Materials Science and Engineering C 30 (2010) 190–195
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The influence of a natural cross-linking agent (Myrica rubra) on the properties ofextruded collagen fibres for tissue engineering applications☆
D.I. Zeugolis a,b,⁎, R.G. Paul c, G. Attenburrow d
a Network of Excellence for Functional Biomaterials (NFB), NFB Building, IDA Business Park, Newcastle Road, Dangan, National University of Ireland, Galway (NUIG), Galway, Irelandb Department of Mechanical & Biomedical Engineering, Nun's Island, National University of Ireland Galway (NUIG), Galway, Irelandc Devro Plc, Glasgow, Scotland, G69 0JE, UKd School of Applied Sciences, The University of Northampton, NN2 7AL, UK
☆ This work was carried out at the School of ApplieNorthampton, UK.⁎ Corresponding author. Network of Excellence for Fun
Building, IDA Business Park, Newcastle Road, Dangan,Galway (NUIG), Galway, Ireland. Tel.: +353 0 9149 3166
E-mail address: [email protected] (D.
0928-4931/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.msec.2009.09.017
a b s t r a c t
a r t i c l e i n f oArticle history:Received 26 April 2009Received in revised form 27 July 2009Accepted 24 September 2009Available online 3 October 2009
Keywords:Plant extractCollagen stabilisationMechanical propertiesThermal propertiesBiomaterial
Extruded collagen fibres have been shown to be a competitive biomaterial for both soft and hard tissuerepair. The natural cross-linking pathway of collagen does not occur in vitro and consequently reconstitutedforms of collagen lack sufficient strength. Numerous cross-linking approaches have been investigatedthrough the years, but still there is no ideal method accepted. The use of plant extracts to cross-link collagenscaffolds has been advocated due to superior mechanical properties. As first herein we investigate thestabilisation effect of Myrica rubra on extruded collagen fibres. Fibres treated with M. rubra exhibited higherdenaturation temperature (p<0.005) and lower enthalpy of denaturation (p<0.034) than formaldehyde ofglutaraldehyde. Uniaxial tensile tests of wet tested fibres revealed j-shape curves similar to those of nativetissues. Thin fibres exhibited high stress/low strain graphs, whilst thick fibres yielded low stress/high straingraphs. Cross-linking reduced significantly the fibre diameter (p<0.005) and increased significantly thestress (p<0.004) and force (p<0.001) at break and the modulus at 2.0% strain (p<0.003). An inverserelationship between stress at break and fibre diameter was observed for every treatment. Overall, ourfindings demonstrate the potential of M. rubra in stabilisation of collagen-based materials for tissueengineering applications.
d Sciences, The University of
ctional Biomaterials (NFB), NFBNational University of Ireland,; fax: +353 0 9156 3991.I. Zeugolis).
ll rights reserved.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Collagen, under appropriate conditions of temperature, pH, ionicstrength, collagen concentration and composition and presence of otherconnective tissue macromolecules will spontaneously self-assemble toform microscopic fibrils, fibril bundles and macroscopic fibres thatexhibit D periodic banding patterns virtually indistinguishable fromnative fibres when examined by electron microscopy [1–9]. Theprinciple of self-assembly has been utilised to fabricate extrudedcollagen fibres that closely imitate extracellular matrix assembliessuitable for both soft and hard tissue repair [9–13]. Moreover, after therecent drawback of electro-spun collagen nano-fibres [14,15], extrudedcollagen fibres constitute the solely engineered fibrous scaffolds thatclosely emulate native tissues. Despite significant strides that have been
achieved, there are still many challenges in the engineering of thisscaffold to achieve functional tissue reconstruction.
In every tissue engineering application, it is essential for the scaffoldto provide a mechanically stable construct upon which cells can attach,migrate and proliferate and therefore allow the formation of functionalneotissue. In vivo, the native cross-linking pathway of lysyl oxidaseimparts desired mechanical characteristics and proteolytic resistance onthe collagen fibres in connective tissues [16–21]. However, the lysyloxidase mediated cross-linking does not occur in vitro and consequentlycollagen constructs lack sufficient strength and may disintegrate uponhandling or collapse under the pressure from surrounding tissue uponimplantation. For this reason, a number of cross-linking approaches(chemical, physical and biological) have been investigated through theyears to control mechanical and thermal properties, biological stability,the residence time in the body and to some extent the immunogenicityand antigenicity of the device [18,22–27]. However, at present there is nocommonly accepted ideal cross-linking treatment for collagen-derivedbio-prostheses. Recent studies have supported the use of plant extractsfor stabilisation of collagen scaffolds [27–33]. Herein we investigate thechanges in structural, thermal and mechanical properties that a novelplant extract,Myrica rubra, can bring about in collagen scaffolds.
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2. Materials and methods
2.1. Materials
All chemicals, unless otherwise stated, were purchased fromSigma-Aldrich, UK. The bovine Achilles tendons were kindly providedby the British Leather Research Centre (BLC; Northampton, UK).
2.2. Collagen preparation
A typical protocol for the extraction of collagen was employedas has been described in detail previously [9]. Frozen bovineAchilles tendons were minced, washed in neutral phosphate buffersand suspended in 0.5 M ethanoic acid in the presence of pepsin(2500 U/mg, Roche Diagnostics, UK) for 72 h at 4 °C. Consequently thecollagen suspension was centrifuged (12,000 g at 4 °C for 45 min;Gr20.22 Jouan refrigerated centrifuge, Thermo Electron Corporation,Bath, UK) and purified by repeated salt precipitation (0.9 M NaCl),centrifugation and acid solubilisation (1 M ethanoic acid). Thefinal atelocollagen collagen solution was dialysed (8000 Mw cut off)against 0.01 M ethanoic acid and kept refrigerated at 4 °C untilused. The collagen purity was determined by SDS-PAGE analysis (90%type I) and its concentration was determined by hydroxyprolineassay (7 mg/ml).
2.3. Micro-fibre fabrication and cross-linking
The procedure for fibre formation has been described in detailpreviously [27] and was based on previous work [9,13,34]. Briefly, a5 ml syringe (Terumo Medical Corporation UK Ltd, Merseyside, UK)containing the atelocollagen was loaded into a syringe pump system(KD-Scientific 200, KD-Scientific Inc., Massachusetts, USA) connectedto silicone tubing (Samco Silicone Products, Ltd., Warwickshire, UK) of30 cm in length and 1.5 mm in internal diameter. The pumpwas set toextrude at 0.4 ml/min. One end of the tube was connected to thesyringe pump with the other end placed at the bottom of a container.The collagen solution was extruded into a “Fibre Formation Buffer”(FFB) comprising of 118 mM phosphate buffer and 20% of polyeth-ylene glycol (PEG), Mw 8000 at pH 7.55 and 37 °C. Fibres wereallowed to remain in this buffer for a maximum period of 10 min,followed by further incubation for additional 10 min in a “FibreIncubation Buffer” (FIB) comprising of 6.0 mM phosphate buffer and75 mM sodium chloride at pH 7.10 and 37 °C. Thereafter, the fibreswere either incubated overnight at room temperature (RT) intodistilled water bath or cross-linked in aqueous 1% of formaldehyde or1% glutaraldehyde or 1% M. rubra (kindly provided by Prof. TonyCovington, University of Northampton, Northampton, UK). Finally, thefibres were washed extensively in phosphate buffer saline (PBS), air-dried under the tension of their own weight and conditioned at RT at65% relative humidity for at least 48 h.
2.4. Denaturation temperature
The hydrothermal stability of the fibres was determined using an822e Mettler-Toledo differential scanning calorimeter (Mettler-Toledo International Inc., Leicester, UK). Differential scanning calo-rimetry is a method widely used to study the thermal behaviour ofmaterials as they undergo physical and chemical changes uponheating. This method measures the heat flow necessary for heating ofthe sample with a constant temperature rate (°C/min) [35–37]. Drycollagen fibres were hydrated overnight at RT in 0.01 M PBS at pH 7.4.The wet fibres were removed and quickly blotted with filter paper toremove excess surface water and hermetically sealed in aluminiumpans. Heating was carried out at a constant temperature ramp (5 °C/min) in the temperature range of 15 to 100 °C, with an empty alu-minium pan as the reference probe. The temperature of maximum
power absorption during denaturation (peak temperature) wasrecorded as the denaturation temperature [23,38–40].
2.5. Mechanical testing and structural evaluation
Dry collagen fibres were hydrated overnight at RT in 0.01 M PBS atpH 7.4. The wet fibres were removed and quickly blotted with filterpaper to remove excess surface water. Stress–strain curves weredetermined in uniaxial tension using an Instron 1122 Universaltesting machine (Instron Ltd, Buckinghamshire, UK) operated at anextension rate of 10 mm/min. The gauge length was fixed at 3 cm andsoft rubber was used to cover the inside area of the grips to avoiddamaging the fibres at the contact points. Results obtained with fibresthat broke at contact points with the grips were rejected. The cross-sectional area of each fibre was calculated by measuring the diameterat four places along its longitudinal axis using a Nikon Eclipse E600optical microscope with a calibrated eyepiece (Nikon Instruments,Surrey, UK). It was assumed that the fibres were circular for the cross-sectional area determinations. Surface and fractured ends of collagenfibres that had been extended to failure were examined using aHitachi S3000N Variable Pressure Scanning Electron Microscope(Hitachi, Berkshire, UK). The following definitions were used tocalculate the mechanical data: stress at break was defined as the loadat failure divided by the original cross-sectional area (engineering-stress); strain at break was defined as the increase in fibre lengthrequired to cause failure divided by the original length and moduluswas defined as the stress at 0.02 strain divided by 0.02.
2.6. Statistical analysis
Numerical data is expressed as mean±SD. Analysis was per-formed using statistical software (MINITABTM version 13.1, Minitab,Inc.). One way analysis of variance (ANOVA) for multiple comparisonsand 2-sample t-test for pair wise comparisons were employed afterconfirming the following assumptions: (a) the distribution fromwhich each of the samples was derived was normal (Anderson–Darling normality test); and (b) the variances of the population of thesamples were equal to one another (Bartlett's and Levene's tests forhomogenicity of variance). Non-parametric statistics were utilisedwhen either or both of the above assumptions were violated andconsequently Kruskal–Wallis test for multiple comparisons or Mann–Whitney test for 2-samples was carried out. Statistical significancewas accepted at p<0.05.
3. Results
3.1. Matrix morphology
Optical microscopic evaluation of the cross-linked fibres revealedchanges in colour for the M. rubra (brown) and the glutaraldehyde(yellow) treated fibres. The formaldehyde treated fibres were similarto the control (grey-white). The control fibres were characterised by asmooth nano-textured surface morphology (Fig. 1a), whilst the cross-linked fibres demonstrated more pronounced ridges and crevicesalong the longitudinal fibre axis (Fig. 1b). Evaluation of the failed endsrevealed a closely packed inter-fibre space (Fig. 1c). Typical fracturemodeswere identified, independent of the fixationmethod employed,that can be classified as: (a) smooth fracture; (b) rough fracture withthe internal structure of the fibre appeared slightly drawn out for theone fraction and pulled in for the other fraction; (c) longitudinal splitfracture; and (d) fibrillation fracture.
3.2. Thermal properties
Table 1 summarises the thermal properties of the fibres producedin this study. All cross-linking approaches resulted in fibres with
Fig. 1. Scanning electron microscopy evaluation revealed a smooth surface structure for the non-cross-linked fibres (a), whilst the cross-linked fibres exhibited a rough surfacestructure constituted of ridges and crevices running parallel to the longitudinal fibre axis (b). A closely packed inter-fibre space was apparent from evaluation of the failed ends offibres extended to failure (c).
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denaturation temperature higher than the control (p<0.001), whilstthe enthalpy of denaturation was reduced significantly for everycross-linking technique employed (p<0.009). Fibres treated with M.rubra exhibited the highest denaturation temperature (p<0.005)and the lowest enthalpy of denaturation (p<0.034).
3.3. Biomechanical analysis
Uniaxial tensile tests revealed a j-shape curve (Fig. 2) thatconsisted of a small toe region, a region of sharply increasing stressup to a knee point where the gradient of the curve reduced, and a longregion of constant gradient that remained until failure. For everytreatment, the stress–strain curves showed a diameter dependentvariation; thin fibres exhibited high stress/low strain graphs and ashort toe region (Fig. 2— I), whilst thick fibres yielded low stress/highstrain graphs and a long toe region (Fig. 2 — II).
Table 1 summarises the physical and mechanical properties of theproduced fibres. Cross-linking reduced significantly the fibre diameter(p<0.005), whilst it increased significantly the stress (p<0.004) andforce (p<0.001) at break and the modulus at 2.0% strain (p<0.003).No significant difference was observed in strain at break betweenglutaraldehyde fixed fibres and the control ones (p>0.05). The strainat break was significantly increased for the formaldehyde treatedfibres (p<0.002), whilst it was significantly decreased for the M. ru-bra fixed fibres (p<0.002) in comparison to the non-cross-linkedfibres. An inverse relationship between stress at break and fibrediameter was observed for every treatment; indeed fitting a linearregression model between the stress at break and fibre diameter,strong correlations were obtained (Fig. 3).
4. Discussion
Natural biopolymers such as collagen are favoured for tissueengineering applications. However, the native cross-linking pathwaythat is responsible for the superior mechanical properties of collagenin vivo does not occur in in vitro assemblies and hence non-cross-
Table 1Physical, mechanical and thermal properties of extruded collagen fibres.
Treatment Diameter ±SD(µm)
Stress at break ±SD(MPa)
Strain at break(%) ±SD
Control (n=5) 298±17 2.97±0.87 33±71% formaldehyde(n=6)
218±19 54.07±13.64 63±13
1% glutaraldehyde(n=7)
240±31 33.86±17.39 40±13
1% Myrica (n=5) 205±10 28.18±8.51 15±4
Cross-linking increased significantly the denaturation temperature (TD; p<0.001), the streswhilst it reduced significantly the enthalpy of denaturation (ΔHD; p<0.009) and the fibre
linked scaffolds are characterised by low mechanical strength.Biological, physical and chemical cross-linking approaches havebeen investigated over the years; however there is still no widelyaccepted technique. Biological (transglutaminase) and physical (UVirradiation, dehydrothermal) approaches, although do not affect cellviability; exhibit a rather poor stabilisation function [18,37,41–46].Multifunctional chemical cross-linking methods such as glutaralde-hyde, 1,6-hexamethylene diisocyanate or epoxides create a three-dimensional network that results in collagen scaffolds with enhancedmechanical properties [27,47–52]. However, glutaraldehyde and 1,6-hexamethylene diisocyanate have been found to be cytotoxic and assuch carbodiimide or acyl azide methods have been introduced[24,53,54]. However, both methods have limited cross-linking abilitydue to their short length structure and inability to polymerise [50]. Inthe quest of the ideal cross-linking agent, plant extracts have beeninvestigated over the years. Recent data demonstrate that plantextracts not only optimally stabilise collagen scaffolds and bring aboutmechanical properties similar to native extracellular matrix assem-blies, but also do not compromise cell attachment, proliferation andgrowth [27–33,55–57]. In this study we investigated for the first timethe potential of M. rubra in stabilisation of collagen fibres and wecorrelated our findings with formaldehyde and glutaraldehyde; eitherof these reagents is customarily used to optimally cross-link collagen,but their cytotoxic effect has decreased their use [49,58–60].
4.1. Matrix morphology
Cross-linking using glutaraldehyde and M. rubra brought aboutcolour changes. This phenomenon has been observed previously withnumerous collagenous structures anddifferent cross-linking approaches[27,39,49,61–66]. Ridges and crevices running along the longitudinalaxis of the fibres were identified for every treatment and were morepronounced for the cross-linked fibres. Such morphological featureshave been shown to facilitate cell attachment, fibroblast migration andultimate alignedneotissue formation [67,68] andhavebeenattributed tothe substructure of the fibres [69,70]. The degree of surface roughness
Force at break(N) ±SD
Modulus at 2% strain(MPa) ±SD
TD (°C) ±SD(n=3)
ΔHD (J/g) ±SD(n=3)
0.20±0.04 3.78±1.10 45.47±1.05 13.48±0.741.97±0.18 16.41±2.91 68.76±0.99 9.66±0.70
1.33±0.38 35.19±12.18 75.56±0.61 6.94±0.37
1.04±0.15 23.10±9.45 82.19±1.92 5.19±0.88
s (p<0.004) and force (p<0.001) at break and the modulus at 2.0% strain (p<0.003),diameter (p<0.005). Sample number (n) in parentheses; SD: standard deviation.
Fig. 2. Typical j-shape stress–strain curves of rehydrated extruded collagen fibres wereobserved. The curves exhibited a diameter dependent variation; thin fibres exhibited ahigh stress/low strain graph (I), whilst thick fibres demonstrated a low stress/highstrain graph (II).
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has also been shown to depend on the cross-linking method employed[27,42,71,72]. A compact inter-fibre space was found for every treat-ment; indeed, it has been reported that very little available inter-fibrillarspace occurs in fibres formed in vitro [5]. The four fracture modesidentified are in accord with previous publications [9,27,70,73–76] andtheir relative occurrence has been attributed to thehandling of thefibreswhilst in the wet state, to the different degree of stretching of thedifferent layers of the fibre, to the strain rate, or even to flawswithin thefibre structure.
4.2. Thermal analysis
When collagen is heated in a hydrated state, the crystalline rigidtriple helical molecule denaturates over a narrow range of tempera-tures, the mid-point of which is referred to as denaturationtemperature (TD) and results in the destruction of its tertiary structureand biological function [77]. The denaturation of the triple helix hasbeen shown to be a two-stage process starting with separation of the
Fig. 3. Fitting a linear regressionmodel between stress at break andwet fibre diameter, strongthe stress at break was apparent for every treatment.
polypeptides followed by the denaturation of the helical form [78].The collagen–gelatin transition is a melting process in which collagenchanges into a disorganised random coil [35,36,79]. Differentialscanning calorimetry is employed to evaluate the denaturationtemperature of fully hydrated biomaterials; scaffolds with meltingprofiles lower than 39–40 °C (body temperature) would indicatematerials unsuitable for biomedical applications since they woulddisintegrate upon implantation. Cross-linked and non-cross-linkedfibres exhibited denaturation temperature higher than the bodytemperature making this scaffold an excellent choice for tissueengineering applications. Non-cross-linked fibres had a denaturationtemperature higher than any other non-cross-linked collagenouspreparation (e.g. gels, films, sponges) due to the increased energy ofcrystallisation derived from the interaction between the closelypacked molecules in the fibre form [58,78,80–82]. The presence ofPEG could also be responsible for the increased denaturationtemperature of the fibres compared with that of collagen sponges orfilms, where no added polymer was present. Indeed, polymers mayincrease the denaturation temperature by shifting the equilibriumbetween native and denaturated forms of collagen towards a morecompact native form by steric exclusions [83–85].
The different cross-linking methods employed in this studybrought about different thermal stabilities. It has been reported thatsamples with higher denaturation temperature have more hydrogenbonds and/or fewer hydrophobic bonds than samples with lowerdenaturation temperatures [86,87]. Moreover, it has been shown thatthe denaturation temperature depends on the size of the co-operatingunits, the larger the unit, the slower the kinetics and the higher theshrinkage temperature [76,88]. Indeed, the complex structure of M.rubra brought about the highest denaturation temperature, whilstbetween the aldehydes, glutaraldehyde conveyed higher denatur-ation temperature. In fact, it has been shown that the nature of thebonds formed and the stability of the cross-links introduced vary withthe aldehyde used, which has been attributed to the structuralchanges associated with the collagen–aldehyde reaction [88]. More-over, inter-fibrillar cross-links have not been described for
correlations were obtained and an inverse relationship between the fibre diameter and
194 D.I. Zeugolis et al. / Materials Science and Engineering C 30 (2010) 190–195
formaldehyde and it does not co-polymerise as demonstrated withglutaraldehyde and subsequently cannot bind side-by-side fibrils [89].Furthermore, formaldehyde does not introduce bulky polymericadducts into the fibril structure, as has been shown for glutaraldehyde[90]. Cross-linking increased the denaturation temperature of thefibres due to the better packing and stabilisation of the helices andconsequently decreased the enthalpy of denaturation in all cases[36,58,78,80–82]. However, this transition is a bulk response and doesnot reflect the exact number or location of the cross-links [38,91].
4.3. Biomechanical evaluation
Tensile testing was performed to analyse the biomechanicalproperties of the collagen matrices. In vivo, the primary mechanicalstrength of individual collagen molecules depends upon the extra-cellular formation of triple helical molecules that self-assemble intocollagen fibrils and are stabilised by intra- and inter-molecular cross-links between the adjacent helical molecules [92]. The collagennetwork is primarily responsible for the mechanical properties ofcollagenous tissues, especially for tissues that are exposed to repeatedtensile forces [20,50,51,93,94]. Uniaxial tensile tests of rehydratedcollagen fibres produced j-shape stress–strain curves that have beenreported for native tissues [95,96] and extruded collagen fibres[27,48,70,97]. Similar curves have also been reported for semi-crystalline polymers that yield and undergo plastic flow [98]. Theyielding mechanism involves some form of flow that occurs withinthe fibre, possibly inter-fibrillar slippage, which plays an importantrole in the tensile deformation of aligned connective tissues such astendons [99]. In all cases, the slope of the stress–strain curve increaseswith strain, a characteristic of native and in vitro producedcollagenous structures [100–102]. The low modulus of the toe regionthat gives rise to a non-linear stress–strain curve in native tissues hasbeen attributed to the reorientation/alignment and un-crimping ofthe collagen fibrils, as well as the initiation of stretching of the triplehelix, the non-helical ends and the cross-links [103–106].
Strong inverse correlations between fibre diameter and stress atbreak were obtained suggesting that by controlling the fibre diameter,extruded collagen fibres can be produced with variable strength tomatch the tissue to be replaced. Three different mechanisms couldbe responsible for the observed correlations: (a) the tensile strengthincreases as the cross-sectional area decreases because there is lesschance for defects in thinner sections [107–109], (b) as the fibrediameter decreases, improved longitudinal alignment takes placethat enhances strong interactions between the collagen fibrils[96,97,110,111]. These strong interactions are manifested in thestress–strain curves with the short toe region observed for the thinfibres, whilst the looser interactionswould tend to give rise to a longertoe region for the thick fibres, (c) since water acts as a plasticiser forbiopolymers [112] and as a mild plasticiser for collagen [113], watermolecules are not removed during air-drying [114] and inflexibility offibres is achieved by removal of water from the fibrous structure [115],it is realistic to presume that thick fibres could have increased watercontent, whilst thin fibres have absorbed less water and that is whythick fibres exhibit longer toe regions than their thinner counterpartswithin the same treatment.
The stress, force and modulus values were increased after cross-linking for all treatments. We propose that residual water moleculeswithin the fibrous structure could be responsible for the observedincreased in the aforementioned values. Indeed, in non-cross-linkedfibres water molecules could break down the hydrogen and theelectrostatic bonds that hold collagen fibrils together [116] and theinter- and intra-molecular hydrogen bonds and that control H–O–H–collagen bonds, i.e. the number and the length of distance betweenthe protein chains [72,117]. It is likely that after cross-linking, thesewater-binding sites are unavailable to bond inter-molecularly and
therefore they stiffen the collagen triple helix and prevent slippageand translation to occur between neighbouring molecules [34].
Table 1 clearly demonstrates that different cross-linking approaches,due to the different chemistry that is involved in the stabilisationprocess, lead to diverse mechanical properties. In fact, it is long knownthat even between aldehydes, their ability to cross-link collagen withrespect to thenumberof cross-links introduced and their stabilitydiffersconsiderably among them [118]. Formaldehyde treatment does notresult in inter-fibrillar cross-links; does not co-polymerise as has beendemonstrated with glutaraldehyde; and does not introduce bulkypolymeric adducts into the fibril structure as has been shown forglutaraldehyde [89,90]. Glutaraldehyde on the other hand is abiofunctional cross-linking agent that can self-polymerise and conse-quently create a three-dimensional network with long-range cross-links spanning larger gaps, which can affect the collagen fibresproperties by stiffening and strengthening the fibres [47,48]. M. rubratannin molecule has highly nucleophilic reaction activity and it can becovalently bonded to amino groups of collagen molecules by reactionwith aldehyde [119,120].
The most significant finding of this study is that extruded collagenfibres were produced with mechanical and structural propertiesclosely matching native tissues. For example, human anterior cruciateligament, rat tail tendon, bovine and rabbit Achilles tendons havebeen shown to have diameter ranging from 20 to 400 μm, that canwithstand mechanical loads from 15 to 53 MPa and exhibit strain atbreak from 7 to 40% [29,48,75,97,110,111,121,122]. Fixation using M.rubra yielded fibres with properties falling within this range. It hasbeen reported that plant extracts exhibit lower cytotoxicity thanglutaraldehyde and remarkable resistance to degradation and as suchas promising strategies for functional tissue engineering applications[30,55–57,62,123].
5. Conclusions
This work investigated the influence of M. rubra on the propertiesof extruded collagen fibres. Results reported demonstrate that theproduced fibres are characterised by thermal, structural, physical andmechanical properties that arematching native tissues such as tendonand anterior cruciate ligament. Therefore, stabilisation using M. rubracould be a valuable alternative to aldehyde approaches for the con-struction of three-dimensional scaffolds that would imitate nativeextracellular matrix assemblies.
Acknowledgments
The authors would like to thank Mrs. P. Potter, Ms. S. Lee, Mrs.T. Hayes and Mr. L. Stathopoulos for excellent technical assistance;Dr. S. Jeyapalina and Dr. P. Antunes for their useful discussion; andProf Tony Covington for sample donation. Dimitrios Zeugolis isgrateful to The University of Northampton and EPSRC for financialsupport.
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