research article effects of artificial ligaments with...

Research ArticleEffects of Artificial Ligaments with Different Porous Structureson the Migration of BMSCs

Chun-Hui Wang Wei Hou Ming Yan Zhong-shang Guo Qi WuLong Bi and Yi-Sheng Han

Department of Orthopaedics Xijing Hospital Fourth Military Medical University No 15 West Changle Road Xirsquoan 710032 China

Correspondence should be addressed to Long Bi bilongfmmueducn and Yi-Sheng Han drhanysyeahnet

Received 14 February 2015 Revised 31 March 2015 Accepted 31 March 2015

Academic Editor Hai-Quan Mao

Copyright copy 2015 Chun-Hui Wang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Polyethylene terephthalate- (PET-) based artificial ligaments (PET-ALs) are commonly used in anterior cruciate ligament (ACL)reconstruction surgery The effects of different porous structures on the migration of bone marrow mesenchymal stem cells(BMSCs) on artificial ligaments and the underlying mechanisms are unclear In this study a cell migration model was utilizedto observe the migration of BMSCs on PET-ALs with different porous structures A rabbit extra-articular graft-to-bone healingmodel was applied to investigate the in vivo effects of four types of PET-ALs and a mechanical test and histological observationwere performed at 4 weeks and 12 weeks The BMSC migration area of the 5A group was significantly larger than that of the otherthree groupsThemigration of BMSCs in the 5A groupwas abolished by blocking the RhoAROCK signaling pathway with Y27632The in vivo study demonstrated that implantation of 5A significantly improved osseointegration Our study explicitly demonstratesthat the migration ability of BMSCs can be regulated by varying the porous structures of the artificial ligaments and suggests thatthis regulation is related to the RhoAROCK signaling pathway Artificial ligaments prepared using a proper knitting method andline density may exhibit improved biocompatibility and clinical performance

1 Introduction

Anterior cruciate ligament (ACL) rupture is the most com-mon ligament disorder in daily life [1] The ACL is anintra-articular ligament that connects the femur to the tibiato maintain the stability of the knee joint [2] Because ofthe poor self-healing ability of an injured ACL surgicalACL reconstruction is usually required [3] In the USAapproximately 6permil of the population suffers pain due to ACLinjury each year half of whom require surgical ACL recon-struction [4ndash7] Autologous and allogeneic grafts are widelyutilized in traditional ACL replacement and reconstructionmethods but they are limited by donor-site morbidity anda long rehabilitation period [8] Since the 1980s artificialligaments such as Gore-Tex and Dacron ligament prosthe-ses have increased in popularity Deterioration is a typicallong-term clinical outcome due to mechanical mismatchand limited graft-to-bone healing [9] In 1992 ligamentadvanced reinforcement system (LARS) ligaments composed

of polyethylene terephthalate (PET) were developed [10 11]LARS ligaments are approved for marketing by the UnitedStates Food and Drug Administration (FDA) and havebeen applied clinically for decades [12 13] but have somedisadvantages [14] Guidoin et al observed that scar tissuemacrophages and giant cells could infiltrate between thefibers resulting in ligament failure [15] The LARS ligamentis not biodegradable complicating tissue ingrowth to form anew nature ligament [16] Although inducing tissue ingrowthis very important for the biocompatibility of implants [17]tissue ingrowth is often random and is affected by the surfacetopography pore diameter and porosity of the materials [1819] For scaffolds of different porosities the cellular responsesto the implant differ [20] After the implantation of artificialligaments bone marrow mesenchymal stem cells (BMSCs)are the primary cell type in the implanted area Implants withappropriate surface topography pore diameter and porositymay guide the migration of BMSCs and encourage bonetissue ingrowth and promote graft-to-bone healing

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 702381 12 pageshttpdxdoiorg1011552015702381

2 Stem Cells International

In general cellular migration is a coordinated cyclicprocess of polarization protrusion attachment contractionand rear release [21 22] The Rho family of small guano-sine triphosphatases (including RhoA Rac and Cdc42) isinvolved in one of the most important molecular signalingpathways mediating cellular migration [23ndash25] Cdc42 isrequired for the cell polarization and filopodia formationthat influence protrusions Rac is essential for lamellipodiaformation and forward movement while RhoA regulatesactin stress fibers and focal adhesion formation [26 27]Rac and Cdc42 can active RhoA to regulate trailing edgesformation at the back of the cell [28 29] These threeproteins cooperate with each other for efficient cellularmigration Rho-associated kinase (ROCK) a downstreameffector of Rho that is activated by RhoA phosphorylatesthe downstream substrate myosin light chain phosphatase(MLCP) directly or indirectly [27 30 31] ROCK can alsodecrease cofilin activity to increase random migration [32]Finally RhoA and ROCK signaling regulate tail retractionand integrin adhesion at the rear of cells [33] Y27632 aspecific inhibitor of ROCKs inhibits the RhoA kinase familyand influences stress fibers vinculin and myosin light chainphosphatase [34]

The purpose of this study was to investigate cellularmigration ability on PET-based artificial ligaments (PET-ALs)with different porous structures prepared using differentknitting methods We hypothesized that cellular migrationis regulated by the porosity of artificial ligaments andthat differences in BMSC migration are attributable to theRhoAROCK signaling pathway Thus selection of a properknitting method and line density for artificial ligaments mayenable improved biocompatibility and clinical performance

2 Materials and Methods

21 Preparation of Grafts and Mode To obtain PET-ALs withdifferent porous structures we employed 500PETfibers (90crystalline diameter 2846plusmn104 120583m) in a bundle with a linedensity of 5000D (5000 grams per 9000 meters of PET fiberbundles under conventional moisture regain) and 300 PETfibers in a bundle with a line density of 3000D (3000 gramsper 9000 meters of PET fiber bundles under conventionalmoisture regain) as materials and weaved them using twodifferent knitting methods

The full-set threading warp knitting method (A) canproduce braided fabrics with a smoother surface and dimen-sional stability For this knittingmethod we used an indepen-dent improved Raschel II two-bar full-width weft insertionwarp knitting machine (Haining Wellington New MaterialCo Zhejiang China) The width was reduced to 120 cmand the horizontal density (119875

119886) and longitudinal density

(119875119887) were set at 4cm and 8cm respectively The front bar

used polyester fiber with 10 elastic module and 20 cNdtexbreaking strength The back bar used two high-purity (90crystalline) PET fibers of different linear densities (3000D5000D)Themachine speed was 3000 rminThe 5000D and3000D PET-ALs weaved using the full-set threading warpknitting method were denoted by 5A and 3A respectively

The part-set threading warp knitting method (B) pro-duces a braided fabric surface with relief and provides moresprang for improved porosity For this knitting method weused an RSJ4 multibar lace warp knitting machine (HainingWellington New Material Co Zhejiang China) The widthwas 340 cm and the horizontal density (119875

119886) and longitudinal

density (119875119887) were set at 4cm and 8cm respectivelyThe front

bar used full-set threading with polyester fiber and the backbar used part-set threading (50) with high-purity (90crystalline) PET fibers of different linear densities (3000D5000D) The 5000D and 3000D PET-ALs weaved using thepart-set threading warp knitting methods were denoted by5B and 3B respectively

The knitted PET-ALs were washed with a chemicalcleaning agent double distilled water acetone and absoluteethanol in an ultrasonic cleaner for 30min each to remove oildirt and chemical residue

The cell migration model was fabricated using 316Lmedical stainless steel (Figures 1(a)-A -B and -C) witha height of 45mm The diameter of the central cylinderwas 10mm and the annular ring at the top of the modelwas 35mm The central cylinder and the annular ring wereconnected using an 8-mm-wide arm through which BMSCscould be seeded

22 Graft Characterization

221 Scanning Electron Microscopy The morphologies andthe pore diameters of the four types of PET-ALs were eval-uated using an SEM (HITACHI-S4800 Scanning ElectronMicroscope Japan) at a voltage of 5 kV

222 Porosity The open porosity of the four types of PET-ALs was evaluated based on the principle of liquid displace-ment (119899 = 6) The following equation was used to calculatethe porosity of the PET-ALs

120593 =

[(119875119879minus 119875119878) minus (119875

119877minus 119875119875)] 120588H

2O

119881119873minus (119875119877minus 119875119875) 120588H

2Otimes 100 (1)

where 119875119879is the weight of the pycnometer when completely

filled with distilled water and the tested PET-ALs 119875119878is the

weight of the dried pycnometer and the tested PET-ALs 119875119877is

the weight of the pycnometer and the residual water after theremoval of the tested PET-ALs 119875

119875is the weight of the dried

pycnometer 119881119873

is the nominal volume of the pycnometer(119881119873= 50mL) and 120588H

2O is the density of distilled water at the

measurement temperature

223 Mechanical Properties The four types of PET-ALs (119899 =6) were rolled up into tube-like mesh structures to thespecifications of an adult ACL The combined length of thetwo free ends was 700 plusmn 02mm and the diameter of theligament section was 70 plusmn 05mmThe PET-ALs were testedusing an Instron testing system (Model 4442 Instron IncMA) During the test both ends of the PET-ALs were fixedwith homemade clamps and the distance between the clampswas 500plusmn02mmThepretensionwas 2N and the elongation

Stem Cells International 3

(A) (B) (C) (D)

1

2

3

(a)

5A 3A 5B 3B

(b)

5A 3A 5B 3B

(c)

Figure 1 Gross view of the cell migration model (a-A a-B and a-C) and cell migration model with sheets (a-D) The gross view and SEMimages of surfacemorphology of four kinds of PET-ALs (b and c) 5A the linear density is 5000D and the knittingmethod is full-set threadingwarp knitting ldquo5000Drdquo indicates 5000 grams weight of the 9000 meters PET fiber bundles under convention moisture regain 3A the lineardensity is 3000D and the knitting method is full-set threading warp knitting 5B the linear density is 5000D and the knitting method ispart-set threading warp knitting 3B the linear density is 3000D and the knitting method is part-set threading warp knitting 1 indicatescentral cylinder (side) 2 indicates annular ring 3 indicates central cylinder (underside) Scale bars 500120583m

rate was 10mmmin The stiffness and ultimate failure loadwere measured

23 In Vitro Tests

231 Isolation and Culture of BMSCs Animal experimentswere performed after approval by the Institutional AnimalCare and Use Committee of The Fourth Military MedicalUniversity Four-week-old Sprague-Dawley (SD) rats (male)were sacrificed by cervical dislocationThe ratswere sterilizedwith 75 alcohol for 5min The bilateral femur and tibiamarrow cavities were exposed under aseptic conditions andwashed with 10mL of DMEM medium (Corning USA)containing 10 fetal bovine serum (FBS Gibco USA)100UmL penicillin and 100UmL streptomycin (Sigma)The cell suspension was collected and centrifuged at 375timesgfor 5min The supernatants were removed and 5mL of newDMEM medium was used to resuspend the sediment forinoculation into 25-cm2 plastic culture flasks The mediumwas first changed at 24 h and then changed every 3 dThe cell

confluencewas 70ndash80 after 7-8 d the cells were subculturedand the first parental generation (119875

1) was marked Then the

BMSCs were subcultured every 3ndash5 d and the numberingof the parental generations of BMSCs increased as thesubculturing proceeded

232 Seeding of BMSCs Before BMSC seeding the fourtypes of PET-ALs were sterilized with cobalt-60 The sheetswere placed in an untreated 6-well plate and washed in PBS(phosphate buffered saline pH 72ndash76) three times and inDMEM another three times BMSCs from the third to fifthparental generation (119875

3ndash1198755) were used for seedingThe adher-

ent BMSCs were digested using 025 trypsin with 002EDTA The cell suspension was collected and centrifuged at375timesg for 5min The sediment was resuspended and seededonto the sheets at 2 times 105mL in 6-well plate and cultured at37∘C in a humidified atmosphere containing 5 CO

2

233 Evaluation of BMSC Adhesion and Morphology Thesheets (119899 = 3) were gently washed in PBS three times and


fixed overnight in 25 glutaraldehyde solution after the cellshad been seeded and cultured for 3 days Then the sheetswere washed in DDW (doubly distilled water) 3 times andthen dehydrated and coated with conductive coating Themorphologies of the BMSCs on the four types of PET-ALswere investigated via scanning electron microscopy (SEMHITACHI-S4800 Japan) and the diameters of the four typesof PET-ALs were measured

For the BMSC adhesion studies the F-actin cytoskeletonsof the cells were stained with TRITC-labeled phalloidin(Cytoskeleton USA) and the nuclei of the cells were stainedwith 410158406-diamidino-2-phenylindole (DAPI SigmandashAldrichSt Louis MO USA) After 3 days of incubation the sampleswere fixed with 4 paraformaldehyde for 30min washed3 times with PBS permeabilized in 01 Triton X-100 for10min and washed 3 times with PBS After blocking nonspe-cific antibody binding with 1 bovine serum albumin (BSA)for 30min at room temperature the cells were stained withTRITC-labeled phalloidin (1 60 dilution in blocking solu-tion) for 20min and counterstained with DAPI for 10minThe samples weremounted on coverslips and examined usinga confocal laser scanningmicroscope (TCS SP5 Leica SolmsGermany) and the areas of cells were measured using ImagePro Plus 60 (Media Cybernetics Silver Spring USA) Sixdifferent substrate fields weremeasured per sample and threeseparate samples were measured for each type of implant

234 Cell Migration Analysis The cell migration model wasplaced in a 6-well plate with PET-ALs and then 2mL of 5 times105mL119875

3ndash1198755BMSCswas seeded on the plateThemodel was

removed after culturing for 24 h Then fluorescein diacetate(FDA) was added to the final concentrations of 100 120583gmLFDA can cross themembranes of living cells and appear greenunder fluorescence microscopy The density of live cells wascounted by Image Pro Plus 170 to confirm that the actual cellseeding densities were the same on different groups of ALsAfter the cellmigrationmodelwas removed and the cells werecultured for an additional 24 h 48 h and 72 h the cells onthe PET-AL sheets were fixed with Carnoy stationary liquid(methanol glacial acetic acid = 3 1) followed by stainingfor 20min with a working solution of Giemsa stain (Catnumber 32884 Sigma)Themigration area of the BMSCs wasestablished and computed using Image Pro Plus 60

235 Western Blotting After the cell migration model wasremoved and the cells were incubated for 72 h the BMSCsfrom the four types of PET-AL sheets were harvested usingpancreatic enzyme and rinsed three times with PBS RIPAbuffer was added to lyse the cells The lysates were cen-trifuged at 104 timesg for 10min at 4∘C and the protein inthe supernatant was extracted Equal amounts of protein(30 120583g) were separated by 10 SDS-PAGE and transferredto a PVDF membrane (poly-vinylidene fluoride Bio-Rad)After blocking with 5 nonfat dry milk in Tris-bufferedsaline with Tween for 1 h the membranes were incubatedwith the following antibodies overnight at 4∘C anti-RhoA(Santa Cruz Biotechnology Inc USA) anti-ROCK1 (Santa

Cruz Biotechnology Inc USA) anti-pMLC (Cell Signal-ing Technology Inc USA) and anti-GAPDH (Santa CruzBiotechnology Inc USA) followed by incubation withhorseradish peroxidase-conjugated antibody for 1 h at roomtemperature Signals were detected using the Western-LightChemiluminescent Detection System (Peiqing China) Theintegrated density values were calculated using Image ProPlus 60

24 In Vivo Tests

241 Implantation Surgery Procedure The animal experi-ments were performed strictly in accordance with the animalprotocol approved by the Institutional Animal Care andUse Committee of The Fourth Military Medical UniversityEighteen adult New Zealand rabbits (males 12 weeks oldand 30 plusmn 04 kg) were subjected to extra-articular graft-to-bone healing surgery The four types of PET-ALs wererandomly implanted into two tunnels (119877 = 2mm) in thedistal femur and two tunnels (119877 = 2mm) in the proximaltibia Gentamicin (5mgkg) and penicillin (50KUkg) wereinjected for 3 consecutive days postoperatively The rabbitswere randomly sacrificed at 4 and 12 weeks after surgery

242 Mechanical Tests Six specimens from each group wereused for mechanical tests Each graft was sutured using aNo 5 ETHIBOND suture and mounted onto a special jigto allow the graft to extend out of the tunnel The testwas performed using a Material Testing System Model 858(MTS Systems Minneapolis MN) at an elongation rateof 2mmmin The ultimate failure load (N) and stiffness(Nmm) were measured

243 Histological Observations All specimens were fixed in80 ethanol for two weeks Specimens were dehydrated ina graded ethanol series (70ndash100) and then embedded ina methylmethacrylate (MMA) solution for 3 weeks Afterpolymerization of the MMA at 50∘C for 24 h pathologicalsections to the longitudinal axis and the horizontal axis wereprepared using a band saw (Leica Microtome Wetzlar Ger-many) stained with Van Gieson and observed under a lightmicroscope (Leica LA Microsystems Bensheim Germany)Bone formation qualitatively analyzed the VG staining ofpathological sections to the horizontal axis using Image ProPlus 60 and the percentage of new bone formation wascalculated using the following formula

119878119886

1205871198772minus 119878119887

times 100 (2)

where 119878119886is the area of the newly formed bone 119878

119887is the area

of the graft and 119877 is the radius of the tunnel The bonding ofthe graft to the adjacent tissue was analyzed by observing theVG staining of pathological sections to the longitudinal axisSix different sections were measured per sample and threeseparate samples were measured for each type of implant ateach time point


Table 1 Characterization of different PET-ALs (n = 6 mean plusmn SD)

Implant Pore diameter(120583m)

Porosity()

Ultimate failure load(kN)

Stiffness(Nmm)

5A 1534 plusmn 102 552 plusmn 09 66443 plusmn 01015 2153 plusmn 1563A 1907 plusmn 121lowast 724 plusmn 32lowast 4886 plusmn 00688lowast 864 plusmn 32lowast

5B 1804 plusmn 159lowast 683 plusmn 15lowast 44210 plusmn 00980lowast 926 plusmn 74lowast

3B 2453 plusmn 173lowastdagger 795 plusmn 22lowastdagger 25668 plusmn 00794lowastdagger 327 plusmn 27lowastdaggerlowastCompared with 5A group 119901 lt 005 daggerCompared with 3A group 119901 lt 005 Compared with 5B group 119901 lt 005ldquo5rdquo indicates that the PET-ALs were weaved with 5000D PET fiber bundles ldquo5000Drdquo indicates 5000 grams weight of the 9000meters PET fiber bundles underconvention moisture regainldquo3rdquo indicates that the PET-ALs were weaved with 3000D PET fiber bundlesldquoArdquo indicates that the PET-ALs were weaved in full-set threading warp knitting methodldquoBrdquo indicates that the PET-ALs were weaved in part-set threading warp knitting method

25 Statistical Analysis The results were analyzed usingGraphPad Prism 5 and are presented as the means plusmn SDfor each group At least three separate experiments wereperformed to obtain each set of results 119901 lt 005 wasconsidered significant in the paired Studentrsquos 119905-test

3 Results

31 Characterization of Different PET-ALs The characteris-tics of the four types of PET-ALs are summarized in Table 1The pore diameter and the porosity of the 3000D PET-ALswere significantly bigger than those of the 5000D PET-ALsproduced using the same knitting method (119901 lt 005) Porediameter and porosity were significantly higher in 3B thanin the other three PET-ALs (119901 lt 005) The pore diametersand porosities of 3A and 5B were not significantly differentThe ultimate failure load and stiffness of 5Awere significantlyhigher than those of the other three PET-ALs (119901 lt 005)but there was no significant difference between 3A and 5Bdespite their different line densities (119901 gt 005) SEM images ofthe four types of PET-ALs (Figure 1(b)) revealed that the twoknitting methods produced different surface morphologies5A and 3A had a more smooth and cramped constructionThese differences could also be observed in the gross photo(Figure 1(b)) of the four types of grafts

32 Effect of Different PET-ALs on BMSC Adhesion andMorphology The morphology of the BMSCs on the PET-ALs was observed after culturing for 72 h Figure 2(a) showsSEM images of BMSCs on the scaffolds In those images theBMSCs on 5A and 3A (Figures 2(a)-A and 2(a)-C) exhibited amore rounded appearance than those on 5B and 3B (Figures2(a)-E and 2(a)-G) Figures 2(b)-A 2(b)-C 2(b)-E and 2(b)-G present the immunostaining of the BMSCs on the scaffoldsNucleus and F-actin were stained blue and red respectivelyAs shown in Figure 2(c) the projected cell area of the BMSCswas significantly smaller on the 5A PET-ALs than on theother three PET-AL types (8846 plusmn 1568 120583m2 versus 10940 plusmn1169 120583m2 11761 plusmn 1765 120583m2 and 14503 plusmn 1339 120583m2119901 lt 005)

33 Effect of Different PET-ALs on Cell Migration Area Afterthe cell migration model was removed and FDA staining

indicated that there were no significant differences amongthe actual cell seeding densities on different group of ALs(live cell density 908 plusmn 42mm2) culturing was performedfor 24 h 48 h and 72 h cell migration was evaluated byGiemsa staining (Figure 3(d)) and the cellmigration areawasmeasured (Figure 3(c)) At the 24-h time point themigrationarea of the 5A PET-ALs (169 plusmn 34mm2) was significantlylarger than that of the other PET-AL types (119901 lt 005)followed by 3A (118 plusmn 13mm2119901 lt 005) 5B (95 plusmn 17mm2119901 lt 005) and 3B (67 plusmn 16mm2 119901 lt 005) The migrationarea of 3Awas significantly greater than that of 5B (119901 lt 005)Themigration area of 5B was significantly greater than that of3B (119901 lt 005)

34 Effect of Different PET-ALs on the Expression of RhoAROCK Pathway Components Western blotting analysis wasperformed after the cell migration model was removed andculturing was performed for 72 h (Figures 3(a) and 3(b))Theexpression levels of RhoA andROCK in the BMSCs on the 5APET-ALwere set as the reference values andwere significantlyhigher for 5A than for the other three PET-AL types (119901 lt005) The RhoA and ROCK expression levels were 1 on the5A PET-ALs and lower expression levels were observed on3A (0831plusmn0024 0718plusmn0032 119901 lt 005) 5B (0729plusmn00170590plusmn0045 119901 lt 005) and 3B (0636plusmn0037 0555plusmn0043119901 lt 005)

35 Effect of Y27632 on Cellular Migration We treated the5A PET-ALs with Y27632 at concentrations of 0 5 10 and20120583molL for 72 h Western blotting analysis (Figures 4(a)and 4(b)) revealed that the expression of ROCK and MLCPon the 5A PET-ALs decreased with increasing concentrationof the inhibitor Y27632 The expression of ROCK andMLCP decreased significantly at Y27632 concentrations of10 120583molL and 20 120583molL (119901 lt 005) but they did notsignificantly decrease when the concentration of Y27632 was5 120583molL (1 versus 0804 plusmn 0155 and 0851 plusmn 0114 119901 gt005) and the ROCK and MLCP expression levels were notsignificantly different between the 10 120583molL and 20 120583molLgroups (0505 plusmn 0031 versus 0393 plusmn 0059 and 037 plusmn 0087versus 0283plusmn0028 119901 gt 005) Cell migration was visualizedby Giemsa staining (Figure 4(d)) and the cell migration areawas measured (Figure 4(c))The cell migration area of the 5A


5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)

Figure 2 SEM images of BMSCs morphology in different groups after incubation for 3 days (a) Confocal laser scanning microscopy images(b) of BMSCs F-actin cytoskeletal (red Rhodamine-phalloidin) morphology and nucleus (blue DAPI) in different groups after 3 days ofincubation (c) Histogram of the cell projected cell area which was analyzed from the fluorescent images P indicates cellular pseudopod Tindicates cellular tail and lowast indicates 119901 lt 005 Scale bars 200 120583m

PET-ALs decreased significantly when the concentration ofY27632 was 10 120583molL or 20120583molL (119901 lt 005) but whenthe concentration of Y27632 was 5120583molL (634 plusmn 77mm2

versus 549plusmn56mm2119901 = 0133) the cell migration areas didnot significantly differ between the 10 120583molL and 20120583molLgroups (227 plusmn 28mm2 versus 174 plusmn 27mm2 119901 = 009)These results indicate that the appropriate concentrationof Y27632 to inhibit cellular migration is 10120583molL to20120583molL We selected 15 120583molL as the concentration ofY27632 to treat BMSCs to observe themorphology of BMSCson different PET-ALs After treatment with 15 120583molL Y27632for 3 days the BMSCs displayed more filopodial extensionsand more podosomes (Figures 2(a)-B 2(a)-D 2(a)-F and2(a)-H) and the projected cell areas (Figure 2(c)) of the fourgroups were significantly augmented (119901 lt 005)

36 Effect of Different PET-ALs on Biomechanical PropertiesAs shown in Figures 5(a) and 5(b) the 5A PET-AL exhibitedan ultimate failure load of 4642 plusmn 425N and stiffness of1632 plusmn 252Nmm at 4 weeks after surgery These valueswere significantly higher than those of the other three groups(119901 lt 005) and were followed by the 3A group (3502 plusmn209N 1233 plusmn 113Nmm) the 5B group (2660 plusmn 253N1043 plusmn 144Nmm) and the 3B group (2015 plusmn 181N 72 plusmn068Nmm) At week 12 after surgery the ultimate failureloads and stiffnesses of the four groups were significantlyincreased (119901 lt 005)The biomechanical properties of the 5APET-AL were still significantly higher than those of the otherPET-ALs (7825 plusmn 252N 3772 plusmn 230Nmm 119901 lt 005)followed by 3A (5748 plusmn 566N 3198 plusmn 171Nmm 119901 lt005) 5B (4195 plusmn 242N 2547 plusmn 177Nmm 119901 lt 005) and3B (3233 plusmn 259N 1815 plusmn 117Nmm)


GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)

Figure 3 Western blot (a) and semiquantitative analysis (b) of RhoA and ROCK expression in BMSCs on different groups after incubationfor 3 days Stereomicroscope images (d) of Giemsa staining of different groups and histogram of cell migration area (c) which was analyzedfrom the stereomicroscope images lowast indicates 119901 lt 005 Scale bars 2mm (white)

37 Effect of Different PET-ALs on Bone Regeneration andOsseointegration Von-Gieson (VG) staining was used toassess the implant-to-bone healing of the four types of PET-ALs at 4 and 12 weeks (Figure 5(d)) After 4 weeks weobserved that more regenerative bone (red) had integratedinto the implant in the 5A PET-AL group (921 plusmn 135)

but little newly formed bone was integrated in the 3A PET-AL group (637 plusmn 053) The least regenerative bone wasobserved in the 5B and 3B PET-AL groups (415 plusmn 054241 plusmn 046) High-resolution microscope images of VGstaining of pathological sections to the longitudinal axis ofthe 5A PET-AL revealed that the regenerative bone bonded


Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A

0120583molL 5120583molL 10120583molL 20120583molL

(d)

Figure 4 Western blot (a) and semiquantitative analysis (b) of ROCK and p-MLC expression in BMSCs on 5A PET-ALs after incubation for72 h in the concentration gradient of 0 5 10 and 20 120583molL of Y27632 Stereomicroscope images (d) of Giemsa staining of 5A PE-ALs treatedwith different concentrations of Y27632 and histogram of cell migration area (c) which was analyzed from the stereomicroscope images lowastindicates 119901 lt 005 Scale bars 2mm

tightly to the 5APET-ALs and no inflammatory reaction wasobserved In the images of the VG staining of pathologicalsections to the horizontal axis of 5A PET-AL we observedmore regenerative bone and less fibrous tissue (dark blue)in the inner region of the PET-AL whereas less regenerativebone andmore fibrous tissue (dark blue) were observed in theinner region of the 3A 5B and 3B PET-ALs After implan-tation for 12 weeks the amount of regenerative bone wassignificantly higher in the 5A PET-AL group (2132 plusmn 337)than the other three groups (1411 plusmn 115 121 plusmn 153876 plusmn 148 119901 lt 005) High-resolution microscope imagesof VG staining of pathological sections to the longitudinalaxis of the 5A PET-AL revealed that new bone was formedbetween the fibers of the 5A PET-ALs while more fibroustissue was observed in the other three PET-AL groups

4 Discussion

Osseointegration into the insertion site is an important factorin artificial ligament use [35] Many studies of materials andsurface modification have sought to improve the osseointe-gration of artificial ligaments [36ndash38] but few have exam-ined the effects of artificial ligaments with different porousstructures on bone regeneration Such studies are necessaryto improve the biocompatibility and clinical performance ofartificial ligaments

In this study four types of artificial ligaments preparedfrom nonbiodegradable and avirulent polyethylene tereph-thalate (PET) fibers were used to investigate the effects ofporous structures on cellular migration We observed that5A PET-ALs were the most suitable for BMSCmigration andbone regeneration the other three groups exhibited smallercellular migration area and more soft tissues (dark blue) thanbone regeneration (Figure 5(d)) Scar tissue infiltration hin-ders the osseointegration of artificial ligaments [39 40] Sincethe regenerative bone bonded tightly to the four types of PET-ALs (Figure 5(d)) the biomechanical properties of the fourPET-ALs groups were positively correlated with the quantityof the newly formed bone Interestingly we did not observesignificant differences in pore diameter and porosity between3A and 5B but the migration area and bone regeneration of3A were significantly higher than those of 5B SEM imagesand gross photos (Figures 1(b) and 1(c)) revealed differencesbetween the surface topographies of the A PET-ALs andthe B PET-ALs the A PET-ALs had a smoother surfacewhereas the B PET-ALs had a more rugged surface Wedemonstrated that a rugged surface has amain negative effecton cellular migration A strong correlation among decreasedpore diameter decreased porosity and increased cellularmigration area was observed in the four PET-AL groups(Table 1) We demonstrated that PET-ALs with lower porediameter and lower porosity had a larger cellular migration


5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)

Figure 5 (a) The ultimate failure load of each group at 4- and 12-week time points (b) The stiffness of each group at 4- and 12-week timepoints (c) Histomorphometric analysis of new bone formation within each kind of implant (d)The light microscope images of pathologicalsections of each kind of group High-resolution microscope VG staining images of pathological sections to the longitudinal axis showed thedetails of graft-to-bone interface The newly formed bone was stained red and fibrous tissue was stained dark blue The black arrows indicatenewly formed bone and the green arrows indicate fibrous tissue lowast indicates 119901 lt 005 Scale bars 500 120583m (blue) and 200 120583m (green)


area (Figure 3(d)) and the inhibition of BMSC migrationby the porous structure of the artificial ligaments increasedas the porosity and pore diameter increased In accordancewith our findings extensive research has demonstrated thatincreasing pore diameter has a negative effect on boneingrowth [41ndash43] Although some studies have suggestedthat increasing the porosity of scaffolds in combination withthe pore diameter provides a larger surface area for theattachment and proliferation of cells [20] cells appeared lessmobile with the combined effect of pore diameter increasing(Figure 3(d)) Although previous studies have demonstratedthat cellular behavior is affected by the surface topographypore diameter and porosity of materials the underlyingbiological mechanisms have remained elusive [44 45]

The RhoAROCK signaling pathway is part of the Rhofamily which is important for limiting membrane protru-sions [46 47]This pathway retracts the rear of the cell by sev-ering existing F-actin filaments and phosphorylating myosinlight chain phosphatase (MLC) to regulate cell migration[48] In the present study western blotting analysis (Figures3(a) and 3(b)) verified that the expression of RhoA andROCKdiffered among the grafts Expressionwas significantlyhigher in the 5A group than the other three groups followedby the 3A group 5B group and 3B group Researcheshad reported that ROCK negatively regulates membraneprotrusion to promote migration [33] Cells with high ROCKexpression exhibit a rounded appearance and the inhibitionof ROCK augments the projected cell area In our study SEMimages (Figure 2(a)) of BMSCs in the A groups exhibited amore rounded appearance than those of the B groups andimmunostaining images revealed that the projected cell areaof BMSCs was significantly smaller in the A group thanthe B group Our results indicate that differences in BMSCmigrationmay be at least partly attributable to the expressionof the RhoAROCK signaling pathway This conclusion isfurther supported by the morphology projected cell areaand migration area of BMSCs treated with Y27632 a specificinhibitor of ROCKs [34]We observed increased lamellipodiaand filopodia formation by BMSCs in the SEM images ofthe four types of PET-ALs immunostaining also revealedincreased F-actin staining after the BMSCs were treatedwith Y27632 and the projected cell areas were significantlyincreased in the four groups

Based on the above findings different weaving methodsthat lead to different surface topographies diameters andporosities of PET-ALsmay regulate BMSCmigration throughthe RhoROCK signaling pathway Weaving PET-ALs usingdifferent methods is a potential strategy for improving thebiocompatibility of PET-ALs An ideal ACL prosthesis shouldbe biocompatible and should mimic the mechanical prop-erties of the native ACL [14 15] In our study the 5A PET-AL was the most suitable for BMSC migration and boneregeneration The ultimate failure load and the stiffness ofnative ACLs are 2160 plusmn 157N and 242 plusmn 28Nmm thusthe 5A PET-AL has mechanical properties similar to those ofthe native ACL We demonstrated that 5A is an ideal ACLprosthesis for ACL reconstruction

Cellular migration can also be promoted by modificationof artificial ligaments by growth factors [49] and bioactive

proteins [50] which effectively improve bone tissue ingrowthto promote graft-to-bone healing [51 52] In future studieswe should use an improved knitting method in combinationwith surface modification to increase the biocompatibility ofthe PET-ALs

5 Conclusions

In this study four types of PET-ALs with different surfacetopographies diameters and porosities were investigatedThe 5A PET-ALs were the most suitable for BMSCmigrationand effectively promoted graft-bone healing Moreover thebeneficial effect of the 5A PET-AL on cellular migrationwas abolished by blocking the RhoROCK signaling pathwaywith Y27632 Our study explicitly demonstrates that themigration ability of BMSCs is regulated by differences in theporous structure of artificial ligaments via the RhoROCKsignaling pathway and establishes a proper knitting methodand line density of artificial ligament to attain improvedbiocompatibility and clinical performance

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Chun-Hui Wang and Wei Hou contributed equally to thisstudy

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (Grant no 81371932 and 81371982)

References

[1] W Widuchowski J Widuchowski B Koczy and K SzylukldquoUntreated asymptomatic deep cartilage lesions associated withanterior cruciate ligament injury results at 10- and 15-yearfollow-uprdquoTheAmerican Journal of Sports Medicine vol 37 no4 pp 688ndash692 2009

[2] M Bourdon-Santoyo I Quinones-Uriostegui V Martınez-Lopez et al ldquoPreliminary study of an in vitro development ofnew tissue applying mechanical stimulation with a bioreactoras an alternative for ligament reconstructionrdquo Revista de Inves-tigacion Clınica vol 66 supplement 1 pp S100ndashS110 2014

[3] C Legnani A Ventura C Terzaghi E Borgo and WAlbisetti ldquoAnterior cruciate ligament reconstruction with syn-thetic grafts A review of literaturerdquo International Orthopaedicsvol 34 no 4 pp 465ndash471 2010

[4] H Sofu T Yildirim S Gursu A Issin and V Sahin ldquoShort-term effects of partial meniscectomy on the clinical results ofanterior cruciate ligament reconstructionrdquo Knee Surgery SportsTraumatology Arthroscopy vol 23 no 1 pp 184ndash187 2015

[5] T Zantop M Wellmann F H Fu and W Petersen ldquoTunnelpositioning of anteromedial and posterolateral bundles inanatomic anterior cruciate ligament reconstruction anatomic


and radiographic findingsrdquo The American Journal of SportsMedicine vol 36 no 1 pp 65ndash72 2008

[6] C L Ardern K EWebster N F Taylor and J A Feller ldquoReturnto sport following anterior cruciate ligament reconstructionsurgery a systematic review and meta-analysis of the state ofplayrdquo British Journal of Sports Medicine vol 45 no 7 pp 596ndash606 2011

[7] D A Shaerf P S Pastides K M Sarraf and C A Willis-Owen ldquoAnterior cruciate ligament reconstruction best practicea review of graft choicerdquo World Journal of Orthopaedics vol 5no 1 pp 23ndash29 2014

[8] V K Goradia M C Rochat M Kida and W A GranaldquoNatural history of a hamstring tendon autograft used foranterior cruciate ligament reconstruction in a sheepmodelrdquoTheAmerican Journal of Sports Medicine vol 28 no 1 pp 40ndash462000

[9] Z Ge F Yang J C H Goh S Ramakrishna and E H LeeldquoBiomaterials and scaffolds for ligament tissue engineeringrdquoJournal of Biomedical Materials Research Part A vol 77 no 3pp 639ndash652 2006

[10] J Chen A Gu H Jiang W Zhang and X Yu ldquoA comparisonof acute and chronic anterior cruciate ligament reconstructionusing LARS artificial ligaments a randomized prospective studywith a 5-year follow-uprdquo Archives of Orthopaedic and TraumaSurgery vol 135 no 1 pp 95ndash102 2015

[11] P Lavoie J Fletcher and N Duval ldquoPatient satisfaction needsas related to knee stability and objective findings after ACLreconstruction using the LARS artificial ligamentrdquo The Kneevol 7 no 3 pp 157ndash163 2000

[12] Z-T Liu X-L Zhang Y Jiang and B-F Zeng ldquoFour-strandhamstring tendon autograft versus LARS artificial ligamentfor anterior cruciate ligament reconstructionrdquo InternationalOrthopaedics vol 34 no 1 pp 45ndash49 2010

[13] G Shen Y Xu Q Dong H Zhou and C Yu ldquoArthroscopicposterior cruciate ligament reconstruction using LARS artificialligament a retrospective studyrdquo Journal of Surgical Research vol173 no 1 pp 75ndash82 2012

[14] N L Leong F A Petrigliano and D R McAllister ldquoCur-rent tissue engineering strategies in anterior cruciate ligamentreconstructionrdquo Journal of Biomedical Materials Research PartA vol 102 no 5 pp 1614ndash1624 2014

[15] M-F Guidoin Y Marois J Bejui N Poddevin M W Kingand R Guidoin ldquoAnalysis of retrieved polymer fiber basedreplacements for the ACLrdquo Biomaterials vol 21 no 23 pp2461ndash2474 2000

[16] F A Petrigliano D R McAllister and B M Wu ldquoTissueengineering for anterior cruciate ligament reconstruction areview of current strategiesrdquoArthroscopy vol 22 no 4 pp 441ndash451 2006

[17] T Nau and A Teuschl ldquoRegeneration of the anterior cruciateligament current strategies in tissue engineeringrdquo World Jour-nal of Orthopedics vol 6 no 1 pp 127ndash136 2015

[18] R J Petrie A D Doyle and K M Yamada ldquoRandom versusdirectionally persistent cell migrationrdquoNature Reviews Molecu-lar Cell Biology vol 10 no 8 pp 538ndash549 2009

[19] M Estevez E Martınez S J Yarwood M J Dalby andJ Samitier ldquoAdhesion and migration of cells responding tomicrotopographyrdquo Journal of Biomedical Materials ResearchPart A vol 103 no 5 pp 1659ndash1668 2015

[20] M B Claase J D de Bruijn D W Grijpma and J Fei-jen ldquoEctopic bone formation in cell-seeded poly(ethylene

oxide)poly(butylene terephthalate) copolymer scaffolds ofvarying porosityrdquo Journal of Materials Science Materials inMedicine vol 18 no 7 pp 1299ndash1307 2007

[21] P Friedl and K Wolf ldquoTumour-cell invasion and migrationdiversity and escape mechanismsrdquo Nature Reviews Cancer vol3 no 5 pp 362ndash374 2003

[22] D A Lauffenburger and A F Horwitz ldquoCell migration aphysically integrated molecular processrdquo Cell vol 84 no 3 pp359ndash369 1996

[23] J W Erickson and R A Cerione ldquoMultiple roles for Cdc42 incell regulationrdquo Current Opinion in Cell Biology vol 13 no 2pp 153ndash157 2001

[24] X-T Xu Q-B Song Y Yao B Xu P Ruan and Z-Z TaoldquoInhibition of RhoAROCK signaling pathway promotes theapoptosis of gastric cancer cellsrdquo Hepato-Gastroenterology vol59 no 120 pp 2523ndash2526 2012

[25] D Epting K Slanchev C Boehlke et al ldquoThe Rac1 regulatorELMO controls basal body migration and docking in multicili-ated cells through interactionwithEzrinrdquoDevelopment vol 142no 1 pp 174ndash184 2014

[26] S Etienne-Manneville ldquoCdc42mdashthe centre of polarityrdquo Journalof Cell Science vol 117 part 8 pp 1291ndash1300 2004

[27] W T Arthur and K Burridge ldquoRhoA inactivation byp190RhoGAP regulates cell spreading and migration bypromoting membrane protrusion and polarityrdquo MolecularBiology of the Cell vol 12 no 9 pp 2711ndash2720 2001

[28] C D Nobes and A Hall ldquoRho GTPases control polarityprotrusion and adhesion during cell movementrdquo Journal of CellBiology vol 144 no 6 pp 1235ndash1244 1999

[29] E E Sander J P ten Klooster S van Delft R A van derKammen and J G Collard ldquoRac downregulates Rho activityreciprocal balance between both GTPases determines cellularmorphology and migratory behaviorrdquo The Journal of CellBiology vol 147 no 5 pp 1009ndash1022 1999

[30] X Li L Liu J C Tupper et al ldquoInhibition of protein geranylger-anylation and RhoARhoA kinase pathway induces apoptosisin human endothelial cellsrdquo Journal of Biological Chemistry vol277 no 18 pp 15309ndash15316 2002

[31] S A Barman S Zhu and R E White ldquoRhoARho-kinasesignaling a therapeutic target in pulmonary hypertensionrdquoVascularHealth andRiskManagement vol 5 pp 663ndash671 2009

[32] D P White P T Caswell and J C Norman ldquo120572v1205733 and 12057251205731integrin recycling pathways dictate downstream Rho kinasesignaling to regulate persistent cell migrationrdquo The Journal ofCell Biology vol 177 no 3 pp 515ndash525 2007

[33] R A Worthylake and K Burridge ldquoRhoA and ROCK promotemigration by limiting membrane protrusionsrdquo The Journal ofBiological Chemistry vol 278 no 15 pp 13578ndash13584 2003

[34] E Novozhilova U Englund-Johansson A Kale Y Jiao and POlivius ldquoEffects of ROCK inhibitor Y27632 and EGFR inhibitorPD168393 on human neural precursors co-cultured with ratauditory brainstem explantrdquo Neuroscience vol 287 pp 43ndash542015

[35] J Yang J Jiang Y Li et al ldquoA new strategy to enhanceartificial ligament graft osseointegration in the bone tunnelusing hydroxypropylcelluloserdquo International Orthopaedics vol37 no 3 pp 515ndash521 2013

[36] J S Mulford and D Chen ldquoAnterior cruciate ligament recon-struction a systematic review of polyethylene terephthalategraftsrdquo ANZ Journal of Surgery vol 81 no 11 pp 785ndash789 2011


[37] U G Longo G Rizzello A Berton et al ldquoSynthetic grafts foranterior cruciate ligament reconstructionrdquo Current Stem CellResearch ampTherapy vol 8 no 6 pp 429ndash437 2013

[38] H Li and S Chen ldquoBiomedical coatings on polyethyleneterephthalate artificial ligamentsrdquo Journal of Biomedical Mate-rials Research Part A vol 103 no 2 pp 839ndash845 2014

[39] S Kawamura L Ying H-J Kim C Dynybil and S A RodeoldquoMacrophages accumulate in the early phase of tendon-bonehealingrdquo Journal of Orthopaedic Research vol 23 no 6 pp1425ndash1432 2005

[40] P L Hays S Kawamura X-H Deng et al ldquoThe role ofmacrophages in early healing of a tendon graft in a bone tunnelrdquoThe Journal of Bone amp Joint SurgerymdashAmerican Volume vol 90no 3 pp 565ndash579 2008

[41] A C Jones C H Arns D W Hutmacher B K Milthorpe AP Sheppard and M A Knackstedt ldquoThe correlation of poremorphology interconnectivity and physical properties of 3Dceramic scaffolds with bone ingrowthrdquo Biomaterials vol 30 no7 pp 1440ndash1451 2009

[42] J A Cooper H H Lu F K Ko J W Freeman and C TLaurencin ldquoFiber-based tissue-engineered scaffold for ligamentreplacement design considerations and in vitro evaluationrdquoBiomaterials vol 26 no 13 pp 1523ndash1532 2005

[43] R T Tran P Thevenot Y Zhang D Gyawali L Tang and JYang ldquoScaffold sheet design strategy for soft tissue engineeringrdquoMaterials vol 3 no 2 pp 1375ndash1389 2010

[44] M Hulsman F Hulshof H Unadkat et al ldquoAnalysis of high-throughput screening reveals the effect of surface topographieson cellular morphologyrdquo Acta Biomaterialia vol 15 pp 29ndash382015

[45] K Kolind KW Leong F Besenbacher andM Foss ldquoGuidanceof stem cell fate on 2D patterned surfacesrdquo Biomaterials vol 33no 28 pp 6626ndash6633 2012

[46] K Kimura M Ito M Amano et al ldquoRegulation of myosinphosphatase by Rho and Rho-associated kinase (Rho-kinase)rdquoScience vol 273 no 5272 pp 245ndash248 1996

[47] R A Worthylake S Lemoine J M Watson and K Bur-ridge ldquoRhoA is required for monocyte tail retraction duringtransendothelial migrationrdquoThe Journal of Cell Biology vol 154no 1 pp 147ndash160 2001

[48] T B Kuhn P J Meberg M D Brown et al ldquoRegulating actindynamics in neuronal growth cones by ADFcofilin and rhofamily GTPasesrdquo Journal of Neurobiology vol 44 no 2 pp 126ndash144 2000

[49] T Kawai T Yamada A Yasukawa Y Koyama T Muneta andK Takakuda ldquoBiological fixation of fibrous materials to boneusing chitinchitosan as a bone formation acceleratorrdquo Journalof Biomedical Materials Research Part B Applied Biomaterialsvol 88 no 1 pp 264ndash270 2009

[50] M M Murray K P Spindler P Ballard T P Welch DZurakowski and L B Nanney ldquoEnhanced histologic repair in acentral wound in the anterior cruciate ligament with a collagen-platelet-rich plasma scaffoldrdquo Journal of Orthopaedic Researchvol 25 no 8 pp 1007ndash1017 2007

[51] J Qu N A van Alphen A R Thoreson et al ldquoEffectsof trypsinization and mineralization on intrasynovial tendonallograft healing to bonerdquo Journal of Orthopaedic Research vol33 no 4 pp 468ndash474 2015

[52] Y Inagaki K Uematsu M Akahane et al ldquoOsteogenic matrixcell sheet transplantation enhances early tendon graft to bonetunnel healing in rabbitsrdquo BioMed Research International vol2013 Article ID 842192 8 pages 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


In general cellular migration is a coordinated cyclicprocess of polarization protrusion attachment contractionand rear release [21 22] The Rho family of small guano-sine triphosphatases (including RhoA Rac and Cdc42) isinvolved in one of the most important molecular signalingpathways mediating cellular migration [23ndash25] Cdc42 isrequired for the cell polarization and filopodia formationthat influence protrusions Rac is essential for lamellipodiaformation and forward movement while RhoA regulatesactin stress fibers and focal adhesion formation [26 27]Rac and Cdc42 can active RhoA to regulate trailing edgesformation at the back of the cell [28 29] These threeproteins cooperate with each other for efficient cellularmigration Rho-associated kinase (ROCK) a downstreameffector of Rho that is activated by RhoA phosphorylatesthe downstream substrate myosin light chain phosphatase(MLCP) directly or indirectly [27 30 31] ROCK can alsodecrease cofilin activity to increase random migration [32]Finally RhoA and ROCK signaling regulate tail retractionand integrin adhesion at the rear of cells [33] Y27632 aspecific inhibitor of ROCKs inhibits the RhoA kinase familyand influences stress fibers vinculin and myosin light chainphosphatase [34]

The purpose of this study was to investigate cellularmigration ability on PET-based artificial ligaments (PET-ALs)with different porous structures prepared using differentknitting methods We hypothesized that cellular migrationis regulated by the porosity of artificial ligaments andthat differences in BMSC migration are attributable to theRhoAROCK signaling pathway Thus selection of a properknitting method and line density for artificial ligaments mayenable improved biocompatibility and clinical performance

2 Materials and Methods

21 Preparation of Grafts and Mode To obtain PET-ALs withdifferent porous structures we employed 500PETfibers (90crystalline diameter 2846plusmn104 120583m) in a bundle with a linedensity of 5000D (5000 grams per 9000 meters of PET fiberbundles under conventional moisture regain) and 300 PETfibers in a bundle with a line density of 3000D (3000 gramsper 9000 meters of PET fiber bundles under conventionalmoisture regain) as materials and weaved them using twodifferent knitting methods

The full-set threading warp knitting method (A) canproduce braided fabrics with a smoother surface and dimen-sional stability For this knittingmethod we used an indepen-dent improved Raschel II two-bar full-width weft insertionwarp knitting machine (Haining Wellington New MaterialCo Zhejiang China) The width was reduced to 120 cmand the horizontal density (119875

119886) and longitudinal density

(119875119887) were set at 4cm and 8cm respectively The front bar

used polyester fiber with 10 elastic module and 20 cNdtexbreaking strength The back bar used two high-purity (90crystalline) PET fibers of different linear densities (3000D5000D)Themachine speed was 3000 rminThe 5000D and3000D PET-ALs weaved using the full-set threading warpknitting method were denoted by 5A and 3A respectively

The part-set threading warp knitting method (B) pro-duces a braided fabric surface with relief and provides moresprang for improved porosity For this knitting method weused an RSJ4 multibar lace warp knitting machine (HainingWellington New Material Co Zhejiang China) The widthwas 340 cm and the horizontal density (119875

119886) and longitudinal

density (119875119887) were set at 4cm and 8cm respectivelyThe front

bar used full-set threading with polyester fiber and the backbar used part-set threading (50) with high-purity (90crystalline) PET fibers of different linear densities (3000D5000D) The 5000D and 3000D PET-ALs weaved using thepart-set threading warp knitting methods were denoted by5B and 3B respectively

The knitted PET-ALs were washed with a chemicalcleaning agent double distilled water acetone and absoluteethanol in an ultrasonic cleaner for 30min each to remove oildirt and chemical residue

The cell migration model was fabricated using 316Lmedical stainless steel (Figures 1(a)-A -B and -C) witha height of 45mm The diameter of the central cylinderwas 10mm and the annular ring at the top of the modelwas 35mm The central cylinder and the annular ring wereconnected using an 8-mm-wide arm through which BMSCscould be seeded

22 Graft Characterization

221 Scanning Electron Microscopy The morphologies andthe pore diameters of the four types of PET-ALs were eval-uated using an SEM (HITACHI-S4800 Scanning ElectronMicroscope Japan) at a voltage of 5 kV

222 Porosity The open porosity of the four types of PET-ALs was evaluated based on the principle of liquid displace-ment (119899 = 6) The following equation was used to calculatethe porosity of the PET-ALs

120593 =

[(119875119879minus 119875119878) minus (119875

119877minus 119875119875)] 120588H

2O

119881119873minus (119875119877minus 119875119875) 120588H

2Otimes 100 (1)

where 119875119879is the weight of the pycnometer when completely

filled with distilled water and the tested PET-ALs 119875119878is the

weight of the dried pycnometer and the tested PET-ALs 119875119877is

the weight of the pycnometer and the residual water after theremoval of the tested PET-ALs 119875

119875is the weight of the dried

pycnometer 119881119873

is the nominal volume of the pycnometer(119881119873= 50mL) and 120588H

2O is the density of distilled water at the

measurement temperature

223 Mechanical Properties The four types of PET-ALs (119899 =6) were rolled up into tube-like mesh structures to thespecifications of an adult ACL The combined length of thetwo free ends was 700 plusmn 02mm and the diameter of theligament section was 70 plusmn 05mmThe PET-ALs were testedusing an Instron testing system (Model 4442 Instron IncMA) During the test both ends of the PET-ALs were fixedwith homemade clamps and the distance between the clampswas 500plusmn02mmThepretensionwas 2N and the elongation


(A) (B) (C) (D)

1

2

3

(a)

5A 3A 5B 3B

(b)

5A 3A 5B 3B

(c)



23 In Vitro Tests








2










24 In Vivo Tests




119878119886

1205871198772minus 119878119887

times 100 (2)


119887is the area





Porosity()


Stiffness(Nmm)





3 Results








5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)






GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(A) (B) (C) (D)

1

2

3

(a)

5A 3A 5B 3B

(b)

5A 3A 5B 3B

(c)



23 In Vitro Tests








2










24 In Vivo Tests




119878119886

1205871198772minus 119878119887

times 100 (2)


119887is the area





Porosity()


Stiffness(Nmm)





3 Results








5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)






GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









24 In Vivo Tests




119878119886

1205871198772minus 119878119887

times 100 (2)


119887is the area





Porosity()


Stiffness(Nmm)





3 Results








5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)






GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Porosity()


Stiffness(Nmm)





3 Results








5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)






GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5A

3A

5B

3B

+minus

PP

PP

P P

P

P

T

T

T T

TT

T

T

(A) (B)

(C) (D)

(E) (F)

(G) (H)

(A)

(C)

(E)

Y27632

(a)

+minus

(B)(A)

(C)

(E)

(D)

(F)

(G) (H)

(b)

5A 3A 5B 3B

2500

2000

1500

1000

500

0Proj

ecte

d ar

ea p

er ce

ll (120583

m2)

Y27632minusY27632+

lowastlowast

lowastlowast lowast

lowastlowast

(c)






GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


GAPDH

RhoA

ROCK

5A 3A 5B 3B

(a)

15

10

05

00

RhoA

RO

CK ex

pres

sion

(fold

incr

ease

of5

A)

RhoA ROCK

lowast

lowast

lowast

lowast

lowast

lowast

5A 3A5B 3B

(b)

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

5A3A 3B

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

24h 48h 72h

5B

(c)

5A

3A

5B

3B

24h

24h

24h

24h

48h

48h

48h

48h

72h

72h

72h

72h

(d)





Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Y27632

ROCK

p-MLC

GAPDH

0 5 15 20

(a)

ROCK p-MLC

5A3B

ROCK

p-M

LC ex

pres

sion

(fold

incr

ease

of5

A)

15

10

05

00

lowastlowast

lowast

lowastlowast

lowast

3A5B

(b)

0 5 15 20

80

60

40

20

0

Cel

l mig

ratio

n ar

ea (m

m2)

lowast

Y27632 (120583molL)

(c)

Y27632

5A


(d)



4 Discussion




5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5A 3A5B 3B

100

80

60

40

20

0

Ulti

mat

e fai

lure

load

(N)

4 12

lowast

lowast

lowast

lowast

lowast

lowast

(Weeks)

(a)

5A 3A5B 3B

4 12

50

40

30

20

10

0

Stiff

ness

at fa

ilure

(Nm

m)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(b)

5A 3A5B 3B

4 12

25

20

15

5

10

0Bone

vol

ume

pore

vol

ume (

)

lowast

lowastlowast

lowast

lowast

lowast

(Weeks)

(c)

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

4W

12W

12W

12W

12W

12W

12W

12W

12W12W

12W

12W

12W

5A

3A

5B

3B

(d)








5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusions






Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article effects of artificial ligaments with...

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