Wired silk architectures provide a biomimetic ACL tissue engineering scaffold

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Wired silk architectures provide a biomimetic ACL tissueengineering scaffold

Xiang Lia,b, Jess G. Snedekera,b,n

aDepartment of Orthopedics, University of Zurich, Balgrist University Hospital, Zurich, SwitzerlandbDepartment Health Sciences and Technology, ETH Zurich, Switzerland

a r t i c l e i n f o

Article history:

Received 2 November 2012

Received in revised form

11 March 2013

Accepted 14 March 2013

Available online 2 April 2013

Keywords:

Silk scaffold

Ligament tissue engineering

ACL reconstruction

Cyclic loading

nt matter & 2013 Elsevie1016/j.jmbbm.2013.03.015

hor. University of Zurichitzerland. Tel.: +41 443 [email protected] (J.G. Sn

a b s t r a c t

Silk has been increasingly investigated as a scaffold for tissue-engineered anterior cruciate

ligament (ACL) grafts, primarily due to a uniquely advantageous combination of biocompat-

ibility and robust biomechanical strength in the short and middle terms. While previous

studies have explored the biomechanical and biological effects of graft geometry, these studies

have largely ignored the effects of repeated loading on long term biomechanical performance

—an important consideration considering the relatively slow rate with which the silk scaffold

is remodeled. In the present study, we utilized a tensile bioreactor to carry out cyclic loading

tests on various silk ACL scaffold designs. Silk scaffolds were fabricated with three different

architectures (wired, braided, and straight fibered). These were tested in static loading, low

cyclic loading to 250 cycles, and high cyclic loading to 100,000 cycles. Different scaffold

conditions including dry, wet, with cells, without seeded cells were tested and compared. The

ultimate tensile strength (UTS), linear stiffness and construct elongation rate were used to

compare the structural behavior of each graft architecture. Based upon this analysis, silk

scaffolds with a wired structure exhibited biomechanical behavior most similar to the native

human ACL. We thus conclude that the wired silk scaffold design we present provides a

biofidelic mechanical basis for tissue engineering strategies for ACL reconstruction.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ligament injuries, typically occurring during sports and otherrigorous physical activities, are among the most commonmusculoskeletal disorders. Due to its anatomical location,the anterior cruciate ligament (ACL) is subjected to high forcesduring even normal daily activities and is predisposed totraumatic injury. Consequently, ACL rupture is among themost frequent and severe of ligament injuries (Parkkari et al.,2008). It has been estimated that more than 250,000 patientsper year suffer ACL disruption in the United States (equivalentto 1 in 3000 from the general population), with approximately

r Ltd. All rights reserved.

, Department of Orthope63 755.edeker).

50,000 of these cases treated by surgical reconstruction(freeman and A.L., 2008; Majewski et al., 2006; Cooper et al.,2005; Vunjak-Novakovic et al., 2004). Although numeroussurgical options exist for ACL reconstruction, including auto-grafts, allografts, xenografts, or synthetic grafts, each is asso-ciated with inherent drawbacks, including donor sitemorbidity in the case of autografts (Bach et al., 1998, 1998),disease transmission (Strickland et al., 2003) and immuneresponse in the case of allografts (Badylak et al., 1995;Milthorpe, 1994), and more universal issues like ligamentlaxity, mechanical mismatch, and others (Maletius andGillquist, 1997; Teh et al., 2011; Miller and Gladstone, 2002).

dics, Balgrist University Hospital, Uniklinik Balgrist, Forchstrasse

dx.doi.org/10.1016/j.jmbbm.2013.03.015

dx.doi.org/10.1016/j.jmbbm.2013.03.015

dx.doi.org/10.1016/j.jmbbm.2013.03.015

mailto:[email protected]

dx.doi.org/10.1016/j.jmbbm.2013.03.015

j o u r n a l o f t h e m e c h a n i c a l b e h a v i o r o f b i o m e d i c a l m a t e r i a l s 2 2 ( 2 0 1 3 ) 3 0 – 4 0 31

Thus despite a wide range of options for graft-based ACLreconstruction, further improvements are required. It is widelyviewed that rapid developments in tissue engineering mayoffer promise in this regard (Vunjak-Novakovic et al., 2004;Liu et al., 2008; Shen et al., 2010; Sahoo et al., 2010; Fan et al.,2009; Ge et al., 2006; Zeugolis et al., 2011).

Particularly in cases for which a graft is immediatelychallenged by high mechanical demands, a wise choice ofscaffold represents a keystone within a tissue engineeringbased strategy. In addition to mechanical stability, an ACLscaffold should also be biodegradable, biocompatible, and besuitably cell permissive to allow cell ingrowth and eventualgraft remodeling (Teh et al., 2011; Liu et al., 2008). Silkworm silkfibroin, a natural biopolymer derived by removing the hyper-allergenic sericin component of raw silk (Teh et al., 2010; Wanget al., 2011), has been used as clinical suture material forcenturies (Moy et al., 1991). Silk fibroin provides an excellentcombination of strong yet adjustable mechanical properties(failure stresses up to 4.8 GPa), remarkable toughness andelasticity (up to 35% failure strains), and a large degree ofbiological stability (Shen et al., 2010; Altman et al., 2003; Wanget al., 2006). As a structural template for eventual tissueremodeling silk fibroin has been shown to be similar tocollagen in supporting cell attachment, inducing appropriatemorphology and cell growth (Minoura et al., 1995; Inouye et al.,1998), with a relatively slow degradation rate that involves agradual loss of tensile strength over 1 year in vivo (Vunjak-Novakovic et al., 2004; Altman et al., 2003). Thus, due to itsestablished biocompatibility and well-suited biomechanicalproperties, silk fibroin has been increasingly investigated as apotential ligament or tendon graft material in the last decade(Ge et al., 2006; Zhang et al., 2010; Sandmann and Tischer, 2010;Panas et al., 2009; Laurencin and Freeman, 2005; Weitzel et al.,2002; Panas-Perez et al., 2013).

Many in vitro studies have been performed on silk basedscaffolds for ligament tissue engineering, evaluating effects ofsurface treatments, biological factors, and cell types (Teh et al.,2011; Liu et al., 2008; Shen et al., 2010; Sahoo et al., 2010; Wanget al., 2011; Min et al., 2004a, 2004b; Fang et al., 2009; Chen et al.,2012; He et al., 2013). There are also numerous studies thathave reported positive results using silk-based ligament scaf-folds in preclinical animal models (Liu et al., 2008; Fan et al.,2009; Chen et al., 2009; Altman et al., 2008). Safety and efficacyoriented clinical trials of silk-based ACL scaffolds in humanknees have also been reported (Horan, 2009). While thepromise of silk-based grafts for ACL reconstruction seemsclear, there is still substantial room for improvement particu-larly at the level of meso-scale (equivalent to collagen fiber andfascicle level) graft architecture (Cooper et al., 2005; Liu et al.,2008). More specifically, the manner in which the silk fibroin isstructured can largely dominate the biomechanical perfor-mance of the graft (and thus downstream, the biologicalperformance as well). The current study thus focuses onoptimizing the architecture of a silk ACL graft. Some progresshas been made in this regard by other groups, for instance inthe development of cabled silk structures for ligament recon-struction (Horan et al., 2006). Other studies have investigatedthe implications of hierarchical organization of silk matrixusing 6-cord wire-rope configurations (Vunjak-Novakovicet al., 2004; Altman et al., 2003, 2002).

One major issue with published studies to date is that theygenerally neglect to account for changes in the mechanicalbehavior of the graft after repeated cycling, particularly underwet conditions. Over the course of rehabilitation, an ACL graftwill undergo many thousands of cycles before it has beenrobustly incorporated within the bone tunnels, and its mid-substance has been populated by fibroblastic cells. Whilemedium to longer term cyclic mechanical behavior of silkbased ligament scaffolds is at least as important as the initialgraft properties, only a few studies have focused on thisaspect (Vunjak-Novakovic et al., 2004; Cartmell, 2011). We arethus lacking a rigorous evaluation of how silk graft architec-ture affects the evolution of mechanical properties undercyclic loading.

Therefore, the purpose of this study is to design a silk ACLscaffold with biofidelic mechanical properties, as character-ized under functionally meaningful test conditions. Wetested different architectures under cyclic loading. A speciallydesigned bioreactor for cyclic loading was implemented.The mechanical properties of different silk ACL scaffoldsafter cyclic loading (up to 100,000 cycles) were measured. Bycomparing the response of various graft architectures withthe mechanical properties of human ACL, we were able toidentify a suitable architecture for the design of silk ACLscaffolds.

2. Material and methods

2.1. Preparation of silk ACL scaffolds

Raw silk fibers (Bombyx mori) were obtained from a commer-cial supplier (Grege 20/22, Trudel Limited, Zurich, Switzer-land). All scaffolds were produced using a specially designedwiring machine. Three different classes of hierarchical ACLgraft architectures were employed—wired, braided andstraight fibered (Laurencin and Freeman, 2005; Seo et al.,2007; Liu et al., 2008). Across all designs, a consistent size(�30 mm) and total number (3456) of fibers (the basic struc-tural unit) was chosen to facilitate comparison with pre-viously reported designs of silk ACL scaffolds (Horan et al.,2006; Altman et al., 2002). The geometries of the differenthierarchical architectures can be described using a labelingconvention of A(a)nB(b)nC(c)nD(d), where A, B, C, D representincreasing hierarchical level—fibers(A), bundles(B), yarns(C)and cords(D)), while a, b, c, d is the twisting level correspond-ing to the length (mm) per turn on each of the hierarchicallevels. For example, a wired ACL scaffold architecture definedas 6(0)n2(2)n144(10)n2(12) indicates 6 fibers in 1 bundle with-out twist (0 means parallel), 2 bundles in 1 yarn with 2 mmper turn, 144 yarns in 1 cord with 10 mm per turn, 2 cords in 1ACL scaffold with 12 mm per turn.

A number of different geometries for each architecturewere screened in pilot studies before finally fixing thegeometry of each architecture for further tests. This pre-selection was based on considerations of scaffold size, initialfailure force and feasibility of fabrication. Specifically, silkACL scaffolds were manufactured in the following designs:wired—6(0)n2(2)n144(10)n2(12), braided— 6(0)n2(2)n96(10)n3(12), and straight—6(0)n2(2)n288(10)n1(0) (Fig. 1).

Fig. 1 – Schematic of silk ACL scaffold with three different architectures.


2.2. Sericin extraction

The silk ACL scaffolds were produced with raw silk yarns.The antigenic protein sericin was removed by immersingthe scaffolds into an aqueous solution of 0.5 wt% Na2CO3 at90–95 1C, using a magnetic stirrer (Basic C, IKA-WERKE,Germany) at 300 RPM for 90 min, then rinsing with runningdistilled water for 15 min, and air drying at 60 1C. Theseprocedures were repeated three times. This protocol wasbased on previous literature (Teh et al., 2010; Wang et al.,2011), but slightly adapted for our study, since the scaffoldswe fabricated were tighter and thicker than those reported inthe literature (Fang et al., 2009). Scanning electron micro-scopy (FEG-SEM, Zeiss LEO Gemini 1530, Germany) with an in-lens detector was used to view the surface of the silk fiber toevaluate the efficacy of sericin extraction. Prior to imaging,the scaffolds were coated with platinum to facilitate imaging.

2.3. Mechanical test

Tension to failure tests (used for screening various designs),and low-cycle-loading tests were performed on a universalmaterial testing machine (Zwick 1456, Zwick GmbH, Ulm,Germany) with a 20 kN force sensor (Gassmann Theiss, Bick-enbach, Germany). Suitable soft tissue fixation clamps wereused (Fessel et al., 2011). The distance between the clamps was3071 mm to simulate the normal ACL length (Beynnon andAmis, 1998; Nurmi et al., 2004). The initial tension to failuretests were applied after a preload of 5 N, and afterwards at adisplacement-controlled loading of 0.5 mm/s. For the low-cycle loading tests, after applying a preload of 5 N, 250 cycles

from 100 N to 250 N, representing the loads of normal walking,were applied using displacement-control and a cycle speed of0.5 mm/s (Coleridge and Amis, 2004; Beynnon and Amis, 1998).

To simulate longer term loading of the graft underapproximately physiological conditions, a specialized bior-eactor was developed. A stepper motor (NA23C60, ZaberTechnologies Inc, Canada) was used to apply cyclical loadswhile measuring applied force on a 1 kN load cell (KMM20,Inelta Sensorsystems, Germany). Custom clamps were devel-oped to fix the silk scaffold at an initial length of 2873 mm.A polysulfon (PSU1000, Quadrant AG, Switzerland) environ-mental chamber was filled with PBS and covered with analuminum foil cap (no contact with the loading piston). Thebioreactors were fixed in an incubator (C150, Binder,Germany) at 37 1C, humidity of 100%, 5% CO2. The bioreactorwas controlled using custom software (LabVIEW 9.1) todetermine gage length at a preload of 5 N, then apply high-cycle loading using strain control (0–3% strain at 1 Hz) for100,000 cycles. Rest intervals of 30 s were applied at incre-ments of 250 cycles, whereupon the gage length was reas-sessed to account for scaffold creep and/or elongation.

2.4. Cell seeding

Human foreskin fibroblasts (HFFs) were purchased fromAmerican Tissue Culture Collection. Cells were maintainedin McCoy's 5A media with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% L-glutamine (all from Invitrogen) with5% carbon dioxide at 37 1C. To assess cell response to variouscompositions of silk scaffolds, HFFs were prelabeled withCalcein AM (Invitrogen) and seeded on scaffolds for 24 h.


Before seeding, silk scaffolds were first immersed into 75%ethanol solution overnight and then sterilized in an autoclaveat 121 1C for 20 min. The sterile scaffolds were brought intothe cell culture hood. Confluent HFF cultures were washedwith PBS (Lonza) and overlaid with Calcein AM solution at5 mM in PBS for 30 min at 37 1C. Cells were then washed withPBS to remove excess solution. Cells suspensions werecreated by overlaying trypsin to detach cells, neutralizingwith media, and centrifuging cells for 5 min at 900 rpm. Thesupernatant was discarded and the cells were resuspended incomplete media. Cell counts were performed, and cells wereseeded at 1.36�106 cells/cm2. Several samples at both 30 minand 24 h post-seeding, were imaged on an upright Leicamicroscope with the appropriate excitation and emissionfilters and a �20 objective. These samples were then

Fig. 2 – Photo of bioreactors

Table 1 – Overview of test conditions, groups, and numbers o

Sample architecture Sample condition

Silk yarn Unextracted, dry

Extracted, dry

Extracted, wet

Straight Extracted, dry

Extracted, wet

Wired Extracted, dry

Extracted, wet

Wet, sterilized

With cells, 24 h

With cells, 7 days

Extracted, wet

Extracted, wet

With cells

Braided Extracted, dry

Extracted, wet

With cells, 24 h

With cells, 7 days

Extracted, wet

Extracted, wet

With cells

dehydrated in graded ethanol series: 1�10 min. in 25%ethanol, 1�10 min. in 50% ethanol, 1�10 min. in 70% etha-nol, 1�10 min. in 85% ethanol, 1�10 min. in 96% ethanol,3�10 min. in 100% ethanol. After that, specimens weresputter-coated with platinum and imaged by SEM as pre-viously described Fig. 2.

2.5. Statistical analysis

Test conditions, groups, and numbers of samples (n) are listedin Table 1. All data were expressed as mean7standarddeviation (SD) in quantification. The data were comparedwith Student's t-test, and statistically significant values weredefined as po0.05 (pointed out with asterisk in the chart).

fixed in the incubator.

f samples.

Test type Number of samples

Tension to failure 10



4

3

250 cycles 6

100,000 cycles 3


3

250 cycles 6

100,000 cycles 3


3. Results

The ACL graft designs described above were able to beconsistently produced in the three architectures (Fig. 1).Scanning electron micrographs indicated that the appliedsericin extraction protocol was effective (Fig. 3). Further, HFFcells were clearly attached to the scaffolds after 30 min ofseeding and well spread and aligned with the silk fibers after24 h (Fig. 4).

Biomechanical analysis of initial properties: The ultimatetensile strength (UTS) of the silk yarns significantly decreasedafter sericin extraction (Fig. 5), from 9.470.3 N for native silkyarn to 7.370.4 N of sericin extracted (dry) and finally to6.070.3 N for sericin extracted silk in wet conditions (PBS),shown in Fig. 5a. The stiffness of silk yarns decreased as wellafter sericin extraction, from 2.070.1 N/mm for native silkyarn to 1.470.2 N/mm for sericin extracted (dry) to 1.070.2 N/mm for sericin extracted in wet conditions, shown in Fig. 5b.The elongation at tensile failure for silk yarns also decreasedafter sericin extraction, from 9.170.3 mm for native silk yarn,to 8.170.3 mm for sericin extracted (dry) and finally to7.070.4 mm for sericin extracted yarns in wet conditions,shown in supplementary materials.

Tension to failure testing of the assembled silk ACL scaffoldsindicated that a straight-fibered architecture had a lower UTSand higher stiffness than the native human ACL (Woo et al.,1991), whereas the wired and braided architectures were morebiofidelic (Fig. 6). The UTS of wired and braided architecturesdecreased significantly (po0.01) from approximately 1900 N indry conditions to approximately 1500 N in wet conditions, with

Fig. 3 – The SEM images of the surface of silk fibers. (A) O

Fig. 4 – Fluorescently labeled HFF cells on s

failure values slightly lower than that of human ACL, shown inFig. 6a. The stiffness of wired and braided architectures alsodecreased significantly (po0.01) from approximately 550 N/mmin dry conditions to around 250 N/mm in wet conditions, fallingclose to values typical of the human ACL, shown in Fig. 6b. Testson the effects of steam sterilization was also investigated(in wired samples only), but indicated little effect on UTS, linearstiffness, and elongation at failure.

Biomechanical analysis of properties after cyclic loading: Focus-ing on the ACL scaffolds yielding the most biofidelic perfor-mance (wired and braided architectures), we then tested theevolution of biomechanical properties after repeated cyclingto 3% strain at 1 Hz (Fig. 7).

Following 250 loading cycles (n¼6), UTS in braided andwired samples decreased �51% and �41%, respectively(po0.01). Stiffness increased �114% and �48% for the braidedand wired specimens, respectively (po0.01). In more limitedtesting to 100,000 cycles (n¼3), UTS in braided and wiredsamples decreased �75% and �66%, respectively (po0.01).Stiffness increased �128% and �70% for the braided andwired specimens, respectively (po0.01). There was no sig-nificant difference on UTS (p¼0.68) and stiffness (p¼0.94) forwired architectures between the silk ACL scaffold with cellsand without cells under 100,000 cycles.

Although both architectures demonstrated similar func-tional performance after 100,000 cycles, the wired architec-ture had generally more stable stiffness and rates ofelongation in comparison to the braided construct (Fig. 8).Analysis of failure mode in the wired architecture revealeddifferences in failure mode after high cyclic loading, with amore brittle rupture being observed (Fig. 9).

riginal raw silk fibers; (B) sericin-extracted silk fibers).

ilk scaffold. (A) at 30 min; (B) at 24 h).

Fig. 5 – UTS and stiffness of silk yarn in different conditions.

(A) UTS; (B) stiffness; po0.05).

Fig. 6 – UTS and stiffness of silk scaffolds with three

architectures. (A) UTS; (B) stiffness; human ACL value

(Woo et al., 1991); po0.05).


4. Discussion

Anterior cruciate ligament reconstruction remains a com-mon, yet often problematic, clinical challenge (Reinhardtet al., 2010; Mascarenhas and MacDonald, 2008). In addressingthis challenge, silk-based tissue engineering approaches toACL reconstruction hold substantial promise (Fan et al., 2009,2008; Zamarripa et al., 2009). De-sericinized silk is highlybiocompatible, with a slow rate of degradation and remark-able mechanical properties. While important progress hasbeen made in advancing the use of this material as an ACLgraft, the range of graft architectures that have been exploredin the scientific literature is limited to a handful of concepts.Further, the functional characterization of these designs hasgenerally neglected important aspects related to postopera-tive, in vivo performance—specifically moderate and highcycle loading within an aqueous environment.

In the present study, we explored three graft architectures:braided, wired, and straight-fibered. Variants of these archi-tectures have been explored using other synthetic materials(Cooper et al., 2005; Lu et al., 2005; Kwansa et al., 2010), but toour knowledge they have not been systematically exploredusing silk constructs to mimic the biomechanical behavior ofthe human ACL. Of these architectures, the straight-fibereddesign produced a very strong, but overly stiff graft that wedid not further explore in cyclic load testing. The otherarchitectures (wired and braided) were able to approximatethe strength and stiffness of the human ACL to varyingdegrees, yet this depended both upon the graft preparationand the testing conditions in which the functional perfor-mance was characterized.

As silk sericin can be a major cause of adverse immunereactions, it is essential to ensure adequate sericin removalbefore usage. The sericin of the silk ACL scaffolds used in thisstudy was effectively removed using slightly adapted proce-dures based on the literature. However, the mechanicalproperties changed remarkably after sericin extraction. Forthe silk yarns used in all of the ACL architectures investigatedin this study, there was a �22% decrease in UTS (from9.4270.33 N to 7.3470.35 N), and a �30% decrease in linearstiffness (from 1.9770.07 N/mm to 1.3770.17 N/mm) asso-ciated with sericin extraction. These data are similar toreported values for single silk fibers after sericin extraction—15.2% decrease in UTS, and 28.3% decrease in linear stiff-ness (Altman et al., 2002). The diameter of silk yarn alsodecreased �29% after sericin extraction. Because of consider-able difference in mechanical properties between raw silkand sericin-extracted silk, any biomechanical characteriza-tion of a silk scaffold design should be performed usingsericin extracted silk.

By adopting both dry and wet testing environments, wefound that wet testing conditions yielded a large drop in UTSand linear stiffness ( �9% decrease in UTS and �35% decreasein stiffness for straight silk ACL scaffold, while �20% and�16% decrease in UTS, �49% and 56% decrease in stiffnessfor wired and braided silk ACL scaffolds, respectively). It hasbeen suggested that this loss of mechanical strength andstiffness is not due to any chemical destabilization of thestructure, but rather that moisture substantially decreases

Fig. 7 – UTS and stiffness of wired and braided silk scaffolds under different loading conditions. (A) UTS; (B) stiffness;

po0.01).


friction between the hydrophobic silk fibers (Horan et al.,2006). This likely explains the fact that mechanical propertiesof the straight-fibered scaffolds were less affected than thoseof the wired and braided scaffolds, which have considerablymore inter-fiber contact and relative movement. Althoughfluid immersion, progressive loss of hydrophobicity, and an

altered state of fiber hydration could explain the slight dropin UTS observed after incubation for 24 h and 7 days, theexact mechanism behind this drop remains ground for futureinvestigation.

While a broad range of sterilization methods could poten-tially be used for silk ACL scaffolds, we investigated the

Fig. 8 – The linear stiffness and elongation of wired and braided silk ACL scaffolds under high cyclic loading.


effects of steam sterilization on the mechanical propertiesof wired silk scaffolds. Here we found slight, but insignificantdifferences in UTS (p¼0.19) and stiffness (p¼0.14) aftersterilization for 20 min at 121C. Further, as expected, wesaw little influence of the sterilization and cell seedingprocess on the mechanical properties of the wired andbraided silk ACL scaffolds. Regarding cell response, we notethat this limited set of experiments was performed using HFFcell lines, rather than primary cells that would better reflectclinical reality. The commercially available lines we usedwere selected for reasons of reproducibility and standardiza-tion, display a uniform phenotype, and can be easily obtainedfor future study to replicate/build on the results. Further, ourprevious experience indicates that HFFs are quite represen-tative of primary tendon fibroblast response behavior regard-ing biocompatibility and early cell attachment, and thussuitable for the focus of this study. Nonetheless, we cannotdraw any conclusion regarding cell invasion, proliferation,and eventual construct remodeling, since such experimentsshould utilize more appropriate cell types at a minimum, andare anyway best studied in vivo.

We note that only one braided sample survived to 100,000cycles (double asterisk in Fig. 7)—precluding any statisticalanalysis. Braided specimen failure was progressive, startingwithin one cord (each braided sample had three cords).In contrast to the wired specimens, slight differences in thelength of the individual cords may have predisposed theshortest cord to higher loading in each cycle. From imageanalysis of scaffold surfaces after cyclic loading (but beforetension to failure), we could not detect differences betweenlow cycle and high cycle sample loading (Fig. 9). Howeveranalysis of internal structures revealed after tension to fail-ure tests did indicate very different failure modes betweenlow and high cycle loading. The failure of scaffold after lowcyclic loading was concentrated within a single cord whilethe high cycle failure was more evenly distributed, indicatingmore diffuse damage.

In the present study we investigated not only the mechan-ical properties of a freshly manufactured graft, but also theevolution of those properties during highly repetitive cyclingin an aqueous environment. We first assessed biomechanicalperformance after 250 cycles, a level of loading identified inpilot testing as already sufficient to reveal significant differ-ences in “mechanical preconditioning” that was dependentupon the architectures of the silk scaffold, and logisticallypossible to achieve before surgical implantation of the graft(to avoid the bulk of post-operative elongation in vivo). Highcyclic loading to 100,000 cycles at 1 Hz with 30 s rest intervalsevery 250 cycles was also applied, roughly corresponding toone month of typical daily loading of the ACL. With this boutof loading, the UTS of scaffolds substantially decreased, likelydue to progressive accumulation of damage within the scaf-fold. In contrast, the linear stiffness of the scaffolds increasedsharply from 0 to 250 cycles (consistent with precondition-ing), but then remained relatively steady until testing con-cluded after 100,000 cycles. We note that the stiffness of thescaffold mainly depends on the degree of fiber twisting, theprimary design variable that can be used to adjust biomecha-nical behavior of the graft—higher degrees of twist yieldlower construct stiffness. Over the course of cyclic stretch,twisting level decreases due to a dynamic creep, correspond-ing to scaffold elongation and the observed increase instiffness.

Perhaps the most important outcome of the present studywas the identification of ACL scaffold designs (wired andbraided) that approximated the biomechanical properties ofthe human ACL in an aqueous test environment. This standsin contrast to the straight-fibered structure yielding a lowerUTS and much higher linear stiffness than the human ACL.Focusing then only on the wired and braided scaffolds asfunctionally biofidelic designs, we observed that the wiredscaffold had relatively higher UTS but a linear stiffness closerto human ACL than the braided structure. Although theelongation of wired scaffold was nearly two-fold higher than

Fig. 9 – Photos and SEM of scaffolds (A) before and (B) after tensile test to failure. Failure mode at the graft level and silk fiber

level are altered by high cycle loading.


the braided scaffold within the first 5000 cycles, after 5000cycles the elongation differences between these structuresdisappeared. Given its relative simplicity (and correspondingease of manufacture), we conclude that a wired silk ACLscaffold may represent an ideal choice as a potential ACLscaffold of the three candidate structures, so long as adequatepre-operative graft preconditioning is performed to minimizein vivo elongation.

Admittedly, there are some limitations in this study. Onlyone specific design for each graft architectural class wasthoroughly tested and compared. Thus no detailed insightto the functional effect of the wiring parameters (twistinglevel, number of fibers per bundle, etc.) could be obtained, aconsideration that may be yield room for further optimiza-tion. Another ground for future work would be an extensivestudy characterizing the architecture dependency of seeded


cell response under different loading conditions (and biolo-gical effects related to local scaffold strain/stress states). Thestructure will also affect cell ingrowth, and eventual scaffoldremodeling—key points that must be eventually consideredwith in vivo study. Thus, our attempt to identify an ‘optimal'structure of a silk scaffold for ACL reconstruction was almostexclusively based on the in vitro biomechanical performanceof a limited range of candidate architectures. Nonetheless, webelieve that this work represents an important step forwardin identifying a promising silk ACL scaffold design, andprovides a necessary functional baseline for furtherimprovements.

5. Conclusion

Silk tissue engineering scaffolds for ACL reconstruction withthree different structures were compared in this study. Thesecomparisons were based on static and cyclic mechanicaltests under different conditions. We found that the tensilestrength and stiffness of silk scaffold markedly decreased inwet condition, but was unaffected by steam sterilization orshort term cell culture. Cyclic loading decreased the tensilestrength of the constructs, with the scaffolds both elongatingand stiffening. Compared to the mechanical properties of thehuman ACL, a wired structure best approximated the biome-chanical behavior of the human ligament. We thereforesuggest a wired silk scaffold geometry as a suitable referencefor future silk ACL scaffold designs, and will adopted thisstructure in our own further studies.

Acknowledgement

The authors wish to thank Trudel Limited (Zurich, Switzerland)for providing raw silk for our research. We also thank Mr.Hansruedi Sommer, Dr. Ram Sharma and Ms. Jingyi Rao fortheir expertise and kind help in the experiments. This studywas partly funded by the Chinese Scholarship Council, and theBonizzi Theler Foundation.

Appendix A. Supporting information

Supplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.jmbbm.2013.03.015.

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Wired silk architectures provide a biomimetic ACL tissue engineering scaffold