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Tissue Engineering and Regenerative Medicine

Stepwise Differentiation of Mesenchymal StemCells Augments Tendon-Like Tissue Formationand Defect Repair In Vivo Q:1; 2

ZI YIN,a,b,c JIAGUO,b TIAN-YIWU,bXIAOCHEN,a,c LIANG-LIANGXU,b SI-ENLIN,b YUN-XINSUN,b

KAI-MING CHAN,b HONGWEI OUYANG,a,c GANG LIb,c,d,e,f

Key Words. Tendon repair x Mesenchymal stem cells x Differentiation x Growth factor

ABSTRACT

Tendon injuries are common and present a clinical challenge, as they often respond poorlyto treatment and result in long-term functional impairment. Inferior tendon healing re-sponses are mainly attributed to insufficient or failed tenogenesis. The main objective ofthis study was to establish an efficient approach to induce tenogenesis of bone marrow-derivedmesenchymal stem cells (BMSCs), which are themost common seed cells in tendontissue engineering. First, representative reported tenogenic growth factors were used asmedia supplementation to induce BMSC differentiation, and the expression of teno-lineage transcription factors and matrix proteins was compared. We found that trans-forming growth factor (TGF)-b1 significantly induced teno-lineage-specific gene scleraxisexpressionand collagenproduction. TGF-b1 combinedwith connective tissue growth factor(CTGF) elevated tenomodulin and Egr1 Q:3expression at day 7. Hence, a stepwise tenogenicdifferentiation approach was established by first using TGF-b1 stimulation, followed bycombinationwith CTGF for another 7 days. Gene expression analysis showed that this step-wise protocol initiated and maintained highly efficient tenogenesis of BMSCs. Finally, re-garding in situ rat patellar tendon repair, tendons treated with induced tenogenic BMSCshad better structural and mechanical properties than those of the control group, as evi-denced by histological scoring, collagen I and tenomodulin immunohistochemical staining,and tendonmechanical testing. Collectively, these findings demonstrate a reliable andprac-tical strategy of inducing tenogenesis of BMSCs for tendon regeneration and may enhancethe effectiveness of cell therapy in treating tendon disorders. STEMCELLS TRANSLATIONAL

MEDICINE 2016;5:1–11

SIGNIFICANCE

The present study investigated the efficiency of representative tenogenic factors on mes-enchymal stem cells’ tenogenic differentiation and established an optimized stepwisetenogenic differentiation approach to commit tendon lineage differentiation for functionaltissue regeneration. The reliable tenogenic differentiation approach for stem cells not onlyserves as a platform for further studies of underlying molecular mechanisms, it also can beused to enhance cell therapy outcome in treating tendon disorders and develop novel ther-apeutics for tendon injury.

INTRODUCTION

Tendons are elastic collagenous tissues thatconnect muscles and bones. The main func-tion of tendon is to transmit forces frommus-cles to bones formaintenance of normal bodymovement. Tendon injuries occur frequentlyduring sports and other rigorous activitiesand are usually associated with significantmorbidity and abnormal joint movement [1].Because of the limited self-repair capacity oftendon tissues, natural tendon healing often

gives rise to inferior mechanical properties,and the healed tendon is susceptible to rein-

jury. To date, functional healing of tendon in-

juries has been a great challenge. Current

treatment is limited and unsatisfying, as the

biochemical properties and functions of

healed tendon tissue never recover to those

of intact tendon [2–5]. Patients often suffer

fromlong-termpain,discomfort, andevendis-

ability. Therefore, more effective therapeutic

techniques for tendon repair are needed.

aDr. Li Dak Sum and Yip Yio Chin Centerfor Stem Cells and RegenerativeMedicine, School of Medicine, ZhejiangUniversity, Hangzhou, People’sRepublic of China; bDepartment ofOrthopaedics and Traumatology,Faculty of Medicine, and eStem Cellsand Regenerative Medicine Laboratory,Lui Che Woo Institute of InnovativeMedicine, Li Ka Shing Institute of HealthSciences, The Chinese University ofHong Kong, Prince of Wales Hospital,Hong Kong, People’s Republic of China;cChina Orthopedic RegenerativeMedicine Group, Hangzhou, People’sRepublic of China; dKey Laboratory forRegenerative Medicine, Ministry ofEducation, School of BiomedicalSciences, Faculty of Medicine, TheChinese University of Hong Kong, HongKong, People’s Republic of China; fTheChinese University of Hong Kong–ChinaAstronaut Research and Training CenterSpace Medicine Centre on HealthMaintenance of MusculoskeletalSystem, The Chinese University of HongKong Shenzhen Research Institute,Shenzhen, People’s Republic of China

Correspondence: Gang Li, Department ofOrthopaedics and Traumatology and Li KaShing Institute of Health Sciences, Prince ofWales Hospital, The Chinese University ofHong Kong, Shatin, Hong Kong, SAR, People’sRepublic of China. Telephone: 852-37636153; Fax: 852-2646 3020; E-Mail:[email protected]

Received August 25, 2015; accepted forpublication March 7, 2016.

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http://dx.doi.org/10.5966/sctm.2015-0215

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Tendon tissue engineering is a most promising approach andopens new possibilities for tendon regeneration. Mesenchymalstem cells (MSCs) are known to be multipotent and self-renewing, andMSCs can be isolated from various tissues and eas-ily cultured. ThusMSCshavebeenextensively studiedandappliedin tissue engineering as seed cells for tendon repair. Despitemanysuccessful studies showing thatMSCapplication improves tendonhealing, there are concerns about the risk of spontaneous chon-drogenesis andectopic bone formationafterMSC transplantationin tendon injurymodels [6]. The chondro-osteogenetic changes intendon tissues lead to the erroneous deposition of extracellularmatrix and contribute to the poor quality of injured tendon.

The inductionofMSCs todifferentiate into tendon-forming cellsinvitrobefore transplantationmaybeausefulapproach forpromot-ing tendon repair. Various strategies for teno-lineagedifferentiationof MSCs have been reported, including mechanical stimulation,tenogenicmastergene transfection, coculturewith tenocytes,phys-ical topography induction, decellularized tendon matrices, and theuse of bioactive factors [7–11].Most of themdepend on specializedequipment, are time-consuming, andoftenyield variable and incon-sistent results. Bioactive molecules, especially recombinant growthfactors, are readily available, dose controllable, and widely used forpotential clinical applications. At present, several growth factorshave been reported to be able to induce tenogenic differentiationin MSCs, including connective tissue growth factor (CTGF, alsoknown as CCN2), growth differentiation factor (GDF) family mem-bers (GDF-5/bone morphogenetic protein [BMP]-14, GDF-6/BMP-13, and GDF-7/BMP-12), and transforming growth factor (TGF)-bfamily members (TGF-b1, TGF-b2, TGF-b3, etc.) [12–20]. However,it is difficult to determine which represents the most efficient andeffective strategy because of the lack of direct comparative studiesunder identical conditions. In this study, we investigated the effi-ciency of representative tenogenic factors onMSC tenogenic differ-entiation, based on levels of teno-lineage marker expression andcollagen production. We also optimized the tenogenic differentia-tion strategy by stepwise treatment of the selected growth factors.The induced MSCs after in vitro tenogenic differentiation were ap-plied in a tendon injurymodel to evaluate efficacy for tendon repaircompared with MSCs without induction.

MATERIALS AND METHODS

Isolation and Culture of Rat Fluorescence-Tagged BoneMarrow-Derived MSCs

All experimentswere approved by the Animal Research Ethics Com-mitteeof theChineseUniversityofHongKong. Four- to six-week-oldmale green fluorescent protein (GFP)-tagged Sprague-Dawley rats,weighing 250–300 g, were used. The procedure of isolation and cul-ture of rat GFP-tagged bone marrow-derived mesenchymal stemcells (BMSCs) was described previously [21]. The cells were sus-pended ina-essential Eagle’s medium (Thermo Fisher Scientific LifeSciences,Waltham,MA, http://www.thermofisher.comQ:4 ) containing10% fetal bovine serum (Thermo Fisher Scientific Life Sciences) and1% penicillin–streptomycin–neomycin (Thermo Fisher Scientific LifeSciences), seeded intoT75 flasks (Corning, Corning,NY, http://www.corning.com), and incubated at 37°C in a humidified atmospherewith 5% CO2.MSCs from passage 2 or 3 were used for experiments.

Upon reaching confluence, BMSCs were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) mediumalone (Ctrl) or supplementedwith (a) 10 ng/ml TGF‐b1 (PeproTech,

Rocky Hill, NJ, http://www.peprotech.com), (b) 100 ng/ml BMP-12 (PeproTech), or (c) 100 ng/ml CTGF (PeproTech) and50mg/mlascorbic acid (Sigma-Aldrich, St. Louis,MO,http://www.sigmaaldrich.com), or all resulting combinations, with conditioned mediumchanged every 3 days. Growth factor concentrations were selectedon thebasis of previouslypublished reports on tendondifferentiationstrategies [12, 14, 15, 18].

Quantitative Polymerase Chain Reaction

Total cellular RNA was isolated by lysis in TRIzol (Thermo Fisher Sci-entific Life Sciences). The expression levels of tendon-specific genesin cells culturedwith differentmedia were assessed by quantitativepolymerase chain reaction (PCR). PCRwas performed using BrilliantSYBR Green QPCR Master Mix (TaKaRa Bio, Shiga, Japan, http://www.takara-bio.com) on a Light Cycler apparatus (ABI 7900HT).The PCR cycling consisted of 40 cycles of amplification of the tem-plate DNA, with primer annealing at 60°C. The relative expressionlevel of each target gene was then calculated using the 22DDCt

method. Rpl19 was used as endogenous reference gene. PCR effi-ciencies of target genes and Rpl19were approximately equal. Dataare presented as fold change relative to the expression level of neg-ative control samples (untreated BMSCs). All primers (Tech Dragon,Hong Kong, People’s Republic of China, http://www.techdragon.com.hk) were designed using primer 5.0 and are summarized insupplemental online Table 1.

Sirius Red Staining

After induction for 7 days, the conditioned medium was removed,and the cells were washed with phosphate-buffered saline. BeforeSirius red staining, cells were fixedwith 70% ethanol for 30minutesandwashed 3 times. The deposited collagenwas stainedwith 0.1%Sirius red in saturated aqueous solution of picric acid. To quantifythe stained nodules, the stain was solubilized with 0.5 ml of 1:1(vol/vol) 0.1%NaOHandabsolutemethanol for 30minutes at roomtemperature. Solubilized stain (0.1ml) was transferred towells of a96-well plate, and absorbance was measured at 540 nm. Data arepresented as mean6 SD, n = 3.

In Vivo Neotendon Formation in Nude Mice

To demonstrate that induced BMSCs can form neotendon in vivo, anudemousemodelwas applied. Briefly, after anesthesia, an incisionwasmadeonthedorsum,andasubcutaneouspocketwascreatedtoexpose the posterior midline. The cell sheet formed by 53 105 in-ducedBMSCsor53105BMSCs in fibrin glue (Beriplast PCombi-Set;CSL Behring, King of Prussia, PA, http://www.cslbehring.com) wassutured to posterior midline at both ends using Ethicon 6-0 suture,and therewas tensile strength on the tendon graft withmovement.At the end of 4 and 6 weeks (n = 4), the implanted tissues were har-vested and subjected to histology for examination of vascularity andcollagen fiber alignment.

Animal Model of Patellar Tendon Injury and Repair

Thirty-four Sprague-Dawley male adult rats (8 weeks old, bodyweight 250–300 g)were used. To create the tendon defect, the cen-tral one-third of the patellar tendon (∼1mm inwidth)was removedfrom the distal apex of the patella to the insertion of the tibia tuber-osity with two stacked sharp blades according to a well-establishedprotocol fromourpreviouswork [12]. The ratsweredivided into twogroups: those treated with (a) BMSCs in fibrin glue and (b) induced

2 Stepwise Inductive Approach for MSC Tenogenesis

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BMSC cell sheets. The engineered tendon tissue was placed in thetendon defect and sutured to the patellar bone and tibia tuberosityusing Ethicon 6-0. The animals were allowed free cage activity untileuthanasia. At weeks 2 and 6 after surgery, 5 animals in each groupwere killed, and the patellar tendons were harvested for ex vivo ex-amination of the presence of transplanted cells by fluorescence im-aging, histology for the examination of cellularity and vascularity ofthe regenerated tissue, and polarization microscopy for the assess-ment of collagen fiber alignment, as well as collagen content deter-mination. At week 6, another 7 animals from each group wereeuthanized, and both contralateral intact and injured patellar ten-dons were harvested for biomechanical tests.

Immunofluorescence

Briefly, cells were fixed in 4% paraformaldehyde for 10 minutes atroom temperature, permeabilized, and blocked for 30minutes with1% bovine serum albumin. Fixed cells were washed and incubatedwith a primary antibody against collagen type I (Abcam, Cambridge,MA, http://www.abcam.com), tenomodulin (SantaCruzBiotechnol-ogy, Santa Cruz, CA, http://www.scbt.com), or control immunoglob-ulinG (BD, Franklin Lakes, NJ, http://www.bd.com) at 4°C overnight.Cellswere incubatedwithAlexa Fluor 555-conjugated secondary an-tibody (ThermoFisher Scientific Life Sciences) for 2 hours, andnucleiwere stainedwith49,6-diamidino-2-phenylindole. Forquantificationof signal intensity, imageswere capturedwith the same gain, offset,magnitude, and exposure time. This was followed by random selec-tion of a minimum of three different images, and intensities werequantified using Image-Pro Plus software as previously reported [9].

Histologic Evaluation and Masson Trichrome Staining

Specimenswere immediately fixed in10%neutral buffered formalin,dehydrated through an alcohol gradient, cleaned, and embedded inparaffin blocks. Histologic sections (5mm)were preparedusing ami-crotome and stained with hematoxylin and eosin (H&E). In addition,Masson trichrome staining was performed according to standardprocedurestoexaminethegeneralappearanceof thecollagenfibers.Polarizing microscopy was used to detect mature collagen fibrils.

Immunohistochemistry

Paraffin sections (5 mm) were incubated in antigen retrieval buffer(100mMTris and5%[wt/vol]urea,pH9.5) at60°C for60minutes forantigenretrieval. Endogenousperoxidasewasblockedby incubationwith 3%hydrogenperoxide inmethanol for 10minutes. Nonspecificprotein bindingwas blockedby incubationwith 10%goat serum.Af-ter overnight incubation at 4°C with antibodies to collagen type I(Abcam) and tenomodulin (Santa Cruz Biotechnology), sectionswere incubated with goat antirabbit horseradish peroxidase(HRP)-conjugatedordonkeyantigoatHRP-conjugatedsecondaryan-tibody (Santa Cruz Biotechnology) for 2 hours at room temperature.The 3,39-diaminobenzidine substrate system (DAB; Dako, Glostrup,Denmark) was used for color development. Hematoxylin stainingwas used to reveal the nuclei.

Mechanical Testing

Mechanical testing was performed using a tension/compressionsystemwith Fast-Track software (Model 5543; Instron, Norwood,MA, http://www.instron.us). The hindlimbs were wrapped ingauze soaked in saline and frozen at280°C for later testing. Be-fore testing, the hindlimbs were gradually thawed to room

temperature, and all soft tissue spanning the knee, except forthe center of the patellar tendon, were sharply transected. Mea-surement of the tendoncross-sectional areawasperformedusingaqueous rapid-curing alginate dental impression paste, digitalphotography, and computerized image analysis as previously re-ported [22]. The femur-patellar tendon-tibia complex (FPTC) wasthen rigidly fixed to custom-made clamps. After applying a pre-load of 0.1 N, each FPTC underwent preconditioning by cyclicelongation of 0–0.5 mm for 20 cycles at 5 mm/min. This was fol-lowed by load-to-failure test at an elongation rate of 5 mm/min.The load-elongation behavior of the FPTCs and failure modeswere recorded. The structural properties of the FPTCwere repre-sented by stiffness (newtons per millimeter), ultimate load (new-tons), energy absorbed at failure (millijoules), and stress atfailure. For each FPTC, the greatest slope in the linear region ofthe load-elongation curve over a 0.5-mm elongation intervalwas used to calculate stiffness.

Statistical Analysis

All quantitativedata sets areexpressedasmean6 SD.One-wayanal-ysisofvarianceandStudent’s t testwereperformedtoassesswhetherthere were statistically significant differences in the results betweengroups. Values of p, .05 were considered to be statistically signifi-cant. The significance level is presented as either p, .05 or p, .01.

RESULTS

Effect of Different Bioactive Factors on BMSCs’Tenogenic Differentiation

The typicalmorphology of tendon cells is spindle-like in shape andlongitudinally oriented to collagen fiber bundles in tendon tissue.Because the morphologic features are crucial for functional tis-sue,weobservedMSCmorphologywhencultured in thedifferentconditionedmedia. It was found that BMSCs elongated notably inthepresenceof TGF-b1, aloneandwithwithother exogenous fac-tors (combination groups) (Fig. 1A). Under fluorescent micros-copy, GFP fluorescence confirmed the cell morphology changesand BMSC growth in clusters under TGF-b1 alone or with otherexogenous factors (Fig. 1B).

Gene expression was measured in BMSCs (P2) before induc-tion and in BMSC culture in the absence (Ctrl) or presence of ex-ogenous factors.Messenger RNA (mRNA) expression is presentedin Figure 2, normalized to BMSCs (P2). Notably, expression oftenogenic gene Scx and tendon matrix genes Col I, Fmod, Fn,TnC, and Thbs4 was significantly increased in the presence ofTGF-b1 alone and with other exogenous factors at day 3 (Fig. 2).Treatment with BMP-12 alone, CTGF alone, or their combinationdid not exhibit any significant difference in teno-lineage gene ex-pression compared to the control group or BMSCs (P2). No signif-icant difference in cell morphologywas observed between TGF-b1alone andwith other growth factors. Although expression levels oftheother transcriptiongeneEgr1and tendonspecificmarkerTnmdat day 3 did not show any significant difference between all 8groups, Egr1expressionwas elevated significantly in BMP12alone,CTGFalone,andTGF-b1/CTGFgroupsatday7comparedwithday3(Fig. 2). The greatest increase in Tnmd gene expression was ob-served in the presence of TGF-b1/CTGF (Fig. 2).

Differentiation toward the tenogenic lineage was character-ized by immunofluorescence staining for tendon matrix proteincollagen type I and tendon-specific marker tenomodulin. The

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results showed that TGF-b1 alone and with other growth factorssignificantly promoted collagen type I production compared withthe control andother groups (Fig. 3A, 3C),which is consistentwithmRNA expression results. Additionally, immunofluorescencestaining of tenomodulin showed higher levels detected in theTGF-b1/CTGF and TGF-b1/BMP12/CTGF combination treatmentgroups (Fig. 3B). Quantification of immunofluorescence stainingshowed that the TGF-b1/CTGF and TGF-b1/BMP12/CTGF groupsshowed the highest levels of both collagen type I and tenomodu-lin expression (Fig. 3C, 3D), whereas no significant difference wasdetected between the two groups. Furthermore, Sirius red stain-ing was conducted to evaluate the extracellular matrix, especiallycollagen secretion andproduction,which is themajor componentin tendon tissue. The intensity of Sirius red staining was signifi-cantly higher with TGF-b1, alone and with other growth factors,than in groups without TGF-b1 (Fig. 3E). Quantification data con-firmed that TGF-b1 is the most potent factor in promoting colla-gen production; no synergetic effect was observed with othergrowth factors (Fig. 3F).

Stepwise Tenogenic Differentiation Strategy for BMSCs

Based on these comparative studies of TGF-b1, BMP12, CTGF, andtheir combinations on tenogenic induction of BMSCs, it was foundthatTGF-b1alonesignificantlyandefficiently induced teno-lineage-specific gene scleraxis expression and collagen production. In addi-tion,TGF-b1combinedwithCTGFelevated tenomodulinmRNAandprotein expression at day 7. Hence, the stepwise tenogenic

differentiation approach was established by first using TGF-b1 stimulation for 3 days, followed by combination with CTGFfor another 7 days. Gene expression analysis during this induc-tive process showed that this stepwise protocol initiated andmaintained the high efficiency of tenogenic differentiation ofBMSCs, as evidenced by significantly higher expression of Scx,Egr1, Col I, Comp, Tnc, Thbs4, Fmod, and Tnmd (Fig. 4). The ef-ficacy of this stepwise tenogenic differentiation approach forBMSCswas further evaluated in amodel of in vivo tendon repairand regeneration.

In Vivo Ectopic Neotendon Formation

In the in vivo ectopic implantationmodel, engineered tendons byinduced BMSCs using the stepwise tenogenic differentiation ap-proach or uninduced BMSCswere implanted subcutaneously intoSCID mice. We found that in the induced BMSC group, a greaternumber of cells exhibited spindle-shaped morphology and orga-nized collagen deposition at both 4 and 6weeks postimplantation(Fig. 5A), whereas the cells in the BMSC group showed roundshapes in random arrangements (Fig. 5A). H&E staining showedthat the matrices of the induced BMSCs were dense, whereasthe BMSC groups were filled with loose, disarranged soft tissue(Fig. 5). Detection of GFP-tracked BMSCs showed that implantedBMSCswerepresent subcutaneouslywithin themice for at least 6weeks andparticipated in ectopic new tissue regeneration inbothgroups (Fig. 5B). Sections were also observed under polarizedlight, and bands of collagen fibers were detected only in the

Figure 1. Morphologic appearance of BMSCs in culture supplementedwith various growth factors. (A): Representative phase-contrast imagesshowing the change of BMSC morphology under different treatments at day 3. (B): GFP fluorescence images showing BMSC growth patternsunder different treatments at day 3. Scale bars = 100 mm.




induced BMSC group at 6 weeks postimplantation. This impliedthat the induced BMSCs could form tendon-like tissue in vivoand better arrangement of extracellular matrices.

Induced BMSC Enhances Rat Patellar Tendon Repair

To further evaluate the tendon repair potential of the inducedBMSCs in situ, a patellar tendon injury model was used.

Histology of the Repaired Tendon

After 2 weeks, histology showed that more cells exhibitedspindle-shaped morphology in the induced BMSC group (Fig.6).Whereas the cell morphology in the BMSC group showed rel-atively round shapes (Fig. 6), Masson trichrome stainingshowed that the collagen fibrils in the induced BMSC groupwere aligned along the axis of tensile load (Fig. 6). In the BMSCgroup, the extracellular matrices in the repair site were

Figure 2. Tenogenic marker gene expression trends in BMSCs cultured in the absence of growth factors (Ctrl) or the presence of TGF-b1 (T),BMP-12 (B), or CTGF (C), and all resulting combinations (TB, TC, BC, and TBC). Gene expression levels are presented relative to that of BMSCs(BMSC-P2). As a single factor, TGF-b1 led to the greatest upregulation of expression of all tenogenic genes (Scx, Col1, Thbs4, TnC, Egr1, andTnmd). Expression of all tenogenicmarker geneswas upregulated in all the combination groupswhere TGF-b1was present. Data are presentedas mean6 SD. All groups not connected by a common letter are significantly different (p , .05).




relatively disorganized and had more immune cell infiltrationand vascular components (Fig. 6).

After 6weeks, thehistologic appearance of theneotendons inthe induced BMSC group was more like native tendon structure,with fewer cell components and apparent bands of dense colla-gen fibers filling in the gap tissues (Fig. 6). There were denser

collagen fibers and continuous tissues at the junction siteformed in the induced BMSC group, as evidenced byMasson tri-chrome staining (Fig. 6). Collagen I immunohistochemistrystaining was more intense in the induced BMSC group at both2 and 6 weeks postimplantation, suggesting enhanced collagentype I production and arrangement (Fig. 7A). Tenomodulin

Figure 3. Expression of collagen and tenomodulin in BMSCs induced with various growth factor treatments. Immunofluorescence staining ofcollagen type I (A) and tenomodulin (B) at day 7was significantly upregulated in the groups treatedwith TGF-b1 and CTGF. Scale bars = 100mm.Quantification data of immunofluorescence staining of collagen type I (C) and tenomodulin (D)was present. (E): Sirius red staining for collagendeposition evaluation in BMSCs cultured in the absence of growth factors (Ctrl) or the presence of TGF-b1 (T), BMP-12 (B), or CTGF (C) and allresulting combinations at day 7, showing the increased collagen production in the groups of T, T+B, T+C, and T+B+C. (F):Quantification data ofSirius red staining. Data are presented as mean6 SD. All groups not connected by a common letter are significantly different (p , .05).




Figure 4. Teno-lineage marker gene expression in BMSCs during the stepwise tenogenic differentiation approach by first using TGF-b1 stim-ulation for 3 days, followedby combinationwithCTGF for another 7 days. The expression of Scx, Egr1,Col I,Comp,Tnc, Thbs4, Fmod, and Tnmd ispresented at days 1, 3, 5, 7, and 9. p, p , .05.




immunohistochemistry staining revealed more positive cells inthe induced BMSC group than in the BMSC group at junctionsand neotendon formation sites (Fig. 7A).We also usedpolarizedlight microscopy to compare the collagen fiber maturation ofthe two groups. The induced BMSC group showedmorematureand organized collagen fibers in the repaired site than the BMSCgroup at both 2 and 6 weeks postimplantation (Fig. 6).

Mechanical Properties of the Repaired Tendons

To further correlate tissue structural features with their mechan-ical properties, harvested tendons (n = 7 for each group) weresubjected to mechanical testing 6 weeks after surgery. The in-duced BMSC group had better mechanical properties than thoseof the controls. The stiffness (18.3862.19N/mmvs. 10.7862.08N/mm) was significantly higher in the induced BMSC group (Fig.7B), which corresponded to 88% of the value obtained for intactrat tendon. The stress at failure in the induced BMSC treatmentgroup was 2.15-fold that of the control group (15.046 2.13MPavs. 6.98 6 1.13 MPa, p , .05) (Fig. 7B), 63% and 29% of that ofintact rat patellar tendon, respectively. The modulus in the in-duced BMSC group was twofold higher than that of the controlgroup (148.75 6 26.15 MPa vs. 72.47 6 10.5 N, p , .05) (Fig.7B), 84% and 41% of that of intact rat patellar tendon.

DISCUSSION

The present study sought to investigate a reliable method to en-hance tenogenic differentiation of BMSCs and generate func-tional neotendon tissue in vivo. First, three potential tenogenicfactors, TGF-b1, BMP12, and CTGF,were chosen to evaluate theirsingle or combined use on tenogenic differentiation of BMSCs.

Notably, TGF-b1was themost potent single factor that stimulatestenogenic geneupregulation andextracellularmatrix production.BMSCs respondedbetter to combinedTGF-b1/CTGFandTGF-b1/BMP12/CTGF stimulation, as evidenced by the significant upregu-lation of tenomodulin and collagen type I protein expression.Thus,weusedTGF-b1 to initiate tenogenesis inBMSCsby increas-ing transcription factor Scx significantly, followed by supplemen-tation of TGF-b1/CTGF to induce and maintain full teno-lineagecommitment. This stepwise tenogenic differentiation approachachieved optimal results within 10 days of induction. Finally,the induced BMSCs by this stepwise strategy had enhanced qual-ities for neotendon formation in both an ectopic tendon forma-tion model and a rat patella tendon injury model.

Despite many studies using growth factor supplementationfor tenogenic differentiation of BMSCs, with varying degrees ofsuccess, efficacy is hard toevaluatebecauseof differences in cells,duration, and readout indices. Hence, it is essential to comparethe potential growth factors shoulder-to-shoulder to concludewhich is the most potent factor for tenogenic differentiation. Be-cause adult tissue regeneration is generally thought to recapitu-late developmental processes, insights from developmentalbiology have been considered important for tendon repair [23].It was demonstrated that TGF-b and fibroblast growth factor(FGF) are the main growth factors involved in tendon develop-ment by embryologic experiments and genetic analyses [20,24]. However, studies showed that there was no positive teno-genic effect of FGF4 on adult stem/progenitor cell differentiation[20, 25]. Application of human recombinant TGF-b ligands led toa significant increase in the mRNA expression level of tenogenicgenes, whereas there was no significant difference between dif-ferent ligands and no synergetic effect [18–20]. Thus, we choseone of the TGF-b ligands as a representative tenogenic factor to

Figure 5. Ectopic neotendon formation by induced BMSCs. (A):H&E staining showing the histology of ectopic neotendon formation at 4 and 6weeks postimplantation. Scale bars = 50 mm. Polarized light microscopy image showing the maturation of the newly-formed collagen fibrils.Scale bars = 50mm. (B): GFP fluorescence images showing implanted BMSC survival in neotissue site at 4 and 6 weeks postimplantation. Scalebars = 25 mm.




compare with other candidates. Additionally, BMP-12, BMP-13,and BMP-14 showed similar effects on inducing MSC differenti-ation, such as enhanced extracellular matrix and tenogenicmarker gene upregulation. CTGF has also been demonstratedto sufficiently promote MSCs and TSPCsQ:5 to differentiate intoteno-lineage both in vitro and in vivo. In this study, TGF-b1 singlestimulus led to the strongest induction of the tendon-specificgenes Scx, Bgn, Col11, Thbs4, Fmod, Tnc, and Fn (p , .01).Among them, Scx, Fmod, and TnCwere particularly highly upre-gulated, to.20-fold.Moreover, the promoting effect of TGF-b1on tenogenic gene expression was detected as early as day 3,and neither of the other two growth factors exhibited any effectat this time point; previous reports showed that the effect ofCTGF on tenogenesis was generally achieved as long as day 14[12, 26]. Taken together, these data indicate that TGF-b1 playsa more profound role in tenogenic differentiation than BMP12and CTGF in mesenchymal stem cells.

Besides the induction of specific differentiation, the mainte-nance of teno-lineage phenotype deserves more attention andconsideration. Maeda et al. [27] examined 11 candidatecytokines/growth factors for their role in maintaining Scx expres-sion in adult tenocytes in vitro, including TGF-b1, -2, and -3;GDF5,7, and4; insulin-likegrowthfactor1;platelet-derivedgrowthfactor; epidermal growth factor; vascular endothelial growthfactor; and BMP2. The results indicated that the addition ofTGF-b1, -2, and -3 andGDF8 results in the retention of Scx-GFP ex-pression [27]. This is consistent with our findings that TGF-b1 notonly induced Scx expression but also maintain its expression for atleast 10 days. However, it was reported that the expression of Egr1and Tnmd was not induced after TGF-b2 exposure in a mouse

mesenchymal stem cell line [28], which was also true in our study.Furthermore, TGF-b1/CTGF and TGF-b1/BMP12/CTGF combina-tion treatment groups showed significant increases in Egr1 andTnmd expression. For cost considerations, we used TGF-b1/CTGF combination treatment to induce maturation of BMSCstoward the teno-lineage, after TGF-b1 treatment.

How MSCs differentiate toward the tenogenic pathway ispoorly understood. TGF-b signaling is critical for tendon develop-ment, asmostof the tendonsand ligaments in the limbs, trunk, tail,and head are lost in Tgfb22/2 and Tgfb32/2 double-mutant em-bryos [24]. Our recent study also demonstrated that Mkx inducesthe expression of Scx, a transcription factor specific for tenocytesand their progenitors, via the TGF-b signaling pathway [18].More-over, it was reported that mechanical forces maintain the expres-sion of Scx also through TGF-b/Smad2/3-mediated signaling [27],Therefore, the TGF-b signaling pathway is essential for tendon dif-ferentiation through Smad2/3 activation. However, TGF-b is alsoessential for chondrogenesis and osteogenesis. Under the condi-tions used in the study, tendon differentiation is dominant overchondrogenesis. The expression of Sox9 increased at day 3 but de-creased gradually during the optimized stepwise tenogenic induc-tionprotocol.However, thematurechondrocytemarker collagen IIwasundetectable inBMSCculture in theabsence (Ctrl) or presenceof exogenous factors at both day 3 and day 7. These results indi-cated that successful chondrogenesis also requires 3D culture con-ditions, a combination of other factors (such as insulin/transferrin/sodium selenite, bovine serum albumin, and dexamethasone inchondrogenic media), or longer induction time.

Hurleandcolleagues reportedthatTGF-b coordinatescartilageand tendon differentiation, which ismediated by stage-dependent

Figure6. Histology result of repairedpatella tendonat2and6weeks after healing. (A):H&Estaining showinghistologyof the junctionbetweennormal tendon and neotendon, as well as the midpoint of neotendon formation at 2 and 6 weeks postimplantation. Scale bars = 100 mm. (B):Masson’s trichrome staining showing deposited collagen at the repaired tissue site at 2 and 6weeks postimplantation. Scale bars = 100mm. (C):Polarized light microscopy image showing maturation of the newly formed collagen fibrils. Scale bars = 200 mm.




regulation of transcriptional signaling repressors [29, 30]. The ex-pression of CTGF has been shown to be involved in tendon healingprocesses and has been shown to favor fibrogenesis in connectivetissue healing over ectopic mineralization [14]. Therefore, CTGFmay help stem cells to follow the tenogenesis pathway rather thanosteogenesis. A recent report suggested that CTGF-induced teno-genic differentiation was regulated by focal adhesion kinase andextracellular signal-regulated kinase 1/2 signaling [26]. The incom-plete description of how CTGF’s signaling pathway crosstalks withtheTGF-b signalingpathway isa limitationof thepresent study thatwarrants future in-depth investigation to unravel the specificmechanisms by which TGF-b1 and CTGF regulate tenogenesis.

In addition to BMSCs, tendon stem/progenitor cells havebeen studied as seed cells for tendon repair. It was reported thattendon stem/progenitor cells not only expressed higher level ofteno-lineage genes but also exhibited higher clonogenicity com-paredwith BMSCs [31]. Further studies should try to evaluate thecurrent stepwise tenogenic differentiation approach on tendonstem/progenitor cells. Another limitation of our study was thatwe did not purposely study the most optimal growth factor

concentrations.We have used reportedworkable concentrationsbased on single-stimulus tenogenic conditions in the currentstudy; optimizing growth factor stimulation duration and concen-tration shall be the subject of future studies.

CONCLUSION

Thepresent study focusedonapproachesusinggrowth factors fortenogenesis, and the key tenogenic growth factors reportedwerecompared with tenogenic differentiation by induced BMSCs. Weestablished a stepwise inductive approach to induce tenogenesisof BMSCs by using TGF-b1 and TGF-b1/CTGF sequentially, whichare efficient and effective for induction of tenogenic differentia-tion both in vitro and in vivo. Moreover, we found that the appli-cation of this stepwise tenogenic differentiation approach onBMSCs before transplantation promoted the functional repairof injured tendons, in contrast toBMSCswithout induction. Thesefindings provide important information for future tendon tissueengineering applications. The reliable BMSC tenogenic differenti-ation approach not only serves as a platform for further

Figure 7. (A): Immunohistochemistry of collagen I and tenomodulin expression of repaired tendon at 2 and 6 weeks. Scale bars = 100mm. (B):Mechanical properties of repaired tendonat6weeks in inducedBMSCandcontrol groups.Data arepresentedasmean6SD.n=7.p,p, .05;pp,p, .01Q:6 .




underlying molecular mechanisms studies, it also can be used toenhance cell therapy outcome in treating tendon disorders.

ACKNOWLEDGMENTS

This work was supported by Zhejiang Provincial Natural ScienceFoundation of China (LR14H060001), NSFC grants (81522029,81401781), and Key Scientific and Technological Innovation TeamofZhejiangProvince (2013TD11). This studywas supported inpartby SMART program, Lui Che Woo Institute of Innovative Medi-cine, Faculty of Medicine, The Chinese University of Hong Kong.This research project was made possible by resources donatedby Lui CheWoo Foundation Limited. Y.Z. was a recipient of a post-doctoral research follow from the Faculty of Medicine, The Chi-nese University of Hong Kong (June 2014 to June 2015).

AUTHOR CONTRIBUTIONS

Z.Y.: manuscript writing, collection and/or assembly of data, dataanalysis and interpretation; J.G., T.W., L.X., S.L., and Y.S.: collec-tion and/or assembly of data; X.C.: collection and/or assemblyof data, data analysis and interpretation; K.M.C.: conceptionand design, financial support; H.O.: final approval of manuscript;G.L.: conception and design, financial support, data analysis andinterpretation, final approval of manuscript.

DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

G.L. has uncompensated intellectual property rights and is an un-compensated consultant. The other authors indicated no poten-tial conflicts of interest.

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