crosslinking is dependenton the primary structure of collagen. The greateramount of lysine and hydroxylysine residues in equinecollagen could explain the different physicochemicalproperties, e.g. the slightly higher shrinkage tempera-ture, with a higher natural crosslinking of equinecompared to bovine collagen. In crosslinked equinecollagen matrices, the cleavage sites of collagenaseappear masked better than in crosslinked bovinecollagen matrices. This could be explained by thedifferent primary structure and the differences in theformation of alpha-chain linkages after EDC/NHS-crosslinking. These linkages would not be expected toaffect shrinkage temperature, but perhaps enzymaticsusceptibility. Alternatively, collagenase cleavage sitescould be blocked because of a higher amount ofattached glycosaminoglycans in equine compared tobovine collagen. A higher extent of bonded glycosami-noglycans would reduce the content of free lysine andhydroxylysine residues. This could explain the contraryresults between equal amounts of free amino groups andthe higher content of these amino acids in natural equinecompared to bovine collagen. Equine and bovinecollagen were harvested from Achilles tendons, preparedand manufactured under the same, good manufacturingpractice (GMP) approved conditions. This is supportedby the comparable ultrastructural morphology of thedifferent collagen origins. However, inferior differencesin collagen purity and glycosaminoglycan contentcannot be excluded.

Another factor that may also affect the enzymaticdegradation susceptibility of equine collagen matrices istheir reduced swelling ratio and lower surface area.Because the enzymatic activity mainly affects the matrixsurface [40], collagenase may degrade less collagen pertime unit in matrices of equine compared to bovinecollagen origin. In summary, small differences in theprimary structure of bovine and equine collagen resultedin significant different physicochemical properties.

ARTICLE IN PRESSP. Angele et al. / Biomaterials 25 (2004) 2831–28412840

Equine collagen-based matrices had a significant in-crease in resistance to collagenase treatment after EDC/NHS-crosslinking and less distinct matrix swelling aftercontact with fluid.

For tissue engineering purposes, the higher enzymaticstability, the higher form stability, as well as the lowerrisk of transmissible disease make the case for consider-ing equine-based collagen. Furthermore, the resultsindicate that data obtained for tissue engineeringwith a collagen of a certain species may not betransferable to scaffolds based on collagen from adifferent species, because of differing physico-chemicalproperties.

Acknowledgements

The authors thank Andrea Havasi, Tanja Weinfurt-ner, Tom B .ottner and Martina Kreuzer for excellenttechnical assistance. We are furthermore thankful toProf. Dr. Joachim Hammer and his coworkers RonnyMai and Johann Fierlbeck regarding the mechanicaltests. This work was supported by grants of DFG (Ne734/2 and He 378/29), ForBioMat, HighTechOffensiveBavaria and NIH-NIAMS (1 R01 AR-48132-01).

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