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CHONDROGENIC PRE-DIFFERENTIATED AND UNDIFFERENTIATED MSCs IN CARTILAGE REPAIR 1337(Invitrogen, Carlsbad, CA). The diluted BM was layered onto3 ml of Ficol Paque (Amersham Biosciences, Uppsala, Sweden)in 15 ml centrifuge tubes and centrifugated at 2,500 rpmfor 25 min. The buffy coat was extracted and washed usingDMEM low glucose supplemented with 1% penicillin/streptomycinand 1% L-glutamine by spinning at 1,600 rpm for10 min. The resulting pellet (mononuclear cells) was suspendedin growth medium DMEM-LG supplemented with10% fetal bovine serum (FBS) and cultured in 75 cm 2 tissueculture flasks (Nunc, Rockside, Denmark). Cell culturemedium was changed every 3 days and the cells were culturedand expanded until passage 3 (P3) in a humidified incubatorat 378C, 5% CO 2 . Cell viability was verified at each passageusing Trypan blue exclusion dye method prior to cell counting.All MSCs used for transplantation were at passage 3.MSCs Seeding in Alginate BeadsMSCs were harvested using TrypLE TM Express at P3 andsuspended in 1.2% sterile alginate solution (Sigma–Aldrich,St. Louis, MO) in 0.15 M sodium chloride at cells densities of1 10 6 cells/ml. The cell suspension was dropped into a102 mM calcium chloride solution using a syringe. Theresulting beads were washed with 0.15 M sodium chlorideafter 10 min of polymerization and transferred into 2 ml ofgrowth medium (DMEM supplemented with 10% FBS). MSCalginate constructs in growth medium were prepared justbefore being transplanted into the knee defects (Group 1). Toprepare CMSCs, the mesenchymal cells were cultured at P3in defined medium containing 40 mg/ml Ascorbate-2-Phosphate(Sigma–Aldrich, St. Louis, MO), 1 mM sodium pyruvate,40 mg/ml proline (Sigma–Aldrich), 0.003 M dexamethasone(Sigma–Aldrich), 1 insulin–transferrin–selenium, 10 ng/mlTGF-b3, and 10 ng/ml BFGF. The cells were maintained incell culture incubator at 378C and 5% CO 2 for 3 weeks(Group 2). Characterization of MSCs and CMSCs wereassessed as described in previous studies. 16 CMSCs used fortransplantations were at passage 3.Creation of Cartilage DefectTwelve (n ¼ 12) rabbits were equally divided into twogroups. Each animal underwent surgery to create fullthickness cartilage defects on the articular surface of theweight-bearing medial femoral condyle of both knees (hindlegs). The defects were created using a custom-made cylindricalchondrotome (Fig. 1A), and standardized according to thefollowing dimensions: 5 mm diameter and approximately1 mm depth (or until cartilage tissue were completelyremoved, as observed under 5 magnification). Care wastaken to not violate the subchondral bone. Total removal ofcartilage tissue was confirmed using observations madeunder the aid of a microsurgical microscope.Transplantation of Cell Alginate ConstructsTransplantations of the MSC and CMSC constructs into thedefective areas (i.e., right knee in each respective group)were conducted approximately 3–4 weeks following theinitial defect creation procedure. The transplanted cells wereobtained only from rabbits of the same litter. The left kneewas left untouched until the end of the experiment,representing the control group of the study. A periosteal flapwas used to hold the alginate beads at the defect area usingmicrosurgical sutures (Nylon 9-0) and fibrin glue.Post-operatively, antibiotics and analgesia were administeredfor 3 consecutive days. No wound infection wasFigure 1. Gross morphology. (A) Defect was created in the medial chondyle of the knee. (B) Right knee (treatment) 3 months afterCMSCs implantation. (C) Left knee (control) defect with an irregular surface observed at 3 months. (D) Right and left knees of CMSCstreated rabbits 6 months after implantation. In the right knee, repair tissue appears smoother and homogenous than the left.JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2011
1338 DASHTDAR ET AL.observed in any of the rabbits. The rabbits moved freelywithin their cages and were sacrificed at 3 and 6 monthsafter transplantation.Morphological AnalysisThe quality of cartilage tissues in both knees were comparedby two independent observer, in accordance to the Brittberg/International Cartilage Research Society (ICRS) morphologicalscore and general histological evaluation. 17,18Histology and Immunohistology ApplicationThe distal femur was cut and the medial condyle was dividedinto two equal parts using a saw. Half of the sample used forhistology. These samples were fixed in 10% buffered formalinfor 48–72 h, and decalcified in 10% formic acid. The specimenswere processed for histological slides, embedded in paraffin,and cut into 5 mm sections. The slides were stained usinghematoxylin and eosin (H&E), Safranin O Fast green andimmunohistochemical staining for collagen type II. Immunohistochemistryfor collagen type II was performed usingprimary antibody anticollagen type II (mouse-anti-rabbit;Merck, Darmstadt, Germany) in 1:100 dilution and accordingto the company protocol (DakoCytomation, Glostrup, Denmark).The histology images were analyzed using O’Driscollhistological scoring as previously described. 19Biochemical AnalysisProtein and glycosaminoglycan (GAGs) were determinedusing Bio-Rad DC protein assay kit (Bio-Rad Laboratories,Richmond, CA) and Blyscan sulfated Glycosaminoglycanassay kit (Biocolor Ltd., Country Antrim, UK) according topreviously established methods 20 or based on the manufacturer’sprotocols. Spectrophotometer absorbance measurementswere performed at 750 and 656 nm for protein andGAG assays, respectively. GAG content was normalizedaccording to the protein contents (mg GAG/mg protein).Statistical AnalysisGrading scores obtained from the Brittberg, O’Driscollmeasures and GAGs contents were statistically analyzedusing Wilcoxon signed-rank test available on the statisticalsoftware package SPSS (version 17.0). To compare thesignificant differences of values between the two experimentgroups and, between treatment and control groups, Mann–Whitney tests were conducted accordingly.RESULTSMorphological ObservationThe gross morphological appearances of the regeneratedcartilage tissues filling in the defects (Fig. 1A) at3 months post-transplantation are shown (Fig. 1B andC). The Brittberg’s scores for the MSC and CMSCgroups were significantly higher than their respectivecontrols (8.17 1.83 vs. 7.00 2.28 for MSC andCMSC, respectively; p < 0.05). There were significantdifferences in the Brittberg Score of transplantedknees in both groups treated with MSCs at 6 months(7.50 1.38 points) to that of the untreated knees(4.50 1.38 points). CMSC-treated knees demonstratedbetter result as compared to MSCs group(9.00 2.00 vs. 7.33 1.53). However, these differenceswere not statistically significant (Table 1).Figure 1D illustrates the gross appearance of theregenerated tissue between the MSC-treated and theuntreated defect at 6 months post-transplantation. Inthe MSC-treated knee, the regenerated cartilagetissues exhibited smooth glistening surface with lessdistinct demarcation of the defect border peripherally.In contrast, the regenerated tissues in the untreatedknee had rough surface and very distinct woundmargins.Histological and Histochemical AnalysisO’Driscoll histological scores of the post-implantationrepair tissue was significantly (p < 0.05) higher in thetransplanted sites when compared to the control sitesat 3 months (10.5 1.38 vs. 7.67 0.82) and 6 monthsTable 1. Summary of the Results Comparing the Treated (Right Knee) and Untreated (Left Knee) Groups , andComparing the MSC- and CMSC-Treated GroupsLeft Knee(Untreated)Knee Sidep-Value Cell Type Used (Comparing Right Knee)(WilcoxonSigned-RankTest)ComparingRight Knee(Treated)Left–RightKneesMSC CMSCp-Value(Mann–WhitneyU Signed-RankTest) ComparingMSC- andCMSC-TreatedKnees3 monthsBrittberg 7.00 2.28 8.17 1.83 0.336 7.33 1.53 9.00 2.00 0.317O’Driscoll 7.67 0.82 10.5 1.38 0.042 10.0 1.00 11.0 1.73 1.000GAG/protein 0.82 0.34 1.23 0.84 0.600 0.77 0.09 0.82 0.18 0.2756 monthsBrittberg 4.50 1.38 7.50 1.38 0.024 7.67 1.00 7.33 1.73 1.000O’Driscoll 8.67 1.86 11.8 1.47 0.026 11.6 2.08 12.0 1.00 0.655GAG/protein 0.82 0.10 1.36 0.16 0.046 1.67 0.14 1.29 0.38 0.052Data presented are mean values 1 SD. Statistical significant values (p < 0.05).JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2011
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