research article study of bone marrow mesenchymal and...

Research ArticleStudy of Bone Marrow Mesenchymal and Tendon-DerivedStem Cells Transplantation on the Regenerating Effect ofAchilles Tendon Ruptures in Rats

Mohanad Kh Al-ani12 Kang Xu1 Yanjun Sun1 Lianhong Pan1 ZhiLing Xu1 and Li Yang1

1Key Laboratory of Biorheological Science and Technology Ministry of Education Bioengineering College Chongqing UniversityChongqing 400030 China2Veterinary College Tikrit University Ministry of Higher Education Tikrit Iraq

Correspondence should be addressed to Li Yang cquliyanggmailcom

Received 13 August 2014 Accepted 23 December 2014

Academic Editor Juan Carlos Casar

Copyright copy 2015 Mohanad Kh Al-ani 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

Comparative therapeutic significance of tendon-derived stem cells (TDSCs) and bone marrow mesenchymal stem cells (BMSCs)transplantation to treat rupturedAchilles tendonwas studiedThree groups of SD rats comprising 24 rats each designated as TDSCsandBMSCs andnontreatedwere studied for regenerative effects throughmorpho-histological evaluations and ultimate failure loadFor possible mechanism in tendon repairregeneration through TDSCs and BMSCs we measured Collagen-I (Col-I) Col-III geneexpression level by RT-PCR and Tenascin-C expression via immunofluorescent assay TDSCs showed higher agility in tendonhealing with better appearance density and well-organized longitudinal fibrous structure though BMSCs also showed positiveeffects Initially the ultimate failure load was considerably higher in TDSCs than other two study groups during the weeks 1 and 2but at week 4 it attained an average or healthy tendon strength of 302N Similar higher tendency in Col-IIII gene expression levelduring weeks 1 2 and 4 was observed in TDSCs treated group with an upregulation of 15-fold and 11-fold than the other two studygroups Immunofluorescent assay revealed higher expression of Tenascin-C in TDSCs at week 1 while both TDSCs and BMSCstreated groups showed detectable CM-Dil-labelled cells at week 4 Compared with BMSCs TDSCs showed higher regenerativepotential while treating ruptured Achilles tendons in rats

1 Introduction

About 30 million ligament and tendon injuries are reportedannually across the globe due to lifestyle recreation workpatterns accidents pharmacological agents and degenera-tive biological variables such as gender age and genetics [1]Anatomical studies are crucial for in-depth understandingof tendon healing and regeneration Tendon is comprised ofparallel and well organized collagen (Col) bundles of whichapproximately 90 are of Col-I while the rest of the 10are Col-III -IV -V and -VI [2] Chronic or acute tendoninjuries are primarily treated with conservative or surgicaltreatments where the former is used for symptomatic reliefonly is ineffective and time-consuming but later involvesthe use of autografts allografts xenografts and prostheticdevices [3 4] However there are considerably high risks of

complications such as infection nerve damage adhesion anddistributed skin sensibility Therefore it is crucial to definesome innovative techniques to treat such tendon injuries

Stem cells are undifferentiated and self-renewing cellsable to differentiate into specialized cells of different typeswith specific functions including the biological healing pro-cess [5 6] and include TDSCs BMSCs adipose-derivedmesenchymal stem cells (AdMSCs) and umbilical cordblood-derived stem cells (UCB-SCs) TDSCs and BMSCshave various advantages and superiority over the other manydifferent stem cell types such as quick proliferation tendonrepair specificity reduced regeneration time and superiorshaped tendon formation [7] hence we used these twostem cell types as a model in the present study Recentlyvarious researchers reported that BMSCs may differentiateto give rise into several connective tissue types including

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 984146 11 pageshttpdxdoiorg1011552015984146

2 Stem Cells International

bone cartilage tendon muscle marrow fat and dermis[8ndash10] BMSCs are involved to facilitate the tendon healingprocess intrinsically by accelerating fibroblast proliferationandmodulation of certain growth factors and cytokinins [11ndash13] On the other hand TDSCs are adult stem cells residingin tendons [14] which are histologically and biochemicallyproven as a prime source of tendon repair [15ndash17] Chengand coworkers demonstrated that TDSCs have high potentialfor colony-formation compared with BMSCs [18] It is alsosaid that TDSCs expresses higher mRNA level of tenogenicmarkers- scleraxis (Scx) tenomodulin (Tnmd) and extra-cellular matrix (ECM) components of tendon that is Col-1A1 Col-1A1Cl3-A1 ratio and decorin (Dcn) than BMSCsHowever a comparison between TDSCs and BMSCs ontreating tendon injury has not yet been doneWe hypothesizethat TDSCs are more favorable for treating Achilles tendoninjuries

In the present study we transplanted TDSCs and BMSCsinto the ruptured area of the Achilles tendon for macroscopicappearance histomorphological analyses and biomechanicalstrength were observed to find the possible mechanism ofthe repair promotion and evaluated the cell transplantationeffects of both stem cell typesThe results indicate that TDSCsexhibit a better-regenerative potential when compared withBMSCs in treating ruptured Achilles tendons and could be abetter alternative cell source for treating Achilles tendon

2 Materials and Methods

21 Ethics Statement Ethics Committee of Chongqing Uni-versity College of Bioengineering and Daping HospitalAnimal Experimental Center approved all experimental pro-tocols using SD rats including collection of Achilles tendonsamples

22 Rats and Treatment Groups Seventy-eight Sprague-Dawley (SD) male rats weighing 200 g obtained from theDaping Hospitalrsquos animal experimental center (ChongqingChina) were used as recipients or donors Six SD rats whichdid not undergo an operation were the source of tendon-derived stem cells bone marrow mesenchymal stem cellsand healthy Achilles tendons The remaining 72 SD rats wereused for Achilles tendon healing experiments The rats weredivided in to three groups TDSC BMSC and nontreatedgroup each group 24 rats The study was carried out at threetime points 1 week 2 weeks and 4 weeks Eight rats wereassigned to each time pointThe ratswere placed in individualcages under slandered feeding system

23 Cell Isolation Six SD rats were used to isolate the variouscells The rats were sedated with pentobarbital sodium in ananesthetic chamber Then using 3 Fluothane in a maskthey were sacrificed The femur and tibia including thetendons attached to them were dissected BMSCs of SDrats were isolated using a modified procedure [18] Brieflyboth femur and tibia were excised and the diaphyses werecut The bone marrow was flushed out with DMEM-LG(Gibco) supplemented with 10 FBS 100UmL penicillin

and 100 120583gmL streptomycin Single cell suspension wasgenerated by aspirating the bone marrow back through thesyringe The samples were then washed and centrifuged at1000 rpm to remove the pieces of debris The cell pellets wereresuspended and expanded in a humidified incubator at 5CO2and 37∘C TDSCs were isolated from rats by removal of

the tendons and rinsed with PBS The TDSCs were isolatedaccording to the previous report [19] Briefly the Achillestendons were then minced into small pieces and digestedwith 5mgmL of type I collagenase (SIGMA) at 37∘C with5 CO

2for two hoursThe undigested tissues were removed

by using 70mm nylon sieve and the remaining cell pelletswere cultured with low glucose Dulbeccorsquos modified Eaglersquosmedium (DMEM Gibco) L-glutamine 10 FBS 100UmLpenicillin and 100 120583gmL streptomycin After 12 days the cellcolonies formed and were selected for further culture After100 confluence cells were subcultured and the mediumwas changed every third day For both BMSCs and TDSCsPassage 3 (P3) cells were adopted for identification

24 Cell Identification and Multidifferentiation Assays Flowcytometry was used for identifying stem cell surface mark-ers CD29 CD44 and CD90 of TDSCs and BMSCs Theosteogenic adipogenic and chondrogenic differentiationpotential of TDSCs and BMSCs were tested according to theprevious report [19 20] After induction Alizarin red Oilred and Toluidine blue staining assays were used to confirmosteogenesis adipogenesis and chondrogenesis respectively

25 Animal Model and Surgical Procedures Seventy-two SDrats weighing 200 g provided by Daping Hospital and theResearch Institute of Surgery of the Third Military MedicalUniversity were used Prior to the study all operations andhandling procedures were approved by the hospital Therats were divided into three groups the BMSCs groupthe TDSCs group and the nontreated group each group24 rats Three rats from each group were tested in eachtime point The left hind legs of the animals were used formicromacro observation while the right hind legs were usedfor measuring gene expression Five rats from each groupfromeach time pointwere used for biomechanical evaluationThe rats were anesthetized with pentobarbital sodium in ananesthetic chamber and then with 3 Fluothane in a maskIn aseptic conditions 10mm longitudinal incision of theright and left hind limb was made directly over the Achillestendon (Figures 1(a) and 1(b)) A segment from the middlepart of the Achilles tendon was cut using a surgical blade5mm from the calcaneal insertion site Clinical sutures wereused to suture the incision and iodine was directly applied(Figure 1(c)) The TDSCs and BMSCs were labeled withCM-DiI (C7000 Invitrogen) after that the donor TDSCs (1times 10601mL DMEM) or BMSCs (1 times 10601mL DMEM)were injected around the Achilles tendon of each rat witha syringe (Figure 1(d)) During the recovery period the ratswere placed in individually sterilized cages under standardfeeding system At the end of each time point the rats weresacrificed by over dose of ether anesthesia

Stem Cells International 3

(a) (b)

(c) (d)

SD rats(Male 200 g)

0 Week 1 Week 2 Week 4

Macroscopic Histological Biomechanical Gene expressions analysis

HE staining Inflorescent testing

Injection of

and BMSCs1 times 10

601mL

DMEM

TDSCsobservations observations testing (PCR)

Figure 1 Experimental protocol A complete transverse incision wasmade 10mm from the calcaneal insertion of the Achilles tendon ((a) and(b)) Clinical suture was used to suture the skin and iodine was directly applied (c) TDSCs (1 times 10601mL DMEM) or BMSCs (1 times 10601mLDMEM) were injected in the Achilles tendon with use of a syringe (d)

26 Macroscopic Assessment At the end of each time pointthe rats were sacrificed and the treated legs were removed formacroscopic observation The appearance of the regeneratedtendonswas observed and compared to that of the nontreatedgroup

27 Histological Evaluation The treated rats were sacrificedand the Achilles tendon between the calcaneus and mus-culotendinous junction was harvested at each time pointThe tendon was immersed in 4 PFA overnight dehydratedand embedded into optimal cutting temperature compound(OCT)The specimens were cut into 10 120583m sections by freez-ingmicrotome (LeicaCM1900) and stainedwith hematoxylinand eosin (HE) for histological evaluation

28 Biomechanical Testing Five rats from each group wereused for biomechanical testing as follows the Achilles ten-don between the calcaneus and musculotendinous junctionresected at 1 week 2 weeks and 4 weeks after incision Theproximal and distal ends of the Achilles tendon were fixedsecurely in serrated grips and mounted on to a mechanicaltesting machine (Instron) With 100N load cell capacity theAchilles tendon was pulled at a constant speed of 10mmminuntil rupture The data was recorded with software (WinTestrsquo7)

29 Quantitative (RT) Polymerase Chain Reaction (qPCR)We examined the expression of Col-III Col-I and GAPDHin every time point Total RNA was extracted using the total


Cou

nt

0

100

101

102

103

104

CD29C

ount

0

100

101

102

103

104

CD44

Cou

nt

0

100

101

102

103

104

CD90

Cou

nt

0

100

101

102

103

104

CD29

Cou

nt

0

100

101

102

103

104

CD44

Cou

nt0

100

101

102

103

104

CD90

(A)

(B)

CD29866 CD44

957

CD90826

CD90937

CD44873

CD29924

(a)

TDSC

sBM

SCs

Alizarin red Oil red Toluidine blue

(b)

Figure 2 Cell identification of TDSCs and BMSCs (a) Stem cell surface markers of FCM (a)-(A) TDSCs (a)-(B) BMSCs (b)Multidifferentiation of TDSCs and BMSCs osteogenesis (Alizarin red) adipogenesis (Oil red) chondrogenic (Toluidine blue)


(A) (B) (C)

(D) (E) (F)

Week 1Nontreated BMSCs TDSCs

Poste

rior-

ante

rior

Late

ral

(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Continued


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Macroscopic findings at 1 w 2 w and 4w after surgery The posterior-anterior appearance of the Achilles tendon in the nontreatedgroup (A) the BMSCs group (B) and the TDSCs group (C) The lateral appearance of the Achilles tendon in the nontreated group (D)BMSCs group (E) and TDSCs group (F)

RNA extraction kit (Bioteke Corporation) according to themanufacturerrsquos instructions RNA was subjected to reversetranscription to complementaryDNA (cDNA) using the FirstStrand cDNA kit (Thermo Scientific Rt-First Strand cDNASynthesis kit K1622) PCR conditions were 65∘C for 5minthen 42∘C for 60min and termination at 70∘C for 5minTheproducts were stored at 80∘C Total cDNA for each samplewas amplified in a final volume of the reaction mixture con-taining SsoAdvanced SYBR Green qRT-PCR supermix (Bio-Rad number 1725264) ready-to-use reaction cocktail and spe-cific primers for Col-I Forward AAGGTGACAGAGGCA-TAAAG Reverse GGAAGCTGAAGTCATAACCA AndCol-III Forward CATGATGAGCTTTGTGCAAT ReverseCTGCTGTGCCAAAATAAGAG The cycling conditionswere the denaturation at 95∘C for 30 sec 39 cycles at 95∘Cfor 5 sec optimal annealing temperature for 20 sec 72∘C for30 sec and 60∘C to 95∘C with a heating rate of 01∘Cs TheCFX 96 Real-Time PCR Detection System (Bio-Rad) wasused to record the results The relative expression level ofthe gene of interest normalized to GAPDH was analyzedaccording to the 2minusΔΔct Method

210 Immunofluorescent Assay The treated Achilles tendonof each group was collected in week 1 and week 4 andperformed frozen sections All the sections were immunestained with Tenascin-C (1 100 Abcam) primary antibodyfollowed by Alexa Fluor 488 dye-labeled secondary antibodyDAPI (Roche) was used to stain cell nuclei and observedunder the immunofluorescent microscope to check the cellstransplantation regenerative processes

211 Statistical Analysis All the data are expressed asmeansplusmnstandard deviations Statistical analysis was performed withone-way ANOVA followed by LSD test for comparisonbetween two groups (Origin Lab Origin V 80 Software) A119875 value of lt005 was considered significant

3 Results

31 Study of Stem Cell Markers for the Confirmation andDifferentiation Ability of TDSCs and BMSCs Flow cytometryanalysis was done to identify the stem cells Both TDSCsand BMSCs were tested positively for CD 29 CD 44 and


lowast

lowast

1 week 2 weeks 4 weeks Healthy

NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)

Figure 4 Results of biomechanical testingThe ultimate failure loadin the nontreated group the BMSCs group and the TDSCs group at1 w 2 w and 4w after surgery Healthy indicates the ultimate failureload in a normal Achilles tendon at thirteen weeks of age lowast119875 lt 005and 119875 lt 005 compared with control and 998779119875 lt 005 compared

with BMSCs were considered significant (data represents mean plusmnSD 119899 = 5)

CD 90 which are indicative of stem cell surface markers(Figure 2(a)) It confirmed that the cells we isolated from theAchilles tendon and bone marrow were stem cells Multid-ifferentiation capacity is one of the universal characteristicsof stem cells The differentiation analyses showed that theadipogenesis (Oil red) chondrogenesis (Toluidine blue) andosteogenesis (Alizarin red) of the isolated TDSCs and BMSCswere emerged after induction (Figure 2(b)) Hence based onthe results of cell identification analyses it became assuredthat the isolated stem cells were TDSCs and BMSCs

32 Week-Wise Macroscopic Assessment of MorphologicalChanges in Repaired Achilles Tendon The skin was suturedwith a clinical suture to inject TDSCs (1times 106 01mLDMEM)andor BMSCs (1 times 106 01mL DMEM) in the Achillestendon using a sterile syringe After transplantation of theTDSCs and BMSCs changes in appearance on the treatedarea were analyzed at weeks 1 2 and 4 after surgeries At week1 the defect area appeared clearly in the nontreated group(Figures 3(A) and 3(D)) while the BMSCs group revealedsome connective tissue in the treated area (Figures 3(B) and3(E)) The TDSCs group revealed a good start of growthin connective tissue around the treated area (Figures 3(C)and 3(F)) On approaching week 2 the growth of connectivetissue was stunted while the defected place area was obviousin the nontreated group which remained as such duringweek 1 (Figures 3(A) and 3(D)) On the other hand BMSCstreated group appeared better than week 1 group and TDSCsshowed the best growth in connective tissue at the treated area(Figures 3(C) and 3(F))

At week 4 the TDSCs group appeared significantlydifferent than the other groups and displayed a complete

Achilles tendon with a normal appearance in the posterior-anterior and lateral views (Figures 3(C) and 3(F))TheBMSCsgroup showed obvious connective tissue growth with a littletransverse notch appearance clearly in the posterior-anteriorand lateral views (Figures 3(B) and 3(E)) The nontreatedgroup showed no tissue growth at all (Figures 3(A) and 3(D))

33 Biomechanical Testing The tensile strength of therepaired Achilles tendon in various study groups was testedby the ultimate failure load method The ultimate failureload in the TDSCs group was considerably higher (102N)than that in BMSCs (67N) and nontreated groups (35N)at week 1 after incision (119875 lt 005) The failure load ofTDSCs and BMSCs was increased 19-fold (119875 lt 005) and09-fold (lowast119875 lt 005) respectively where the former has05-fold higher change (998779119875 lt 005) The ultimate failureload at week 2 after incision was significantly higher for theTDSCs (235N) than the BMSCs (165N) and nontreated(108N) by 12-fold (119875 lt 005) for TDSCs and the BMSCsgroup by 05-fold (lowast119875 lt 005) The failure load of TDSCsexceeded 05-fold (998779119875 lt 005) versus BMSCs At week 4the ultimate failure load of the TDSCs group again showeda higher value (302N) than the BMSCs group (2845N) andthe nontreated group (253N) It is important to note thatamong these three groups BMSCs were also higher than thenontreated group However there was no statistically obviousdifference between TDSCs and BMSCs In addition at week 4after surgery the ultimate failure load of TDSCs and BMSCsreached nearly that of a healthy (Figure 4)

34 Histological Study of the Healing Achilles Tendon Thehistological analyses of Achilles tendon sections were madeand gone through hematoxylin and eosin staining for thenontreated group the BMSCs group and the TDSCs groupat three time points (weeks 1 2 and 4) (Figure 5) Denseconnective tissue was observed in the TDSCs and BMSCstreated groups at week 1 after surgery For the TDSCs groupvisible longitudinal fibrous tissue had already emerged alongwith well-organized cell structures not seen in other twogroups At week 2 after surgery both TDSCs- and BMSCs-treated and particularly TDSCs-treated tendons exhibitedmore ECMdeposition and obvious longitudinal fibrous tissuethan that of nontreated tendons with a greater number ofspindle-shaped cells alignedorganized along the longitudi-nal (tensile) axis of the tendon A similar trend was observedat week 4 after surgery TDSCs given improved tendonstatus than BSMCs and hence both TDSCs and BSMCsshowed a spindle-shaped morphology distributed along thelongitudinal fibrous tissue of the tendon On the contrarythe cells of the loose and thin longitudinal fibrous tissue thatbegan to appear in the nontreated group made little organi-zation with higher vascularization In order to examine thetransplanted labeled TDSCs in the excised Achilles tendonfrozen sections were prepared and analyzed by fluorescentmicroscopy The CM-DiI positive cells (red) were detectablearound the tendon atweek 4 after transplant (Figures 6(a) and6(b)) indicating that those transplanted cells were still aliveand may participate in the process of regeneration


Week 1 Week 2 Week 4

Non

treat

edBM

SCs

TDSC

s

Figure 5 Histological analysis of the Achilles tendon HE staining of the nontreated group the BMSCs group and the TDSCs group at threetime points (1 week 2 weeks 4 weeks) Bar 200 120583m

35 Collagen I and Collagen III Gene Expression Analysis Todetect host ECM (collagen) deposition conditions quantita-tive real-time PCR was performed to investigate rat-specificCol-I and Col-III (Figure 6(a)) At the first week TDSCs andBMSCs Col-I gene expression levels were upregulated up to62-fold (119875 lt 005) and 42-fold (119875 lt 005) respectively Thelevels of Col-III were upregulated by 42-fold (119875 lt 005) inTDSCs and 22-fold (119875 lt 005) in BMSCs compared withnontreated group In addition the expression of Col-I and-III was higher in the TDSCs group than the BMSCs groupand it showed significant differences (119875 lt 005) At week 2after surgery Col-I gene expression levels were upregulatedby 41-fold (119875 lt 005) and 23-fold (119875 lt 005) in theTDSCs and BMSCs groups respectively In addition in bothTDSCs and BSMCs the Col-III levels were upregulated by23-fold (119875 lt 005) and 12-fold (119875 lt 005) respectivelycompared with nontreated group In addition the expressionof Col-I and -III was higher in the TDSCs group comparedwith the BMSCs group and it showed significant differences(119875 lt 005) At week 4 after surgery time TDSCs Col-I levelwas higher than BMSCs it showed a significant differencebut only the TDSCs Col-I gene expression level showed asignificant difference and was upregulated by 15-fold (119875 lt005) compared with nontreated group and by 11-fold (119875 lt005) in the BMSCs-treated group (Figure 6(a)) AdditionallyCol-III in all groups showed almost the same expression level(Figure 6(a)) It is obvious that both TDSCs and BMSCs havethe ability to boost the ECM gene expression But hereinwe observed that TDSCs compared to BMSCs have moreagility to trigger the genes involved in expression of ECM andaccelerated tendon repair many folds

36 Immunofluorescent Assay of Injured Achilles Tendon fol-lowed by TDSCs and BMSCs Transplantation The organiza-tion by immunofluorescence staining found that after 1 weekof implantation TDSCs and BMSCs in the injured Achillestendon cells were found in many parts of the distributionof CM-Dil labelled (Figure 6(b)) Following implantation ofTDSCs and BMSCs around the injured Achilles tendon alarge portion with Tenascin-C staining was detected in bothtreated groups (Figure 6(b)) where TDSCs treated groupof mouse showed higher expression of Tenascin-C thanthe BMSCs group In addition 4 weeks after implantationTDSCs and BMSCs were still able to detect CM-Dil labelledcells (Figure 6(c)) In short the results suggest that in theearly postimplantation TDSCs and BMSCs can promote theexpression of Tenascin-C Additionally the implanted cellscan survive for at least 1 month in the Achilles tendon injuryand are involved in the tendon reconstruction

4 Discussion

Achilles tendon rupture accounts for about 35 of all ten-don injuries due to low blood supply and low metabolicactivity of tendon fibroblastic cells in addition to these lowhealing potentials seen in the ruptured tendons Previousstudies reportedTDSCs to be immune-privileged cells havingpotential for allogeneic transplantation [2 14 16] which ledto cell banking and can be used during emergencies Tissueregeneration depends primarily on the rate of proliferationand differentiation of endogenous stem cells to produce ahigh amount of ECM It has been reported that TDSCshave higher colony-formation ability proliferate rapidly and


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs

Week 1 Week 2 Week 4 Week 1 Week 2 Week 4

Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s

DAPI CM-Dil Ten-C Merge

(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)

Figure 6 The analysis of tendon-related ECM expression (a) Rat-specific gene expression analysis of tendon-related ECM genes collagen Iand collagen IIIThe gene transcript levels were relative to GAPDH and normalized to nontreated group lowast119875 lt 005means compared with thecontrol group 119875 lt 005means compared with BMSCs group were considered significant (data represents mean plusmn SD 119899 = 3) (b) Tenascin-Cimmunofluorescent testing in the injured Achilles tendon (c) Cell tracking of TDSCs and BMSCs after transplant at 4 weeks the nuclei werestained by DAPI (blue spots) the CM-Dil was red spots Bar 200 120583m

possess some universal stem cell characteristic compared toBMSCs depending on the age and origin of the stem cells[17 19ndash21] Studies also show that TDSCs express highermRNA level of tenogenic markers scleraxis tenomodulinand ECM components of tendon compared to BMSCs [1417 22] By using mouse model Bi et al [14] found thatcompared to BMSCs TDSCs have more ability to expresshigher mRNA level for Sox9 Comp Runx2 and Scx whilein humans TDSCs express increased level of tenomodulin(TNMD) compared to human BMSCs does

TDSCs as compared to BMSCs are thought to be a potenttherapeutic cell treasure which take an active part in properand enhancedmusculoskeletal repair including tendon repair[21 23] TDSCs showed high potential for chondrogenic andosteogenic differentiation compared to BMSCs and hence atappealing candidate for tendon-bone junction regeneration[21] Keeping in view the superiority of DMSCs over otherstem cell lines in our study we chose two different stem cells

namely TDSCs and BMSCs and transplanted them into theAchilles tendon injured area of rats After transplantation theanimals were allowed to heal for four weeks Macroscopicappearance histomorphology and biomechanical strengthwere used to evaluate animal performance and tissue integritythroughout the healing process At four weeks into theexperiment the treated (TDSCs and BMSCs) groups showedbetter results than the nontreated group In the early stagesof regeneration TDSCs showed a prompt stimulatory effecton tissue remodeling in both macromicro appearance andbiomechanical strength At one week after transplantationsmall changes were observed regenerated tissue was coveringthe treated region in the TDSCs and BMSCs groups whilethere was no visible connective tissue in the injured area ofthe nontreated group At two weeks the TDSCs were betterin macromicro appearance than the other two groups Fourweeks after transplantation the histological evaluation ofTDSCs detected more fibroblastic cell presence arranged in


parallel rows and positive collagen fiber in the treated area asseen in the TDSCs group (Figure 5) and the Achilles tendondisplayed almost normal appearance (Figures 3(C) and 3(F))

The higher mechanical strength can be explained bythe increasing production of collagen Higher mechanicalstrength suggests that the cell transplantation promotes theorganization and synthesis of the various ECM componentsresponsible for the structural and functional repair of tendontissue during different stages of healing The ultimate failureload in the TDSCs groupwas considerably higher than that ofthe BMSCs group and the nontreated group At one week andtwo weeks TDSCs rapidly improved biomechanical strengthin the early stages of healing However after four weeks theultimate failure load of the TDSCs group still showed betterresults than other groups but was not significantly differentIn addition the TDSCs and BMSCs groups showed a highervalue of reached almost the healthy (Figure 4)

In short we supposed that after cell transplantationTDSCs are the first to adapt to the microenvironment in theruptured Achilles tendon Because of the fast proliferationand high affinity of tendon niches TDSCsmight differentiaterapidly into the functional tenocytes to synthesize a greateramount of ECM for remodeling

In addition we investigate the possible mechanism ofthe repair promotion by cell transplantation and test it bythree experiments RT-PCR to check the collagen expres-sion immunofluorescence to analyze the Tenascin-C proteinexpression and CM-Dil to locate the stem cell Initiallythe gene expression of both Col-I and Col-III genes wereupregulated after transplantation of TDSCs and BMSCs inAchilles tendon and TDSCs showed a higher enhancingeffect than BMSCs We considered that in the short timesince the injury the host needs a lot of cells to concentrate intothe wound After cell transplantation TDSCs and BMSCsquickly joined in the regenerative process and revealed ahigh gene expression level of collagen However at 4 weeksthe stimulatory effect of BMSCs for collagen type I geneexpression was gone In contrast TDSCs still showed theenhanced effect which means TDSCs provide continuousstimulation of collagen type I for connective tissue formationin the injured area In addition atweek 4 all the enhancementof Col-III gene expression in the TDSCs and BMSCs wasgone Because of the complicated signal mechanism of themicroenvironment after four weeks Col-III synthesis mightnot play the most important role in the regenerative processThe explanationmay be that Col-III ismore important duringthe earliest stages of tendon healing because it can rapidlyform crosslinks and stabilize the precarious repair site but lessso in the late stages of tissue remodeling [15 22]

Tenascin-C is reported as an important ECM proteinin providing elasticity to the musculoskeletal tissues [1524] This feature is of great importance in the degenerativeand regenerative processes where the normal biomechanicalenvironment of the musculoskeletal tissues is disturbed byinjury In our study we found that in the early stageboth TDSCs and BMSCs implantation groups can promoteTenascin-C protein synthesis in the Achilles tendon rupturedlocation and even former was observed with high proteinexpression than the latter group

The present study led us to speculate that stem cells trans-plantation promotes regenerative processes and TDSCs arethe first to adapt within the microenvironment in rupturedAchilles tendon In addition we found the CM-Dil labeledtransplanted cells were detectable around the tendon at week4 after surgery It can be interpreted that the transplanted cellswere still alive and were involved in the tendon remodelingprocess quickly and efficiently as compared to BMSCs andnontreated group hence proven to be the best choice

5 Conclusion

This study provides evidence that TDSC and BMSC trans-plantation improves the healing potential of rupturedAchilles tendon in rats In addition TDSCs exhibited abetter regenerative potential when compared with BMSCsin treating ruptured Achilles tendons and may be a betteralternative cell source for treatment during Achilles tendoninjuries

Conflict of Interests

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

Acknowledgments

This work was supported by the National Innovation andAttracting Talents Project (ldquo111rdquoProject) (B06023) NationalNatural Science Foundation of China (11032012 10902130and 30870608) and Fundamental Research Funds for theCentral Universities (CQDXWL-2014-007)

References

[1] M N Doral M Bozkurt E Turhan et al ldquoAchilles tendonrupture physiotherapy and endoscopy-assisted surgical treat-ment of a common sports injuryrdquo Open Access Journal of SportsMedicine vol 1 pp 233ndash240 2010

[2] S MacLean W S Khan A A Malik M Snow and S AnandldquoTendon regeneration and repair with stem cellsrdquo Stem CellsInternational vol 2012 Article ID 316281 6 pages 2012

[3] J C-H Goh H-W Ouyang S-H Teoh C K C Chan andE-H Lee ldquoTissue-engineering approach to the repair andregeneration of tendons and ligamentsrdquo Tissue Engineering vol9 supplement 1 pp S31ndashS44 2003

[4] P-O Bagnaninchi Y Yang A J El Haj and N MaffullildquoTissue engineering for tendon repairrdquo British Journal of SportsMedicine vol 41 no 8 p e10 2007

[5] M Denham B Conley F Olsson T J Cole and R MollardldquoStem cells an overviewrdquo in Current Protocols in Cell Biologychapter 23 Unit 23 21 2005

[6] L V Gulotta S Chaudhury and D Wiznia ldquoStem cells foraugmenting tendon repairrdquo Stem Cells International vol 2012Article ID 291431 7 pages 2012

[7] S A Reed and E R Leahy ldquoGrowth and development sym-posium stem cell therapy in equine tendon injuryrdquo Journal ofAnimal Science vol 91 no 1 pp 59ndash65 2013


[8] Y Jia DWu R Zhang et al ldquoBonemarrow-derivedmesenchy-mal stem cells expressing the Shh transgene promotes func-tional recovery after spinal cord injury in ratsrdquo NeuroscienceLetters vol 573 pp 46ndash51 2014

[9] S F Yuan T Jiang L H Sun et al ldquoUse of bone mesenchymalstem cells to treat rats with acute liver failurerdquo Genetics andMolecular Research vol 13 no 3 pp 6962ndash6980 2014

[10] H-L Tsai W-T Chiu C-L Fang S-M Hwang P F RenshawandW-F T Lai ldquoDifferent forms of tenascin-C with tenascin-Rregulate neural differentiation in bone marrow-derived humanmesenchymal stem cellsrdquo Tissue Engineering Part A vol 20 no13-14 pp 1908ndash1921 2014

[11] T-F Huang T-L Yew E-R Chiang et al ldquoMesenchymal stemcells from a hypoxic culture improve and engraft achilles tendonrepairrdquo American Journal of Sports Medicine vol 41 no 5 pp1117ndash1125 2013

[12] N Okamoto T Kushida K Oe M Umeda S Ikehara andH Iida ldquoTreating achilles tendon rupture in rats with bone-marrow-cell transplantation therapyrdquo The Journal of Bone andJoint Surgery American Volume vol 92 no 17 pp 2776ndash27842010

[13] L Lacitignola F Staffieri G Rossi E Francioso and ACrovace ldquoSurvival of bone marrow mesenchymal stem cellslabelled with red fluorescent protein in an ovine model ofcollagenase-induced tendinitisrdquo Veterinary and ComparativeOrthopaedics and Traumatology vol 27 no 3 pp 204ndash2092014

[14] Y Bi D Ehirchiou T M Kilts et al ldquoIdentification of tendonstemprogenitor cells and the role of the extracellular matrix intheir nicherdquoNatureMedicine vol 13 no 10 pp 1219ndash1227 2007

[15] J Zhang B Li and J H-C Wang ldquoThe role of engineeredtendon matrix in the stemness of tendon stem cells in vitroand the promotion of tendon-like tissue formation in vivordquoBiomaterials vol 32 no 29 pp 6972ndash6981 2011

[16] W Shen J Chen Z Yin et al ldquoAllogenous tendonstemprogenitor cells in silk scaffold for functional shoulderrepairrdquo Cell Transplantation vol 21 no 5 pp 943ndash958 2012

[17] H Thaker and A K Sharma ldquoEngaging stem cells for cus-tomized tendon regenerationrdquo Stem Cells International vol2012 Article ID 309187 12 pages 2012

[18] M-T Cheng C-L Liu T-HChen andOK Lee ldquoComparisonof potentials between stem cells isolated from human anteriorcruciate ligament and bone marrow for ligament tissue engi-neeringrdquo Tissue EngineeringmdashPart A vol 16 no 7 pp 2237ndash2253 2010

[19] Z Zhou T Akinbiyi L Xu et al ldquoTendon-derived stemprogenitor cell aging defective self-renewal and altered faterdquoAging Cell vol 9 no 5 pp 911ndash915 2010

[20] Y Sakaguchi I Sekiya K Yagishita and T Muneta ldquoCompar-ison of human stem cells derived from various mesenchymaltissues superiority of synovium as a cell sourcerdquo Arthritis andRheumatism vol 52 no 8 pp 2521ndash2529 2005

[21] P P Y Lui and K M Chan ldquoTendon-derived stem cells(TDSCs) frombasic science to potential roles in tendon pathol-ogy and tissue engineering applicationsrdquo Stem Cell Reviews andReports vol 7 no 4 pp 883ndash897 2011

[22] H Tempfer A Wagner R Gehwolf et al ldquoPerivascular cells ofthe supraspinatus tendon express both tendon- and stem cell-related markersrdquoHistochemistry and Cell Biology vol 131 no 6pp 733ndash741 2009

[23] M Ni P P Y Lui Y F Rui et al ldquoTendon-derived stem cells(TDSCs) promote tendon repair in a rat patellar tendonwindow

defectmodelrdquo Journal of Orthopaedic Research vol 30 no 4 pp613ndash619 2012

[24] A Pajala J Melkko J Leppilahti P Ohtonen Y Soini and JRisteli ldquoTenascin-C and type I and III collagen expression intotal Achilles tendon rupture An immunohistochemical studyrdquoHistology andHistopathology vol 24 no 10 pp 1207ndash1211 2009

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


bone cartilage tendon muscle marrow fat and dermis[8ndash10] BMSCs are involved to facilitate the tendon healingprocess intrinsically by accelerating fibroblast proliferationandmodulation of certain growth factors and cytokinins [11ndash13] On the other hand TDSCs are adult stem cells residingin tendons [14] which are histologically and biochemicallyproven as a prime source of tendon repair [15ndash17] Chengand coworkers demonstrated that TDSCs have high potentialfor colony-formation compared with BMSCs [18] It is alsosaid that TDSCs expresses higher mRNA level of tenogenicmarkers- scleraxis (Scx) tenomodulin (Tnmd) and extra-cellular matrix (ECM) components of tendon that is Col-1A1 Col-1A1Cl3-A1 ratio and decorin (Dcn) than BMSCsHowever a comparison between TDSCs and BMSCs ontreating tendon injury has not yet been doneWe hypothesizethat TDSCs are more favorable for treating Achilles tendoninjuries

In the present study we transplanted TDSCs and BMSCsinto the ruptured area of the Achilles tendon for macroscopicappearance histomorphological analyses and biomechanicalstrength were observed to find the possible mechanism ofthe repair promotion and evaluated the cell transplantationeffects of both stem cell typesThe results indicate that TDSCsexhibit a better-regenerative potential when compared withBMSCs in treating ruptured Achilles tendons and could be abetter alternative cell source for treating Achilles tendon

2 Materials and Methods

21 Ethics Statement Ethics Committee of Chongqing Uni-versity College of Bioengineering and Daping HospitalAnimal Experimental Center approved all experimental pro-tocols using SD rats including collection of Achilles tendonsamples

22 Rats and Treatment Groups Seventy-eight Sprague-Dawley (SD) male rats weighing 200 g obtained from theDaping Hospitalrsquos animal experimental center (ChongqingChina) were used as recipients or donors Six SD rats whichdid not undergo an operation were the source of tendon-derived stem cells bone marrow mesenchymal stem cellsand healthy Achilles tendons The remaining 72 SD rats wereused for Achilles tendon healing experiments The rats weredivided in to three groups TDSC BMSC and nontreatedgroup each group 24 rats The study was carried out at threetime points 1 week 2 weeks and 4 weeks Eight rats wereassigned to each time pointThe ratswere placed in individualcages under slandered feeding system

23 Cell Isolation Six SD rats were used to isolate the variouscells The rats were sedated with pentobarbital sodium in ananesthetic chamber Then using 3 Fluothane in a maskthey were sacrificed The femur and tibia including thetendons attached to them were dissected BMSCs of SDrats were isolated using a modified procedure [18] Brieflyboth femur and tibia were excised and the diaphyses werecut The bone marrow was flushed out with DMEM-LG(Gibco) supplemented with 10 FBS 100UmL penicillin

and 100 120583gmL streptomycin Single cell suspension wasgenerated by aspirating the bone marrow back through thesyringe The samples were then washed and centrifuged at1000 rpm to remove the pieces of debris The cell pellets wereresuspended and expanded in a humidified incubator at 5CO2and 37∘C TDSCs were isolated from rats by removal of

the tendons and rinsed with PBS The TDSCs were isolatedaccording to the previous report [19] Briefly the Achillestendons were then minced into small pieces and digestedwith 5mgmL of type I collagenase (SIGMA) at 37∘C with5 CO

2for two hoursThe undigested tissues were removed

by using 70mm nylon sieve and the remaining cell pelletswere cultured with low glucose Dulbeccorsquos modified Eaglersquosmedium (DMEM Gibco) L-glutamine 10 FBS 100UmLpenicillin and 100 120583gmL streptomycin After 12 days the cellcolonies formed and were selected for further culture After100 confluence cells were subcultured and the mediumwas changed every third day For both BMSCs and TDSCsPassage 3 (P3) cells were adopted for identification

24 Cell Identification and Multidifferentiation Assays Flowcytometry was used for identifying stem cell surface mark-ers CD29 CD44 and CD90 of TDSCs and BMSCs Theosteogenic adipogenic and chondrogenic differentiationpotential of TDSCs and BMSCs were tested according to theprevious report [19 20] After induction Alizarin red Oilred and Toluidine blue staining assays were used to confirmosteogenesis adipogenesis and chondrogenesis respectively

25 Animal Model and Surgical Procedures Seventy-two SDrats weighing 200 g provided by Daping Hospital and theResearch Institute of Surgery of the Third Military MedicalUniversity were used Prior to the study all operations andhandling procedures were approved by the hospital Therats were divided into three groups the BMSCs groupthe TDSCs group and the nontreated group each group24 rats Three rats from each group were tested in eachtime point The left hind legs of the animals were used formicromacro observation while the right hind legs were usedfor measuring gene expression Five rats from each groupfromeach time pointwere used for biomechanical evaluationThe rats were anesthetized with pentobarbital sodium in ananesthetic chamber and then with 3 Fluothane in a maskIn aseptic conditions 10mm longitudinal incision of theright and left hind limb was made directly over the Achillestendon (Figures 1(a) and 1(b)) A segment from the middlepart of the Achilles tendon was cut using a surgical blade5mm from the calcaneal insertion site Clinical sutures wereused to suture the incision and iodine was directly applied(Figure 1(c)) The TDSCs and BMSCs were labeled withCM-DiI (C7000 Invitrogen) after that the donor TDSCs (1times 10601mL DMEM) or BMSCs (1 times 10601mL DMEM)were injected around the Achilles tendon of each rat witha syringe (Figure 1(d)) During the recovery period the ratswere placed in individually sterilized cages under standardfeeding system At the end of each time point the rats weresacrificed by over dose of ether anesthesia


(a) (b)

(c) (d)

SD rats(Male 200 g)




Injection of

and BMSCs1 times 10

601mL

DMEM








Cou

nt

0

100

101

102

103

104

CD29C

ount

0

100

101

102

103

104

CD44

Cou

nt

0

100

101

102

103

104

CD90

Cou

nt

0

100

101

102

103

104

CD29

Cou

nt

0

100

101

102

103

104

CD44

Cou

nt0

100

101

102

103

104

CD90

(A)

(B)

CD29866 CD44

957

CD90826

CD90937

CD44873

CD29924

(a)

TDSC

sBM

SCs


(b)



(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Continued


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral





3 Results



lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

SD rats(Male 200 g)




Injection of

and BMSCs1 times 10

601mL

DMEM








Cou

nt

0

100

101

102

103

104

CD29C

ount

0

100

101

102

103

104

CD44

Cou

nt

0

100

101

102

103

104

CD90

Cou

nt

0

100

101

102

103

104

CD29

Cou

nt

0

100

101

102

103

104

CD44

Cou

nt0

100

101

102

103

104

CD90

(A)

(B)

CD29866 CD44

957

CD90826

CD90937

CD44873

CD29924

(a)

TDSC

sBM

SCs


(b)



(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Continued


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral





3 Results



lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cou

nt

0

100

101

102

103

104

CD29C

ount

0

100

101

102

103

104

CD44

Cou

nt

0

100

101

102

103

104

CD90

Cou

nt

0

100

101

102

103

104

CD29

Cou

nt

0

100

101

102

103

104

CD44

Cou

nt0

100

101

102

103

104

CD90

(A)

(B)

CD29866 CD44

957

CD90826

CD90937

CD44873

CD29924

(a)

TDSC

sBM

SCs


(b)



(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Continued


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral





3 Results



lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral

Figure 3 Continued


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral





3 Results



lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(A) (B) (C)

(D) (E) (F)


Poste

rior-

ante

rior

Late

ral





3 Results



lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowast


NontreatedBMSCs

TDSCsHealthy

40

35

30

25

20

15

10

5

0

Ulti

mat

e fai

lure

load

(N)











Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Non

treat

edBM

SCs

TDSC

s




4 Discussion



Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen I

lowast

lowast

lowast

lowast

lowast

ControlMSCs

TDSCs ControlMSCs

TDSCs


Rela

tive q

uant

ifica

tion

(fold

chan

ge)

Collagen III

lowast

lowast

lowast

lowast

(a)

Non

treat

edBM

SCs

TDSC

s


(b)

BMSC

sTD

SCs

DAPI

DAPI

CM-Dil

CM-Dil

Merge

Merge

(c)












5 Conclusion




Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusion




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 study of bone marrow mesenchymal and...

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