research article immobilized lentivirus vector on...

Research ArticleImmobilized Lentivirus Vector on ChondroitinSulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold forTissue-Engineered Ligament-Bone Junction

Liguo Sun1 Hongguo Li1 Ling Qu2 Rui Zhu3 Xiangli Fan1 Yingsen Xue1

Zhenghong Xie1 and Hongbin Fan1

1 Institute of Orthopedic Surgery Xijing Hospital The Fourth Military Medical University Xirsquoan 710032 China2Department of Clinical Laboratory Xijing Hospital The Fourth Military Medical University Xirsquoan 710032 China3 College of Science Air Force Engineering University Xirsquoan 710051 China

Correspondence should be addressed to Hongbin Fan fanhbfmmueducn

Received 4 April 2014 Accepted 26 May 2014 Published 12 June 2014

Academic Editor Guo-Xian Pei

Copyright copy 2014 Liguo Sun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament(ACL) reconstruction The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer The scaffold was fabricated bylyophilizing the CHS mixture with braided silk cables The scanning electronic microscopy (SEM) showed that microporous CHSsponges were formed around silk cables Each end of scaffold was modified with lentiviral-mediated transforming growth factor-1205733 (TGF-1205733) gene The cells on scaffold were transfected by bonded lentivirus In vitro culture demonstrated that mesenchymalstem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagenThe transcription levels of cartilage-specificgenes also increased with culture time After 2 weeks theMSCswere distributed uniformly throughout scaffold Deposited collagenwas also found to increase The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-1205733 genes inmRNA and protein level Histology also confirmed the production of cartilage extracellular matrix (ECM) componentsThe resultsdemonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiationto reconstruct the cartilage layer of interface

1 Introduction

The rupture of the anterior cruciate ligament (ACL) is oneof the most common injuries of the knee with an incidenceof 1 in 3000 [1] which can result in severe limitations inmobility pain and inability to participate in sports andexercise Due to the limited capacity to regenerate ACL healspoorly in response to the repair by suturing the injured tissueback together So the grafts are required for ACL recon-struction [2] Currently there are approximately 125000ACL reconstruction surgeries performed worldwide eachyear most using biological grafts (autografts and allografts)However there are still drawbacks including the risk ofdisease transmission lack of appropriate donors immuneresponse and high costs [3 4] Recently tissue engineering

has emerged as a promising strategy for the regeneration ofinjured ligamentwith similar biomechanical and biochemicalproperties [4 5] The scaffold plays an important role inconstructing the tissue-engineered ligament by providingappropriate mechanical integrity and biochemical stimula-tion

The integration between soft graft and hard bone-tunnelis particularly critical for biological fixation The nativestructure of ACL-bone insertion has four distinct yet contin-uous regions of ligament noncalcified fibrocartilage calcifiedfibrocartilage and bone Interface tissue engineering is a newstrategy aiming at regeneration of interface and ultimatelyenabling the biological fixation of soft grafts in bone-tunnel[6] The intricate multitissue organization of insertion indi-cates that interface scaffold design must consider the need to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 816979 10 pageshttpdxdoiorg1011552014816979

2 BioMed Research International

regenerate more than one type of tissue as well as exercisingspatial control over the respective cell populations indigenousto different interface regions Initial attempts to improveligament graft to bone fixation focused on augmenting thesurgical graft with a material that could encourage bonetissue ingrowth In previous studies the scaffold modifiedby calcium phosphate or tricalcium phosphate cement wasfound to enhance healing and promote integration [7ndash9]Additional approaches to improve osteointegration includedthe addition of periosteum grafts to the region of graft thatinteracted with the bone and growth factors such as rhBMP-2 [10ndash14] Although these methods have improved osteoin-tegration between graft and bone-tunnel the efforts do notresult in regeneration of fibrocartilage layer of interfaceMoreover single-phase scaffold does not fully mimic thecomplexity of natural interfaceThe ideal biomimetic scaffoldshould be designed to recapitulate the inherent complexity ofmultilayered ligament-to-bone interface It might direct thegrowth of the multitissue and overcome shortcomings men-tioned above This multilayer interface scaffold was reportedto be loaded with different cells (fibroblast chondrocyte andosteoblast) for reconstructing insertion But it is difficult toconduct due to complicated process

In recent years silk has been increasingly studied asthe scaffold for ligament tissue engineering due to its bio-compatibility slow degradability and remarkablemechanicalcapability [15 16] In our previous work the knitted scaffoldwas demonstrated to possess good mechanical strength andinternal connections It could facilitate nutrientsrsquo transmis-sion tissue infiltration and matrix deposition After implan-tation the scaffold could successfully regenerate ACL insmall and large animal models However the typical fourlayers of insertion were not observed which might decreasethe stability of graft [17] Growth factors have long beendelivered to cells to promote cell growth proliferation anddifferentiation Transforming growth factor-1205733 (TGF-1205733)was substantiated to help to maintain a round or polygonalshape of chondrocytes and stimulate total collagen synthesiswhich is crucial in maintaining the cartilage functions [1819] Recombinant TGF-1205733 protein has also been delivered tomesenchymal stem cells (MSCs) for in vitro chondrogenesisIn our previous work the immobilized TGF-1205733 on scaffoldefficiently induced the chondrogenic differentiation of MSCs[20] In this study the bone-tunnel part of scaffold was mod-ified with lentivirus carrying TGF-1205733 gene We hypothesizethat MSCs transfected by lentivirus can regenerate cartilage-like tissue between ligament and bone-tunnel to resemblecartilage layer of native insertion

In order to prove this hypothesis the study was designed(1) to prepare a silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) scaffold modified withlentiviral-mediated TGF-1205733 gene (2) to observe cell prolif-eration and collagen production in modified area of scaffoldand (3) to examine expressions of cartilage related genes inmRNA and protein level with real-time quantitative RT-PCRwestern bolt and histological stains The purpose of currentstudy is to find whether the cartilage layer of ligament-bonejunction can be reconstructed in vitro using gene-modifiedscaffold and MSCs

2 Materials and Methods

21 Scaffold Fabrication Four sericin-free silk fibers werebraided into one bundle and then two bundles were braidedinto one cable Twelve cables placed in parallel were fixedacross a customized polypropylene cylinder mold (diameter6 millimeters length 60 millimeters)The raw BombyxMorisilk fibers were immersed in 002M NaHCO

3solution at

90∘C for 1 hour to remove sericin The fibers were rinsed for20 minutes 3 times squeezed out excess water and allowedto dry overnight To prepare silk fibroin (SF) solution thesericin-free silk fibers were added to 93M lithium bromide(BrLi) solution on top of silk fibers and incubated at 60∘C for4 hours [21] After dialysis with SnakeSkin (Thermo ScientificCo 3500 MWCO USA) the final concentration of SF was20 wt The 01 g of chondroitin sulfate sodium (Sigma CoSt Louis) was mixed with 5mg of hyaluronate (C P FredaPharmacy Co Shandong China) and 12mL of 20 wt SFsolution and then poured into the cylinder mold containingthe 12 silk cables Before lyophilization the cylinder moldwas kept at minus80∘C for 1 hour and then put into lyophilizer(Christ Alpha 1-2 LD Germany) for 24 hours The freeze-dried scaffold was immersed in 90 methanol solutionfor 30 minutes to induce 120573-sheet structural transition andthen washed several times to remove the residual chemicalsThereafter the end of scaffold (length 20 millimeters) wasimmersed in phosphatidylserine (PS) chloroform solutionsfor 5 minutes Finally the scaffold was dried overnight inhood The process was schematically depicted in Figure 1

22 Scaffold Characterization The structures and mechan-ical properties of scaffold were characterized Mercuryporosimetry (Pascal 140 GA) was used to assess the poresize porosity and total surface area of scaffold (119899 = 4)Scanning electronic microscopy (SEM) (HITACHI S-4800Japan) was used to observe the morphology of microporesTo measure hydrophilicity the dried scaffolds (119899 = 6)were hydrated in phosphate buffered saline (PBS) after beingweighed Then the swollen scaffolds were weighed againand the degree of swelling was calculated as the ratio ofthe weight of PBS absorbed by scaffolds normalized tothe initial dry weight [22] Mechanical test was performedusing Shimadzu mechanical testing system (AGS-10kNGShimadzu Inc Japan) with a maximum loading capacity of500N After being hydrated in PBS the 12 cable-reinforcedCHS hybrid scaffolds (119899 = 6) were performed with a gaugelength of 20 millimeters at a loading speed of 2mmminThe maximum tensile load (max-load) maximum tensiledistance (max-disp) break tensile load (break-load) andbreak tensile distance (break-disp) were determined

23 Isolation and Expansion of MSCs MSCs were generatedfrom bone marrow aspirates of rabbits (12 weeks old 25ndash30 kg) According to previous methods [20] mononuclearcells were separated by centrifugation in a Ficoll-Paquegradient (Sigma Co St Louis) and suspended in 20mL ofDulbeccorsquos modified eagle medium (DMEM) supplementedwith 15 fetal bovine serum (FBS) (HyClone Logan Utah)

BioMed Research International 3

PS coatingIm

mersed by PS

Dried overnight

Silk bundle

(4 braided silk fibers)

Silk cable

(24 braided silk bundles)

Mixture of chondroitin sulfate

hyaluronate acid and silk fibroin

PS coating

Freeze-drying for 24 hours

Figure 1 Schematic outline of scaffold fabrication procedure

Cultures were incubated at 37∘C and 5 carbon dioxidefor 72 hours then the nonadherent cells were removedby changing medium When reaching 70ndash80 confluenceadherent cells were detached from the flask using 025trypsin and subcultured A homogenous MSCsrsquo populationwas obtained after 2 weeks of culture and cells of passage 3were harvested for further use The adipogenic osteogenicand chondrogenic differentiations of cells were tested toensure the multilineage potential

24 Cell Adhesion Metabolism Viability and TransfectionCell adhesion to scaffold was examined as reported [23]The hybrid scaffolds were sliced into circular disc (diameter6mm length 4mm) sterilized by brief treatment with 75ethanol The lentivirus vector (1 times 108 copiesmL) witha promoter encoding both TGF-1205733 and enhanced greenfluorescent protein (eGFP) was prepared by GeneCopoeiaCo

Then the scaffold was incubated with transfectionmedium (40 times 106 of virus in 1mL of DMEM) at 37∘C and5 CO

24 hours for immobilization of lentivirus vector The

scaffold was placed into a polypropylene tube (length 25millimeters inner diameter 60 millimeters) loaded with1 times 106 MSCs (passage 3) and incubated At different timepoints (ie 0 05 1 2 4 and 8 hours) the MSCsscaffolds(119899 = 6 for each time point) were gently rinsed with PBS Thedetached cells collected from rinsing solution and mediumwere counted using light microscopy

The metabolism of MSCs on scaffold was evaluated at 13 5 7 9 11 13 and 15 days by alamar blue assay followingthe vendorrsquos instructions (ABD Serotec Oxford USA) Thealamar blue assay is a useful method for cell monitoring on3Dporous scaffold [17] A brief descriptionwas as follows theMSCsscaffold (119899 = 6 for each culture period) was incubatedin culture medium supplemented with 10 (vv) alamar bluefluorescent dye for 2 hours Then 100120583L of medium wasextracted from each sample and measured at 570600 nm ina microplate reader (Sunnyvale CA) The culture mediumsupplemented with 10 alamar blue was used as a negativecontrol

The viability of MSCs seeded on scaffolds (119899 = 4)was examined at culture periods of 1 and 2 weeks usinglivedead cells assayThe assay was based on the combinationof the fluorescein diacetate (FDA) which stains living cellsgreen and propidium iodide (PI) which stains dead cellsred Briefly the MSCsscaffolds were thoroughly rinsed withPBS in a six-well plate Then 2mL of PBS supplemented with2 120583L of PI (2mgmL) and 6120583L of FDA (5mgmL) was addedinto each well The system was allowed to incubate at roomtemperature for 10 minutes Then each sample was washedwith PBS twice and observed with confocal microscopy(Olympus Fluo View FV-1000 Japan)

The transfection of MSCs was examined after 1 and2 weeks using fluorescence microscope (Leica DMI6000BInverted Microscope Germany) The MSCsscaffolds (119899 = 4for each time point) were gently rinsed with 1mL of PBSadded to 3mL of trypsin-EDTA solution (Beyotime CoChina) and shaken for 20 minutes The detached cells werecollected resuspended in DMEM and transferred into flaskfor cells adhesion After 4 hours of culture the transfectedcells which expressed the enhanced green fluorescent protein(eGFP) were observed with fluorescence microscope

25 Collagen Production One million MSCs were loadedonto scaffold and cultured in vitro for periods of 1 and 2weeksThe collagen deposited on scaffoldwas then quantifiedusing sircol collagen dye binding assay kit (Biocolor LtdNewtownabbey Ireland) The dye reagent specifically bindsto the [Gly-X-Y]n helical structure of collagen but not tounwound triple helix or random chains of gelatin Brieflythe samples (119899 = 6) were incubated with 500120583L of pepsinsolution (025mgmL) and shaken for 2 hours Then 1mLof dye reagent was added to 300 120583L of soluble collagen andmixed for 30minutes at room temperatureThe pellet of dyedcollagen was precipitated by centrifugation for 5 minutes andthen dissolved with 1mL of releasing reagentThe absorbancewas measured at 540 nm The standard curve was set up onthe basis of collagen standard provided by the vendor Thecollagen produced was presented as amount of collagen perscaffold


(a)

E E

(b)

(c) (d)

Figure 2 (a) Customized polypropylene cylinder mold containing 12 silk cables (b) Gross morphology of scaffold and the zone encoded Ewas coated with PS (c) SEM image of microporous structure of scaffold (times85) and hybrid microsponge around silk cable (d) The scaffolddisplayed a highly porous microstructure with a high degree of interconnectivity (SEM times 150)

26 Histological Assessment MSCs seeded on the scaffolds(119899 = 6) were cultured and harvested at the end of 1and 2 weeks The samples were washed with PBS andfixed in 10 neutral buffered formalin Thereafter theywere dehydrated through a series of graded alcohols andembedded in paraffin Sections of 5120583m thickness were cutand collected on slides For immunohistochemistry stainsthe slides were incubated with collagen II (Sigma Co)and TGF-1205733 (Sigma Co) antibodies Then detection wasapplied using streptavidin-biotin immunoenzymatic antigendetection system (UltraVision Detection System LabVisionUSA) The results were assessed by three individuals whowere blinded to the treatment

27 Real-Time Quantitative RT-PCR Analysis Total RNAwas extracted from MSCsscaffold (119899 = 6) constructsat the end of 1 and 2 weeks using the RNeasy mini kit(Qiagen Valencia CA) RNA concentration was determinedby using NanoDrop (NanoDrop Technologies WilmingtonDE) and 200 ng of RNA was used to synthesize cDNA withIscript cDNA synthesis kit (Biorad Laboratories HerculesCA) Quantitative real-time PCR measurement was carriedout using the Stratagene Mx3000P system (Stratagene LaJolla CA USA) QuantiTect SYBR Green PCR kit (QiagenValencia CA USA) was used to quantify the transcriptionlevel of collagen II and TGF-1205733 Sequences of all the primersfor real-time PCRwere as follows (51015840ndash31015840) TGF-1205733 TGGCTG

TTG AGA AGA GAG TCC TGC TTC AGG GTT CAGAGT GTT collagen II AAC ACT GCC AAC GTC CAG ATCTG CAG CAC GGT ATA GGT GA cDNA of 1mL fromeach sample wasmixedwith 10 120583L ofQuantiTect SYBRGreenPCR master mix 025 120583L of primer and 85 120583L of RNase-freewater The total reaction volume was 20120583L Real-time PCRreactions were performed at 95∘C for 15 minutes followedby 40 cycles of amplification consisting of denaturation stepat 95∘C for 15 seconds and extension step at 60∘C for 1minute The transcription level normalized to GAPDH wasthen calculated using the 2ΔCt formula with reference to theundifferentiated MSCs

28 Western Blot Analysis Proteins were extracted fromMSCsscaffold (119899 = 4) at the end of 1 and 2 weeks with pepsin(200120583gmL in 008M acetic acid Sigma) for 72 hours at 4∘CThepepsinwas subsequently inactivatedwith 1MNaOHTheextract was concentrated using aNanosep 30 centrifugal filter(30000Mw cutoff Pall Life Sciences USA) Samples werethen separated by electrophoresis in NuPAGE Novex Tris-acetate mini gels (Invitrogen USA) and electrophoreticallytransferred to a supported nitrocellulose membrane (BioradLaboratories) The membranes were tested using westernblot kit (Invitrogen USA) according to the manufacturerrsquosinstructions A brief description was as follows the mem-branes were blocked with buffer for 1 hour and incubatedovernight at 4∘Cwithmonoclonal antibodies against collagen


(a) (b)

0 1 2 3 4 5 6 7 8 9 10

Distance (mm)

180

160

140

120

100

80

60

40

20

0

Load

(N)

(c)

Figure 3 Mechanical properties of regenerated ligaments (a) The scaffold was firmly fixed on machine to perform mechanical test (b) Thescaffold broke in the midsubstance during mechanical test (c) The load-deformation curves of scaffold

II and TGF-1205733 diluted to 1 500 in blocking buffer Themembranes were then washed five times with washing bufferand incubated for 30 minutes with secondary antibodiesdiluted to 1 200 in blocking buffer The membranes wererinsed with washing buffer again and incubated with ECLworking solution for 5minutesThe signal was detected usingVersaDoc Imaging System (Biorad Laboratories) and relativeintensities of positive bands were compared between groups

29 Statistical Analysis The mean and standard deviationwere used to describe the data The data analysis was per-formed using SPSS Statistics 200 statistical software packageA statistical analysis of quantitative results was carried outwith the unpaired Studentrsquos t-test and one-way analysis ofvariance (ANOVA) The statistical significance level was setat 005

3 Results

31 Characterization of Scaffold Twelve cables placed in par-allel were fixed across a customized polypropylene cylindermold (Figure 2(a))The cable-reinforced CHS hybrid scaffoldexhibited an elastic texture and porousmorphology Each endof scaffold (length 20millimeters) in the E areas (Figure 2(b))was immersed in PS chloroform solutions The porosity of

the microporous sponge was found to be 634 plusmn 42 and theaverage pore diameter was 1723 plusmn 526 120583mThe surface areaof the hybrid scaffold was 21 plusmn 03m2g The SEM imagesalso indicated that the pore size ranged from 80 to 230120583m(Figures 2(c) and 2(d)) The swelling ratio of the scaffold wasmeasured to be 6357plusmn 483 Figure 3 showed the summaryof mechanical properties of the scaffold The max-load was1519plusmn117N and themax-disp was 71plusmn06mmThe break-load was 513 plusmn 10N and the break-disp was 80 plusmn 06mm

32 Cell Adhesion Metabolism Viability and TransfectionThe results indicated that the number of nonadherent cellsdecreased proportionately with increasing culture time Thenumber of nonadherent cells at the 4 h group was 77 plusmn 14 times104 This was significantly lower than cell number of 0 hgroup (119875 lt 005) Although the cell number continued todecrease after 4 hours there was no significant differencebetween 4 h and 8 h groups Therefore 4 h incubation wasdeemed sufficient for MSCs to attach onto hybrid scaffold(Figure 4(e))

The metabolism of MSCs seeded on the hybrid scaffoldincreased rapidly with culture time at early stage The valueof alamar blue test increased from 99 plusmn 16 to 708 plusmn 51after 5 days of culture Subsequently it increased gradually


(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)

Figure 4 (a b) The viability of MSCs seeded on scaffold evaluated by confocal microscope at 1-week (a) and 2-week (b) culture period(FDAPI staining green represents live cells red represents dead cells) (c d)The transfection of MSCs observed by fluorescence microscopeat 1-week (c) and 2-week (d) culture period (times40) (e) Nonadherent cells count at 0 05 1 2 4 and 8 h time points after cell seeding (mean plusmnSD 119899 = 6 lowast119875 lt 005) (f) The value of alamar blue assay on days 1 3 5 7 9 11 13 and 15 after cell seeding (mean plusmn SD 119899 = 6)

and reached a plateau The value after 15 days was 857 plusmn34 (Figure 4(f)) Viability of MSCs seeded on scaffold wasevaluated by confocal microscope Based on livedead assayno significant cell death was observed (Figures 4(a) and4(b))The transfected cells expressing eGFPwere observed inthe interconnective pores with fluorescence microscope Thenumber of transfected cells at 2-week time points significantlyincreased compared to that of 1-week ones (Figures 4(c) and4(d))

33 Collagen Production Collagen deposition on hybridscaffold was found to increase proportionately with culture

time The MSCs produced an average of 792 plusmn 45 and1121 plusmn 63 120583g collagen after culture periods of 1 and 2 weeksrespectively The difference between these two groups wasfound to be significant (119875 lt 005) (Figure 5)

34 Histological Analysis Histological examination revealedthat microporous structure of the hybrid scaffold was wellpreserved and few micropore walls collapsed Thus theexchange of nutrients between scaffold and environmentwas ensured The MSCs proliferated robustly along the wallof micropores and exhibited fibroblast morphology withelongated nucleus The 2-week group showed higher cell


140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks

Figure 5 Amount of collagen produced by MSCs seeded on hybridscaffold at 1-week and 2-week culture periods (mean plusmn SD 119899 = 6lowast119875 lt 005)

density and more ECM production when compared with 1-week group Immunohistochemistry staining for collagen IIand TGF-1205733 was found to be positive after 1 week ECMformation was observed to increase with culture time Thestaining of collagen II and TGF-1205733 was more intense at 2weeks when compared with that at 1 week (Figure 6)

35 Transcription Level of Cartilage-Specific Genes MSCscultured on hybrid scaffold were harvested to assess theirgene transcription level using real-time quantitative RT-PCRThe transcription levels of collagen II gene were 0290 plusmn0046-fold and 0462 plusmn 0053-fold at 1 and 2 weeks (119875 lt005) respectively The results showed that TGF-1205733 genetranscription was 0048 plusmn 0005-fold and 0086 plusmn 0006-foldat at 1-week and 2-week time points The difference at thesetwo time points was found to be significant (119875 lt 005)(Figures 7(a) and 7(b)) The results suggested that the hybridscaffold could support the differentiation of MSCs towardschondrocytes

36 Western Blot Analysis The protein expressions of TGF-1205733 and collagen II were compared between groups after1 week and 2 weeks of culture Collagen II and TGF-1205733 were expressed more prominently in 2-week groups incomparison with 1-week groups The relative intensities ofpositive staining band for collagen II were 542 plusmn 44 and762 plusmn 86 after 1 week and 2 weeks respectively There wasa significant difference (119875 lt 005) The relative intensitiesof positive staining band for TGF-1205733 were 465 plusmn 74 and759 plusmn 68 after 1 week and 2 weeks respectively There wasa significant difference (119875 lt 005) (Figures 7(c) and 7(d))

4 Discussion

This study demonstrated that the lentivirus vector couldbe immobilized by PS on scaffold which transfected MSCscontinuously The transfected cells could differentiate intochondrocyte-like cell This tissue-engineered cartilage layermimicked the chondral part of natural ligament-bone junc-tion It might reduce the stress concentration between hard

bone and elastic ligament The results provide potentialapplication of gene-modified scaffold for constructing thecartilage-zone of ligament-bone junction

The adherence between vector and material was achievedby the binding between lentivirus and PS coating a com-ponent of the plasma membrane Shin et al reported thatPS could be incorporated into PLG microspheres whichwas subsequently combined with scaffold Large numbersof transduced cells and increased gene expression wereobserved on the gene-modified scaffold [24] In this studylentiviral vector was immobilized on scaffold by incorpora-tion with PS

The integration of gene therapy into tissue engineeringto control differentiation and direct tissue formation is nota new concept however successful delivery of nucleic acidsinto primary cells progenitor cells and stem cells has provenexceptionally challengingThe gene deliverymethods includeviral and nonviral methods The usually used physical non-viral methods aremicroinjection ballistic gene delivery elec-troporation sonoporation laser irradiation magnetofectionand electric field-induced molecular vibration However theclinical application of nonviral methods is still restrictedby some limitations including low transfection efficiencyand poor transgene expression Viral vectors are generallyhighly effective at delivering nucleic acids to a variety ofcell populations The commonly used vectors mainly includeadenovirus adeno-associated virus (AAV) and retrovirusesAdenovirus and AAV are hardly integrated into target cellgenome Lentivirus belongs to a genus of retrovirus it caneffectively integrate exogenous gene into the host chromo-some so as to achieve the persistence [25] It is very importantthat the lentivirus transfection system does not produce celldamage and immune response [26]

A significant role of PS is to increase the amount of virusthat associates with the material Thus a higher dosage ofvector can be delivered locally For scaffold modification weonce demonstrated an increase in lentivirus activity whencompared with virus alone (unpublished data) In this studywe found very high transduction efficiency of MSCs withan MOI (multiplicity of infection) of 40 on the scaffoldimmobilized by PS At this MOI over 90 of the cellswere identified to be transfected by fluorescence microscopeDespite these high transfection efficiencies there was noevidence of cell death at this MOI Lentiviral vectors cantransfect both replicating and nonreplicating cells It canbe incorporated into the host genome thereby theoreticallyoffering prolonged protein production Although long-termprotein production may enhance cartilage repair there arealso concerns about the formation of potential oncogeniceffects on surrounding cells

Several methods have been used to immobilize viruson scaffold Although these approaches have been effectivevector biotinylation can influence its activity [27 28] PShas a specific interaction with the VSV-G protein whichis influenced in part by electrostatic interactions PS isa relatively low molecular weight lipid that is soluble inorganic solvents which is relatively easy compared with theimmobilization of antibodies or avidin for virus binding


(a) (b)

(c) (d)

Figure 6 Histological evaluation of MSCs on hybrid scaffold at 1-week (a b) (times200) and 2-week (c d) (times100) culture period byimmunohistochemistry staining specific for TFG-1205733 (a c) and collagen II (b d) The MSCs proliferated robustly and produced abundantextracellular matrix The thick arrow indicates silk cable and the thin arrow indicates the hybrid scaffold CHS microsponges

In general a scaffold with a minimal pore size of 150120583mis suggested for bone tissue engineering For soft tissueengineering the pore size of the scaffold usually ranges from150 to 250120583mThe newly formed ECM on the surface wouldfurther prevent the cells from infiltrating into the scaffold[29] It has been estimated that the ligament typically bearspeak loads of about 169N during normal ambulation witha threefold increase from 400N to 500N during strenuousathletic activity [30] In this study the average pore sizeof the hybrid scaffold was 1723 plusmn 526 120583m which wassuitable for ligament tissue engineering The enlarged poresand high porosity of scaffold facilitated tissue ingrowth asdemonstrated by histology examination The hybrid scaffoldhas a microporous structure with interconnecting poresconsequentially enlarging the surface area for better celladhesion However microporous structure can result in poormechanical properties To overcome this drawback braidedsilk cables were introduced to increase tensile strength In thisstudy the hybrid scaffold was designed to have an averagemax-load of 1519 plusmn 117NThis would meet the mechanicalrequirement of ACL for activities of daily living Becausethe max-load transmittable through the hybrid scaffold isproportional to the number of silk cables the tensile strengthof scaffold can be easily adjusted by changing the number ofsupporting silk cables

An important aspect of ECM is its ability to store waterwhich is necessary to support various cell activities and

nutrients exchange The weight swelling ratio of silk cable-reinforced CHS scaffold was 6357plusmn483This is about fourtimes higher than that of sericin-free silk fiber (137 plusmn 12)[31] The discrepancy may be due to different compositionratio and fabricatingmethod used [32]The excellent swellingproperty of scaffold facilitated cell seeding and cell adhesionThe number of detached cells in the rinsing solution andmedium dropped steeply from 960 plusmn 19 times 104 to 495 plusmn24 times 103 within 2 hours Then it continued to decreasewith culture time Therefore the scaffold had both excellentbiomechanical properties and enlarged surface area for cellsadhesion tissue ingrowths and nutrients supply [33]

The ligament-bone junction plays a key role in ACLreconstruction The lack of biological fixation remains theprimary cause of graft failure Due to the complicatedstructure and various cells the ligament-bone junction isdifficult to successfully reconstruct In our previous workthe structure of interface was not successfully reconstructedwith knitted silk scaffold and MSCs in bone-tunnel In thisstudy the gene-modified scaffold was used to regenerate theligament-bone junction

MSC is known to possess the ability of self-renewaland differentiation into various lineages [34] In this studyMSCs seeded on the hybrid scaffold proliferated robustly andshowed good viability The expression of cartilage-specificmarkers (collagen II) was upregulated at high levels withthe increase of TGF-b3 This is correlated with other reports


lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)

Figure 7 The transcription levels of collagen type II gene (a) and TGF-1205733 gene (b) in RT-PCR assay at 1-week and 2-week time pointsthe relative intensities of positive staining band for collagen II (c) and TGF-1205733 (d) in western blot assay at 1-week and 2-week time points(lowast119875 lt 005)

[35]The collagen production was also found to increase withculture time

5 Conclusions

This study has successfully developed a gene-modified silkcable-reinforced CHS hybrid scaffold with potential applica-tion in reconstructing the cartilage layer of ligament-bonejunction Lentivirus vector was imported by PS coating onscaffold TGF-1205733 released by transfected MSCs could inducethe chondral differentiation of MSCs The reinforced silkcables significantly increased the tensile strength of scaffoldto meet the mechanical requirements Future study will focuson introducing multivectors into the hybrid scaffold in orderto reconstruct the structure of ligament-bone junction

Conflict of Interests

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

Authorsrsquo Contribution

Liguo Sun Hongguo Li and Ling Qu contributed equally tothis work

Acknowledgment

Theauthors gratefully acknowledge the funding support fromthe National Natural Science Foundation of China (nos31170936 and 30900311)

References

[1] C B Frank andDW Jackson ldquoThe science of reconstruction ofthe anterior cruciate ligamentrdquo Journal of Bone and Joint SurgeryA vol 79 no 10 pp 1556ndash1576 1997

[2] D M Doroski K S Brink and J S Temenoff ldquoTechniquesfor biological characterization of tissue-engineered tendon andligamentrdquo Biomaterials vol 28 no 2 pp 187ndash202 2007

[3] A J Krych J D Jackson T L Hoskin and D L Dahm ldquoAmeta-analysis of patellar tendon autograft versus patellar ten-don allograft in anterior cruciate ligament reconstructionrdquoArthroscopy vol 24 no 3 pp 292ndash298 2008


[4] F A Petrigliano D R McAllister and B M Wu ldquoTissue engi-neering for anterior cruciate ligament reconstruction a reviewof current strategiesrdquo Arthroscopy vol 22 no 4 pp 441ndash4512006

[5] J A Cooper Jr J S Sahota W J Gorum II J Carter S B Dotyand C T Laurencin ldquoBiomimetic tissue-engineered anteriorcruciate ligament replacementrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no9 pp 3049ndash3054 2007

[6] K L Moffat I-N E Wang S A Rodeo and H H Lu ldquoOrtho-pedic interface tissue engineering for the biological fixation ofsoft tissue graftsrdquo Clinics in Sports Medicine vol 28 no 1 pp157ndash176 2009

[7] X Huangfu and J Zhao ldquoTendon-bone healing enhancementusing injectable tricalcium phosphate in a dog anterior cruciateligament reconstruction modelrdquo Arthroscopy vol 23 no 5 pp455ndash462 2007

[8] H Mutsuzaki M Sakane H Nakajima et al ldquoCalcium-phosphate-hybridized tendon directly promotes regenerationof tendon-bone insertionrdquo Journal of Biomedical MaterialsResearch A vol 70 no 2 pp 319ndash327 2004

[9] Y-C Tien T-T Chih J-H C Lin C-P Ju and S-D LinldquoAugmentation of tendon-bone healing by the use of calcium-phosphate cementrdquo Journal of Bone and Joint Surgery B vol 86no 7 pp 1072ndash1076 2004

[10] C-H Chen W-J Chen C-H Shih C-Y Yang S-J Liu andP-Y Lin ldquoEnveloping the tendon graft with periosteum toenhance tendon-bone healing in a bone tunnel a biomechanicaland histologic study in rabbitsrdquo Arthroscopy vol 19 no 3 pp290ndash296 2003

[11] H-S Kyung S-Y Kim C-W Oh and S-J Kim ldquoTendon-to-bone tunnel healing in a rabbit model the effect of periosteumaugmentation at the tendon-to-bone interfacerdquo Knee SurgerySports Traumatology Arthroscopy vol 11 no 1 pp 9ndash15 2003

[12] K Ohtera Y Yamada M Aoki T Sasaki and K-I YamakoshildquoEffects of periosteum wrapped around tendon in a bonetunnel a biomechanical and histological study in rabbitsrdquoCritical Reviews in Biomedical Engineering vol 28 no 1-2 pp115ndash118 2000

[13] I Youn D G Jones P J Andrews M P Cook and J-K F SuhldquoPeriosteal augmentation of a tendon graft improves tendonhealing in the bone tunnelrdquo Clinical Orthopaedics and RelatedResearch no 419 pp 223ndash231 2004

[14] S A Rodeo K Suzuki X-HDeng JWozney andR FWarrenldquoUse of recombinant human bone morphogenetic protein-2 toenhance tendonhealing in a bone tunnelrdquoTheAmerican Journalof Sports Medicine vol 27 no 4 pp 476ndash488 1999

[15] F A Carneiro M L Bianconi G Weissmuller F Stauffer andA T Da Poian ldquoMembrane recognition by vesicular stomatitisvirus involves enthalpy-driven protein-lipid interactionsrdquo Jour-nal of Virology vol 76 no 8 pp 3756ndash3764 2002

[16] GH Altman F Diaz C Jakuba et al ldquoSilk-based biomaterialsrdquoBiomaterials vol 24 no 3 pp 401ndash416 2003

[17] H Fan H Liu Y Wang S L Toh and J C H Goh ldquoDevel-opment of a silk cable-reinforced gelatinsilk fibroin hybridscaffold for ligament tissue engineeringrdquo Cell Transplantationvol 17 no 12 pp 1389ndash1401 2008

[18] K Na S Kim D-GWoo et al ldquoSynergistic effect of TGF120573-3 onchondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid byin vivo testrdquo Journal of Biotechnology vol 128 no 2 pp 412ndash422 2007

[19] K Yun andH TMoon ldquoInducing chondrogenic differentiationin injectable hydrogels embedded with rabbit chondrocytes andgrowth factor for neocartilage formationrdquo Journal of Bioscienceand Bioengineering vol 105 no 2 pp 122ndash126 2008

[20] H Fan H Tao Y Wu Y Hu Y Yan and Z Luo ldquoTGF-1205733immobilized PLGA-gelatinchondroitin sulfatehyaluronic acidhybrid scaffold for cartilage regenerationrdquo Journal of BiomedicalMaterials Research A vol 95 no 4 pp 982ndash992 2010

[21] D N Rockwood R C Preda T Yucel X Wang M L LovettandD L Kaplan ldquoMaterials fabrication fromBombyxmori silkfibroinrdquo Nature Protocols vol 6 no 10 pp 1612ndash1631 2011

[22] T J Koob and D J Hernandez ldquoMechanical and thermalproperties of novel polymerized NDGA-gelatin hydrogelsrdquoBiomaterials vol 24 no 7 pp 1285ndash1292 2003

[23] N Iwasaki S-T Yamane T Majima et al ldquoFeasibility ofpolysaccharide hybrid materials for scaffolds in cartilage tissueengineering evaluation of chondrocyte adhesion to polyioncomplex fibers prepared from alginate and chitosanrdquo Biomacro-molecules vol 5 no 3 pp 828ndash833 2004

[24] S Shin H M Tuinstra D M Salvay and L D Shea ldquoPhos-phatidylserine immobilization of lentivirus for localized genetransferrdquo Biomaterials vol 31 no 15 pp 4353ndash4359 2010

[25] Y Liu J Kong B-H Chen and Y-G Hu ldquoCombinedexpression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type II inrhesusmonkey lumbar intervertebral disc cells in vitrordquoChineseMedical Journal vol 123 no 15 pp 2082ndash2087 2010

[26] J M Mcmahon S Conroy M Lyons et al ldquoGene transfer intorat mesenchymal stem cells a comparative study of viral andnonviral vectorsrdquo Stem Cells and Development vol 15 no 1 pp87ndash96 2006

[27] R J Levy C Song S Tallapragada et al ldquoLocalized adenovirusgene delivery using antiviral IgG complexationrdquo Gene Therapyvol 8 no 9 pp 659ndash667 2001

[28] W-W Hu M W Lang and P H Krebsbach ldquoDevelopment ofadenovirus immobilization strategies for in situ gene therapyrdquoJournal of Gene Medicine vol 10 no 10 pp 1102ndash1112 2008

[29] G H Altman R L Horan H H Lu et al ldquoSilkmatrix for tissueengineered anterior cruciate ligamentsrdquo Biomaterials vol 23no 20 pp 4131ndash4141 2002

[30] S-G Kim T Akaike T Sasagaw Y Atomi and H KurosawaldquoGene expression of type I and Type III collagen by mechanicalstretch in anterior cruciate ligament cellsrdquo Cell Structure andFunction vol 27 no 3 pp 139ndash144 2002

[31] H Liu Z Ge Y Wang S L Toh V Sutthikhum and J CH Goh ldquoModification of sericin-free silk fibers for ligamenttissue engineering applicationrdquo Journal of Biomedical MaterialsResearch B Applied Biomaterials vol 82 no 1 pp 129ndash138 2007

[32] E S Gil D J Frankowski R J Spontak and S M HudsonldquoSwelling behavior and morphological evolution of mixedgelatinsilk fibroin hydrogelsrdquo Biomacromolecules vol 6 no 6pp 3079ndash3087 2005

[33] L Meinel O Betz R Fajardo et al ldquoSilk based biomaterials toheal critical sized femur defectsrdquo Bone vol 39 no 4 pp 922ndash931 2006

[34] G Vunjak-Novakovic G Altman R Horan and D L KaplanldquoTissue engineering of ligamentsrdquoAnnual Review of BiomedicalEngineering vol 6 pp 131ndash156 2004

[35] B Shen A Wei H Tao A D Diwan and D D F Ma ldquoBMP-2 enhances TGF-1205733-mediated chondrogenic differentiation ofhuman bone marrow multipotent mesenchymal stromal cellsin alginate bead culturerdquo Tissue Engineering A vol 15 no 6 pp1311ndash1320 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


regenerate more than one type of tissue as well as exercisingspatial control over the respective cell populations indigenousto different interface regions Initial attempts to improveligament graft to bone fixation focused on augmenting thesurgical graft with a material that could encourage bonetissue ingrowth In previous studies the scaffold modifiedby calcium phosphate or tricalcium phosphate cement wasfound to enhance healing and promote integration [7ndash9]Additional approaches to improve osteointegration includedthe addition of periosteum grafts to the region of graft thatinteracted with the bone and growth factors such as rhBMP-2 [10ndash14] Although these methods have improved osteoin-tegration between graft and bone-tunnel the efforts do notresult in regeneration of fibrocartilage layer of interfaceMoreover single-phase scaffold does not fully mimic thecomplexity of natural interfaceThe ideal biomimetic scaffoldshould be designed to recapitulate the inherent complexity ofmultilayered ligament-to-bone interface It might direct thegrowth of the multitissue and overcome shortcomings men-tioned above This multilayer interface scaffold was reportedto be loaded with different cells (fibroblast chondrocyte andosteoblast) for reconstructing insertion But it is difficult toconduct due to complicated process

In recent years silk has been increasingly studied asthe scaffold for ligament tissue engineering due to its bio-compatibility slow degradability and remarkablemechanicalcapability [15 16] In our previous work the knitted scaffoldwas demonstrated to possess good mechanical strength andinternal connections It could facilitate nutrientsrsquo transmis-sion tissue infiltration and matrix deposition After implan-tation the scaffold could successfully regenerate ACL insmall and large animal models However the typical fourlayers of insertion were not observed which might decreasethe stability of graft [17] Growth factors have long beendelivered to cells to promote cell growth proliferation anddifferentiation Transforming growth factor-1205733 (TGF-1205733)was substantiated to help to maintain a round or polygonalshape of chondrocytes and stimulate total collagen synthesiswhich is crucial in maintaining the cartilage functions [1819] Recombinant TGF-1205733 protein has also been delivered tomesenchymal stem cells (MSCs) for in vitro chondrogenesisIn our previous work the immobilized TGF-1205733 on scaffoldefficiently induced the chondrogenic differentiation of MSCs[20] In this study the bone-tunnel part of scaffold was mod-ified with lentivirus carrying TGF-1205733 gene We hypothesizethat MSCs transfected by lentivirus can regenerate cartilage-like tissue between ligament and bone-tunnel to resemblecartilage layer of native insertion

In order to prove this hypothesis the study was designed(1) to prepare a silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) scaffold modified withlentiviral-mediated TGF-1205733 gene (2) to observe cell prolif-eration and collagen production in modified area of scaffoldand (3) to examine expressions of cartilage related genes inmRNA and protein level with real-time quantitative RT-PCRwestern bolt and histological stains The purpose of currentstudy is to find whether the cartilage layer of ligament-bonejunction can be reconstructed in vitro using gene-modifiedscaffold and MSCs

2 Materials and Methods

21 Scaffold Fabrication Four sericin-free silk fibers werebraided into one bundle and then two bundles were braidedinto one cable Twelve cables placed in parallel were fixedacross a customized polypropylene cylinder mold (diameter6 millimeters length 60 millimeters)The raw BombyxMorisilk fibers were immersed in 002M NaHCO

3solution at

90∘C for 1 hour to remove sericin The fibers were rinsed for20 minutes 3 times squeezed out excess water and allowedto dry overnight To prepare silk fibroin (SF) solution thesericin-free silk fibers were added to 93M lithium bromide(BrLi) solution on top of silk fibers and incubated at 60∘C for4 hours [21] After dialysis with SnakeSkin (Thermo ScientificCo 3500 MWCO USA) the final concentration of SF was20 wt The 01 g of chondroitin sulfate sodium (Sigma CoSt Louis) was mixed with 5mg of hyaluronate (C P FredaPharmacy Co Shandong China) and 12mL of 20 wt SFsolution and then poured into the cylinder mold containingthe 12 silk cables Before lyophilization the cylinder moldwas kept at minus80∘C for 1 hour and then put into lyophilizer(Christ Alpha 1-2 LD Germany) for 24 hours The freeze-dried scaffold was immersed in 90 methanol solutionfor 30 minutes to induce 120573-sheet structural transition andthen washed several times to remove the residual chemicalsThereafter the end of scaffold (length 20 millimeters) wasimmersed in phosphatidylserine (PS) chloroform solutionsfor 5 minutes Finally the scaffold was dried overnight inhood The process was schematically depicted in Figure 1

22 Scaffold Characterization The structures and mechan-ical properties of scaffold were characterized Mercuryporosimetry (Pascal 140 GA) was used to assess the poresize porosity and total surface area of scaffold (119899 = 4)Scanning electronic microscopy (SEM) (HITACHI S-4800Japan) was used to observe the morphology of microporesTo measure hydrophilicity the dried scaffolds (119899 = 6)were hydrated in phosphate buffered saline (PBS) after beingweighed Then the swollen scaffolds were weighed againand the degree of swelling was calculated as the ratio ofthe weight of PBS absorbed by scaffolds normalized tothe initial dry weight [22] Mechanical test was performedusing Shimadzu mechanical testing system (AGS-10kNGShimadzu Inc Japan) with a maximum loading capacity of500N After being hydrated in PBS the 12 cable-reinforcedCHS hybrid scaffolds (119899 = 6) were performed with a gaugelength of 20 millimeters at a loading speed of 2mmminThe maximum tensile load (max-load) maximum tensiledistance (max-disp) break tensile load (break-load) andbreak tensile distance (break-disp) were determined

23 Isolation and Expansion of MSCs MSCs were generatedfrom bone marrow aspirates of rabbits (12 weeks old 25ndash30 kg) According to previous methods [20] mononuclearcells were separated by centrifugation in a Ficoll-Paquegradient (Sigma Co St Louis) and suspended in 20mL ofDulbeccorsquos modified eagle medium (DMEM) supplementedwith 15 fetal bovine serum (FBS) (HyClone Logan Utah)


PS coatingIm

mersed by PS

Dried overnight

Silk bundle


Silk cable




PS coating













(a)

E E

(b)

(c) (d)







(a) (b)

0 1 2 3 4 5 6 7 8 9 10

Distance (mm)

180

160

140

120

100

80

60

40

20

0

Load

(N)

(c)




3 Results






(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)







140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PS coatingIm

mersed by PS

Dried overnight

Silk bundle


Silk cable




PS coating













(a)

E E

(b)

(c) (d)







(a) (b)

0 1 2 3 4 5 6 7 8 9 10

Distance (mm)

180

160

140

120

100

80

60

40

20

0

Load

(N)

(c)




3 Results






(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)







140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

E E

(b)

(c) (d)







(a) (b)

0 1 2 3 4 5 6 7 8 9 10

Distance (mm)

180

160

140

120

100

80

60

40

20

0

Load

(N)

(c)




3 Results






(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)







140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

0 1 2 3 4 5 6 7 8 9 10

Distance (mm)

180

160

140

120

100

80

60

40

20

0

Load

(N)

(c)




3 Results






(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)







140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

150

100

50

0

Cel

l num

ber (times10

4)

lowastlowast

lowast lowast

0 05 1 2 4 8

Culture time (h)

(e)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Culture time (day)

100

80

60

40

20

0

Ala

mar

blu

e red

uctio

n (

)

(f)







140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















140

120

100

80

60

40

20

0

lowast

Col

lage

n sy

nthe

sis (120583

g)

1week 2weeks





4 Discussion








(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)








lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

1week 2weeks

06

04

02

0

Fold

chan

ges (

colla

gen

II)

(a)

010

008

006

004

002

01week 2weeks

lowast

Fold

chan

ges (

TGF-1205733

)

(b)

100

80

60

40

20

01week 2weeks

lowast

Relat

ive i

nten

sity

(col

lage

n II

)

(c)

lowast

1week 2weeks

100

80

60

40

20

0

Relat

ive i

nten

sity

(TG

F-1205733

)

(d)



5 Conclusions






Acknowledgment


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article immobilized lentivirus vector on...

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