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Evaluation of the effectiveness of a bMSC and BMP-2 polymeric trilayer system in cartilage

repair

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2017 Biomed. Mater. 12 045001

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Biomed.Mater. 12 (2017) 045001 https://doi.org/10.1088/1748-605X/aa6f1c

PAPER

Evaluation of the effectiveness of a bMSC and BMP-2 polymerictrilayer system in cartilage repair

Raquel Vayas1,2, RicardoReyes3,4,María Rodríguez-Évora1, Carlos del Rosario1, Araceli Delgado1,3 andCarmenÉvora1,3

1 Department of Chemical Engineering andPharmaceutical Technology, Universidad de La Laguna, E-38200 La Laguna, Spain2 Servicio deCirugíaOrtopédica y Traumatología, ComplejoHospitalarioUniversitarioNtra. Sra. deCandelaria, E-38010 Santa Cruz de

Tenerife, Spain3 Institute of Biomedical Technologies (ITB), Center for Biomedical Research of theCanary Islands (CIBICAN), Universidad de La Laguna,

E-38200 La Laguna, Spain4 Department of Biochemistry,Microbiology, Cell Biology andGenetics. Cell Biology section, Universidad de La Laguna, E-38200 La

Laguna, Spain.

E-mail: cevora@ull.es

Keywords: cartilage repair, scaffold, BMP-2,MSC, electrospinning

AbstractIn this study a poly(lactide-co-glycolide) acid (PLGA) tri-layer scaffold is proposed for cartilage repair.The trilayer system consists of a base layer formed by a tablet of PLGAmicrospheres, a second layercomposed of amicrosphere suspension placed on top of the tablet, and the third layer, whichconstitutes an external electrospunPLGA thin polymericmembrane. Combinations of bonemorphogenetic protein-2 (BMP-2) encapsulated in themicrospheres of the suspension layer, andbonemarrowmesenchymal stem cells (bMSC) seeded on the electrospunmembrane, are evaluated byhistologic analyses and immunohistochemistry in a critical size osteochondral defect in rabbits. Fiveexperimental groups, including a control group (empty defect), a blank group (blank scaffold), abMSC treated group, two groups treatedwith 2.5 μg or 8.5 μg of BMP-2 and another two groupsimplantedwith bMSC-BMP-2 combination are evaluated. The repair area increases throughout theexperimental time (24weeks). The repair observed in the treated groups is statistically higher than incontrol and blank groups. However, the bMSC-BMP-2 combination does not enhance the BMP-2response. In conclusion, BMP-2 andbMSC repaired effectively the osteochondral defect in the rabbits.The bMSC-BMP-2 combination did not produce synergism.

1. Introduction

Articular cartilage defects can be a consequence oftraumatic lesions or degenerative diseases amongothers, and often lead to degenerative arthritis andprogressive loss of function affecting millions ofpeople worldwide. Adult articular cartilage shows apoor ability for auto-repair [1]. To date, the availabletreatments include microfracture, osteochondralautograft transplantation or mosaicplasty and freshstored osteochondral allograft transplantation [2],implantation of free autologous chondrocyte (ACI) [3]or embedded in a collagen matrix (MACI) [4]. Alter-natively, mesenchymal stem cells (MSC) from differ-ent sources are currently being investigated and/oreven clinically applied [5, 6]. Despite the fact that thesetherapeutic strategies temporarily improve the lesion,

they have no long-term efficiency; therefore, theyremain a challenge for orthopedic therapy [7, 8].

Thus, alternative strategies associated with cell-laden scaffolds or scaffolds providing controlledrelease of active substances such as growth factors orcytokines, are intensively investigated [9–11]. Mem-bers of the TGF-β superfamily are especially impor-tant to restore and maintain the chondrogenicphenotype and in the induction of chondrogenesis oftheMSC fromdifferent sources [12, 13].

Bone morphogenetic proteins (BMPs) regulatevarious stages of cartilage and bone development. Spe-cifically in cartilage, BMPs are involved in all phases ofchondrogenesis, they directly regulate the expressionof several chondrocyte specific genes and also have astrong effect on chondrocyte proliferation and matrixsynthesis [14–18]. The therapeutic use of BMPs has

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17November 2016

REVISED

7April 2017

ACCEPTED FOR PUBLICATION

25April 2017

PUBLISHED

4 July 2017

© 2017 IOPPublishing Ltd

been addressed not only by direct application of theseproteins, but also by applying MSC transfected toexpress certain BMPs, such as BMP-2, or TGF-β3 [6].Knowledge of the factors involved in the developmentand maintenance of the chondrogenic phenotype andthe role of BMPs in the regulation of anabolic path-ways provide strong justification for its application incartilage regeneration and repair. Although previousstudies justify the use of BMPs to stimulate chon-drogenesis and cartilage repair, the use of cell therapyusing MSC has also been the subject of different stu-dies [19–23]. Furthermore, the combination of bothstrategies has been proposed to improve the outcomes.However, discrepancies of results can be found in theliterature: some authors observed an improved perfor-mance [24, 25] whereas others did not detect anyrepair enhancement [20]. Thereby, in order to helpclarify the potential effect of the combination ofBMP-2 and cells in an osteochondral defect, a tri-layersystem was fabricated. The system consisted of a poly(lactide-co-glycolide) (PLGA 50:50) scaffold of threelayers: a microsphere tablet for the bone layer, ahydrogel suspension of microspheres oriented to thechondral area and an external electrospun film of thepolymer. Two different doses of BMP-2, one relativelylow of 2.5 μg and another of 8.5 μg as the high dose,were pre-encapsulated inmicrospheres to increase theresidence time in the defect. Bone marrow mesenchy-mal stem cells (bMSCs) were pre-seeded on the elec-trospunfilm.

2.Material andmethods

2.1.MaterialsSystem fabrication and surgical procedures werecarried out under aseptic conditions. Except for BMP-2, all liquid components, polymers and lab instru-ments were sterilized. BMP-2 with ED50 of 0.68,measured by its ability to induce alkaline phosphataseproduction by C2C12 cells, was purchased from Gen-Script (Piscataway,USA).

2.2. Preparation and characterization of the systemThe three-layer system consisted of a bone-directedlayer formed by a cylindrical tablet of blank micro-spheres, a cartilage-directed layer of microspheresuspension, 10 mg in 10 μl of Pluronic® F-127 (5%),and an external thin polymeric membrane(figure 1(a)). The systemwas assembled in a cylindricalmould which had been preloaded with themembrane.Then, the suspension of microspheres was poured, thetablet was introduced and the whole systemwas closedwith themembrane.

Microspheres were prepared by a double emulsion(w/o/w) method [26]. Briefly, 200 μl of 0.07%poly-vinyl alcohol (PVA) was the aqueous internalphase and 2 ml of a PLGA (Resomer® RG504, Evonik,Germany) methylene chloride solution (50 mgml−1)

the organic phase. The first emulsion was preparedwith a vortex for 1 min and poured into the externalphase of PVA (0.1%w/v). BMP-2 was incorporated inthe internal phase at different concentrations depend-ing on the dose to be administrated. To determineencapsulation efficiency 125I-BMP-2 [26] was used.Scanning electron microscopy (SEM, Jeol JSM-6300)was used for microspheres observation and laser dif-fractometry (Mastersizer 2000, Malvern Instruments)for microsphere size. 18 mg of blank microsphereswere compressed at 37 MPa for 2 min (Carver Inc.model 4120, USA) to fabricate a tablet. The tablet por-osity was measured by mercury intrusion porosimetry(Autopore IV 9510 Porosimeter;Micromeritics).

The membrane was fabricated by electrospinninga Resomer® RG504 20% (p/v) solution in hexa-fluoroisopropanol as previously [27]. Membrane wasvisualized by SEMand images used for fiber diameters.Stereo microscopy (Leica M205 C, Leica LAS, v3 soft-ware) was used for membrane thickness. Real densitywas measured by helium pycnometer (Micromeritics,AccuPyc 1330) to calculate the porosity by the gravi-metricmethod as previously [27].

2.3. Rabbit bMSCbMSC, isolated as previous described [20], wereexpanded at approximately 80% of confluence andkept frozen until use.

Depending on the animal group, some electro-spun membranes were pre-seeded with bMSC. Oncethe membrane was introduced in the syringe, 20 μl ofa cell suspension (1 × 105 cells) in phosphate buffer(pH, 7.4)were dropped on the bottommembrane cir-cle of 4 mm (width of the syringe). Then, as detailed insection 2.2, the system was assembled and 20 μl of thesame cell suspension were placed on the other side ofthe membrane. The system was incubated 2 h prior toimplantation, to allow cell adhesion. To check mem-brane cell adhesion, XTT colorimetric assay (Roche,Madrid, Spain) was assessed at 492 nm/reference690 nm [27]. To visualize viable cells, a Zeiss (Jena,Germany) Axiovert 40 CFL inverted microscope wasused and photographs were taken (Canon PowerShotA620 digital camera Tokyo, Japan) after incubation incalceinAM (Fluka, Sigma-Aldrich,Madrid, Spain).

2.4. Animal surgeryAll the experiments were carried out in conformitywith the EC (2010/63/UE) and Spanish guidelines oncare and use of animals in experimental procedures (RD 1201/2005). Furthermore, the animal experimentswere previously approved by the local committee ofthe University of La Laguna. The surgery procedurewas carried out in aseptic conditions as described [28].The system was placed in an osteochondral defect of4.5 mm of diameter and 4 mm of depth created in theproximal area of the intercondylar space.

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2.5. In vivo release assayThe BMP-2 in vivo release was carried out using125I-BMP-2 as previously described [28]. The125I-BMP-2 system was implanted in the defect of fiverabbits. Then, the remaining radioactivity at the defectsite was measured for four weeks using an externalprobe-type gamma counter (Captus®, NuclearIberica).

2.6.Histological and immunohistochemistryevaluationFive experimental groups were considered for histolo-gical and histomorphometrical evaluation:

- GroupC: control group (empty defect)

- Group B: blank (unloaded scaffolds)

- Group bMSC: scaffolds loadedwith bMSC

- Group BMP-2.5: scaffolds loaded with 2.5 μg ofBMP-2

- Group BMP-8.5: scaffolds loaded with 8.5 μg ofBMP-2

- Group bMSC-BMP-2.5: scaffolds loaded withbMSC and 2.5 μg of BMP-2

- Group bMSC-BMP-8.5: scaffolds loaded withbMSC and 8.5 μg of BMP-2.

The defect bearing femurs from three animals perexperimental group and time point (6, 12, and 24weeks) were fixed in 10% formalin solution (pH =7.4), decalcified, dehydrated, and embedded inParaplast®. Procedure was as previously described[28]. Haematoxylin-erythrosine and toluidine bluewere used for cartilage repair evaluation and scoring.The histological findings (light microscopy, LEICADM 4000B) were scored by two independent evalua-tors using the Wakitani scoring system [29, 30] with amaximum score of 18.

Ten sections of three animals per group werequantitatively analyzed for collagen II (Col II), col-lagen X (Col X), and aggrecan immunoreactivecells [28].

2.7. Statistical analysisStatistical analysis was performed with SPSS.18 soft-ware using one-way analysis of variance (ANOVA)with a Tukey multiple comparison post-test. Signifi-cance was set at p < 0.05. Results are given asmeans± SD.

Figure 1.Characteristics of the trilayer scaffold. Graphical representation of assembled system (a): (1) the cartilage-directed layerformed by amicrospheres suspension; (2) the bone-directed layer formed by amicrospheres tablet and (3) the electrospunmembranewrapping the system. Picture ofmicrospheres tablet (b). SEM images of the electrospunmembrane separately (c) and as part of thesystem in contact with themicrospheres suspension (d). Picture of thewhole system inside the cylindricalmould, ready to beimplanted (e). Fluorescence photomicrograph of calcein-stained, viable bMSC adhered to the electrospunmembrane (f). Scale bars:(b), (e) 1 mm, (c) 10 μm, (d) 40 μm, (f) 180 μm.

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3. Results

3.1. System characteristics and cell adhesion. In vivorelease profileThe mean volume diameter of the microspheres was185 μm. 80% of the particles were in the range of104–270 μm. BMP-2 encapsulation efficiency was75% ± 6%. The characteristics of the membranefabricated by electrospinning as well as the character-istics of the tablet of microspheres are detailed intable 1.

The tablet of microspheres showed a median porediameter in volume of 0.376 μm as determined bymercury intrusion. The weight of the system was 38.5± 3.6 mg.

The components of the system are showed infigure 1. Pictures of the tablet of microspheres(figure 1(b)) and SEM image of the electrospun mem-brane composed of uniform and randomly orientedfibers (figure 1(c)). SEM image of the electrospunmembrane as part of the system and picture of the sys-tem ready to be implanted are showed in figures 1(d)and (e), respectively.

Calcein AM staining images revealed that viablebMSC were on the membrane (figure 1(f)). XTT mea-sures indicated that 26.8% of the 2× 105 initially see-ded cells were adhered to the membrane after 2 hincubation.

In vivo release assay showed a moderate burstrelease during the first 24 h of approximately 17% of

Table 1.Morphological and physical systemparameters.

Parameter Electrospunmembrane Tablet ofmicrospheres

Porosity (%) (gravimetricmethod) 78.1± 5.7 22.2± 0.02

Porosity (%) (mercury porosimetry) — 22.7± 2.1

Weight (mg) 2.07± 0.32 17.6± 0.4

Diameter (fiber, nm/tablet,mm) 806± 180 3.1± 0.02

Thickness (μm)/height (mm) 145.3± 21 2.3± 0.1

Figure 2.Representative images from the control and blank groups 6 (a), (d), 12 (b), (e) and 24 (c), (f)weeks postimplantation showingthe osteochondral defect. The arrows indicate the approximate limits of the defect. AC: adjacent cartilage, DS: defect site, SB:subchondral bone, FC: fibrocartilage. FT:fibrous tissue,Ms:microspheres. Scale bars: (a)–(f) 1 mm.

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BMP-2. Then, the release rate decreased with time.After burst, BMP-2 was delivered at a release rate of5.3% per day during the first week. Then, the releaserate was 3.6%/day, 1.9%/day and 1.4%/day duringthe following 2, 3 and 4 weeks, respectively. A totalrelease of approximately 95% of the loaded BMP-2was achieved at the end of the fourth week, the totalperiod assayed.

3.2.Histological and immunohistochemistryevaluationHistological analysis was performed at 6, 12 and 24weeks postimplantation.

Control (empty defect) and blank groups showedno signs of repair throughout the experimental period(figure 2). In both groups the defect was evident at 6weeks (figures 2(a), (d)), while at 12 and 24weeks post-implantation mild signs of repair, such as fibro-cartilage, were seen (figures 2(c), (e), (f)). Fibroustissue was observed 6, 12 and 24 weeks postimplanta-tion (figures 2(d)–(f)). In some animals residues of

microspheres embedded in connective tissue weredetected (figure 2(e)). In most specimens at six weeks,in particular in the blank group, zones of intense cellactivity, ectopic cartilage formation, endochondralossification and osteosynthesis were observed in thelower region of subchondral bone.

Six weeks postimplantation (figure 3) variousdegrees of repair were observed in the treated groups.In general, all groups (figures 3(b)–(f)) showed a lowerthickness cartilage repaired and less calcified cartilagearea compared with normal cartilage (figure 3(a)). TheBMP-2.5 group induced the minor repair (figure 3(b))whereas the maximum was observed in the bMSC-BMP-8.5 group (figure 3(f)). The degree of integrationinto the adjacent cartilage was variable, the defectmar-gins being evident in some of the animals (figures 3(b),(c)). The subchondral bone showed in all experimentalgroups a high repair degree at this time point(figures 3(b)–(f)). There was a case in the BMP-2.5group where the repaired cartilage appeared inter-rupted by a cleft that crossed throughout its thickness

Figure 3.Representative images showing the osteochondral defect repair at sixweeks postimplantation. A semipanoramic view of thenormal articular cartilage (a) is included for comparisonwith the different treated groups (b)–(f). The arrows show the approximatelimits of the defect. AD: adjacent cartilage, ArC: articular cartilage, BMa: bonemarrow, cc: calcified cartilage, SB: subchondral bone,RC: repaired cartilage. Scale bar: (a)–(f) 1 mm.

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to the subchondral bone. In another case a deficientrepair process, with proliferation and ectopic cartilageformation in subchondral regionwas detected.

Twelve weeks postimplantation (figure 4) a slightincrease in the degree of repair in most experimentalgroups was observed, being more evident in the BMP-8.5 group (figure 4(c)) and in groups treated with thecombination of BMP-2 at both doses with bMSC(figures 4(e), (f)). In these groups a well-integratedrepaired cartilage and variable thickness along thedefect was observed in most of the specimens. Thegreater thickness was observed in the BMP-8.5 group(figure 4(c)) compared to the other groups(figures 4(b), (d)–(f)). In the bMSC (figure 4(d)) andBMP-2.5 groups (figure 4(b)) a thinner and poorly

integrated cartilage with clefts along it was seen. In thebMSC group, the presence of hypocellular repairedcartilage with hypertrophic chondrocyte groups in theregion closest to the subchondral bone (figures 4(g),(h)) and the presence of fibrotic cysts in the region ofthe bone underlying the defect (figure 4(d)) must behighlighted. The subchondral bone had a high level ofrepair in all treated groups (figures 4(b)–(f)).

Twenty-four weeks postimplantation (figure 5) aslightly increased level of repair over 6 (figure 3) and 12weeks (figure 4) was observed. This increase was moreevident in the BMP-2 and BMP-2 combined withbMSC (figures 5(b), (c), (e), (f)) than in the group trea-ted with bMSC (figure 5(d)). Most groups, showed arepaired cartilage slightly thinner than normal

Figure 4.Representative images showing the osteochondral defect repair at 12weeks postimplantation. A semipanoramic view of thenormal articular cartilage (a) is included for comparisonwith the different treated groups (b)–(f). The images from the bMSC group(g) and (h) showhypertrophic chondrocyte groups (arrows) in the deeper zone of the repaired cartilage. The arrows in the images (b)–(f) show the approximate limits of the defect. AD: adjacent cartilage, ArC: articular cartilage, BMa: bonemarrow, cc: calcified cartilage,FCy: fibrous cystic, SB: subchondral bone, RC: repaired cartilage, HCG: hypertrophic chondrocyte groups. Scale bars: (a)–(f) 1 mm,(g) 75 μm, (h) 60 μm.

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cartilage (figure 5(a)), except in bMSC-BMP-8.5group (figure 5(f)), where it was slightly thicker. Thedegree of integration into the adjacent cartilage wasgood in all experimental groups (figures 5(b)–(f)). Thesurface regularity of the repaired cartilage was smooth,although in the groups with bMSC and bMSC-BMP-2.5 (figures 5(d), (e)) small clefts were found in the sur-face of the repaired cartilage. In all experimentalgroups, subchondral bone showed full extent of repair(figures 5(b)–(f)).

Histomorphometrical analysis showed score valuesbetween 2.75 and 6 in the control and blank groupsthroughout the experimental period (figure 6(a)). Bycontrast, the score in the treated groups (figure 6(a))wasbetween 9.25 and 11.75 six weeks postimplantation,between 10.25 and 13.75 twelve weeks postimplantationand in the range of 12.25–14.25 twenty-four weeks post-implantation. Significant differences between the treatedgroups compared to the control and blank groups wereobserved throughout the experimental period.Nodiffer-ences were observed among the treated groups at anytime point of analysis. The scores obtained in the evalua-tion of each of the osteochondral parameters analyzedare shown infigure 6(b).

In order to corroborate the observations oncell morphology and matrix staining, number of immu-noreactive cells for different molecules of mature hyalinecartilage, Col II and aggrecanwas determined throughoutthe experimental period, showing a significantly highernumber of immunoreactive cells for both markers in thetreated groups than in the control and blank groups(figures 7(a), (b)). The expression of collagen X, hyper-trophic cartilage molecule, showed a low number ofimmunoreactive cells in the defect site in all groups,restricted to the cartilage-subchondral bone interface(figures 7(a), (b)).

4.Discussion

Given the difficulty in repairing damaged cartilagewhich lacks capacities for self-regeneration, an alter-native strategy of cell combinations, scaffolds, andbioactive agents has emerged. In this study, weprepared a three-layer system, cell-laden with bMSCand able to control the BMP-2 release. The systemgenerated new tissue using BMP-2, bMSC andthe combination of both. As the structure andcomposition of the systems play a pivotal role, several

Figure 5.Representative images showing the osteochondral defect repair at 24weeks postimplantation. A semipanoramic view of thenormal articular cartilage (a) is included for comparisonwith the different treated groups (b)–(f). The arrows show the approximatelimits of the defect. AD: adjacent cartilage, ArC: articular cartilage, BMa: bonemarrow, cc: calcified cartilage, SB: subchondral bone,RC: repaired cartilage. Scale bar: (a)–(f) 1 mm.

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types of materials (naturals and synthetics) for scaf-folds fabrication have been proposed for cartilagerepair.

In this study, the system was exclusively fabricatedwith PLGA, a FDA approved biodegradable syntheticpolymer useful for fabrication of drug delivery systems. Infact, bMSC were adhered to the PLGA electrospun filmand BMP-2 encapsulated in PLGA microspheres wasreleased at least four weeks post-cartilage-implantation.BMPs are potent cartilage-inducing factors, which in vitrohave been found capable of generating cartilage by indu-cing differentiation ofMSC into chondrocytes [17]. How-ever, to date, there are few studies in which BMP-2 hasbeenused for cartilage repair [6, 28, 31–33].Other authorsused BMP-2 for subchondral bone repair [34, 35]. How-ever, as some authors reported, the protein may diffuse

into chondral space and cause subchondral bone, extend-ing beyond the tidemark affecting the structure of the car-tilage [34]. In the present study, this effect was notobserved since BMP-2may induce chondral or bone dif-ferentiationdependingon the tissue environment.

Histological analysis of the specimens six weekspostimplantation showed the presence of repaired car-tilage, similar to those of hyaline cartilage tissue char-acteristics. Although significant differences were notobserved, the groups treated with bMSC alone or incombination with BMP-2 showed better histologicalfeatures, in terms of similarity to normal adjacent car-tilage, both in cell morphology and in the stainingcharacteristics of extracellular matrix. Likewise, thementioned groups had an acceptable degree of inte-gration compared to the groups treated with BMP-2

Figure 6. (a)Overall histological scores for the osteochondral repair process. (b)Histological scores of cellmorphology,matrixstaining, surface regularity, thickness of cartilage, integration of donor to host adjacent cartilage and thickness of subchondral bone inthe different experimental groups; the same letter over different histograms indicates significant differences between them p< 0.05,N= 4.

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Figure 7. (a)Representative images from the groups B, BMP-8.5, bMSC and bMSC-BMP-8.5 showing immunolabeling for thedifferentmarkers inside the osteochondral defect 24weeks postimplantation. The inset in the first image for Col X represents apositive control for collagenXdemonstrating immunoreactivity in the growing cartilage during endochondral ossification. cc:calcified cartilage, RC: repaired cartilage. (b)Number of immunoreactive cells for Col II, aggrecan andCol X in the differentexperimental groups 6 (6w), 12 (12w) and 24 (24w)weeks postimplantation. Histograms representmeans of immunolabelled cells perunit area (500 μm2)± SD at different time points. The same letter over different histograms indicates significant differences betweenthem p< 0.05,N= 4. Scale bar= 80 μm.

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alone. These results were confirmed by immunohisto-chemical detection of collagen type II and aggrecan inthe tissue repaired. The number of chondrocytes-likeimmunoreactive cells for both markers was slightlyhigher at 6 weeks in groups containing bMSC alone orin combinationwith BMP-2.

Analysis of 12 weeks postimplantation showedincreased histological scores in the groups treatedwith thecombination of bMSC and BMP-2 at doses of 8.5μg,comparedwith the rests of thegroups.Twenty-fourweekspostimplantation, the response observed in the treatedgroups matched with the histological values around 14,except the group of bMSC alone with score of 12. Theresults 24 weeks postimplantation clearly showed a con-solidation of the response seen at 12 weeks. In our study,the repaired cartilage observed at 12 weeks is stable with-out histological signs of hypertrophy or ossification. Simi-lar results have been recently observed using autologousbone marrow coagulates with adenoviral vectors encod-ing BMP-2 [33]. However, the endochondral ossificationreported was probably due to an excessive and continueexposure to BMP-2. By contrast, in the present study thisundesired effectwas not detected even after 24weeks. Theexperimental design of the scaffold, and the encapsulationof BMP-2, allows a controlled release over a period ofmore than four weeks [28, 31], providing repair con-centrations in the defect site. Furthermore, the stability ofthe repaired cartilagewas testedby immunohistochemicalanalysis, showing the absence of collagen type X and thepresence of mature chondrogenic markers, collagen typeII and Aggrecan. In accordance with previous studies[36, 37], the repaired cartilage with normal structure ismechanically stable. Therefore, the repaired cartilageobserved in the present study can be assumed to be fullyfunctional.

The importance of the controlled release of BMP-2 aswell as other growth factors with chondrogenic activityhas been demonstrated previously, using different scaf-folds [28, 31]. Despite bMSC groups initially producing asimilar or slightly better repair response with good histo-logical score, the response did not improve at the samerate throughout the experimental period. Contrary, bothdoses of BMP-2 showed a notable improvement of theinitial response over the experimental period. Although at12 weeks it seemed that the combination of bMSC withBMP-2 tended to increase the response, ultimately thecartilage repair observed in the treated groupswas similar,with no synergistic effect being observed with the combi-nationof both elements. These results agreewithpreviousstudies [20]. However, they do not match with thoseobserved by Kuroda et al [24] who observed a greaterresponse in the group of mMSC (muscle derived MSC)transfected to express BMP-4 compared with mMSCalone.More recently,Mifune et al [25] shows the effect ofplatelet-rich plasma, enhancing cartilage repair bymMSC-BMP-4. Despite the synergism observed by theseauthors [24, 25], the score repair measured was lowerthan thatobserved in thepresent study.

In our study, the absence of synergistic or cumulativeeffect in the groups treated with bMSC and BMP-2 atbothdoses couldbe explainedby the fact that in theosteo-chondral defects there is an endogenous contribution ofbMSC from the animal itself. Therefore, the responseobserved in the combination groups and the BMP-2groups was not different. Furthermore, this was corrobo-ratedby the lowbMSCgroup response.

The use of cell therapy in the repair of osteochon-dral lesions has been intensively studied in the lastdecade with promising results, although with con-troversy [19, 20, 37–39]. Discrepancies are mainlydue to variability in the use of cells, but also due toother factors such as types of scaffolds, experimentaldesign, cell sources, animal model, and histologicalscore systems.

5. Conclusion

There is no doubt about the efficacy of BMP-2 andcells, particularly MSC, in repairing cartilage defects.However, the combination of both strategies did notproduce the expected synergism. From our point ofview, the use of growth factors involves less variabilityand handling leading to a standarized quality productthat could be marketed. Therefore, BMP-2 might bethe best alternative in the treatment of osteochondrallesions leaving the use of cell therapies for specificcases.

Acknowledgments

This work was supported by the Ministry of Scienceand Technology (MAT2011-23819 and MAT2014-55657-R). María Rodríguez-Évora was granted withthe ‘Beca CajaCanarias para posgraduados.’ Theauthors would like to thank DrMaría Rosa Arnau, theveterinary of animal facility of La Laguna University,for assistancewith animal care.

Conflicts of interest

The authors declare no conflict of interest.

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