ARTHRITIS

ORTHOPEDICS August 2011;34(8):382.

Age-related Biological Characterization of Mesenchymal Progenitor Cells in Human Articular Cartilage

by Hong-Xing Chang, MD; Liu Yang, MD, PhD; Zhong Li, MD, PhD; Guangxing Chen, MD, PhD; Gang Dai, MD, PhD

DOI: 10.3928/01477447-20110627-06

Abstract

Adult articular cartilage has a low regeneration capacity due to lack of viable progenitor cells caused by limited blood supply to cartilage. However, recent studies have demonstrated the existence of chondroprogenitor cells in articular cartilage. A critical question is whether these mesenchymal progenitor cells are functionally viable for tissue renewal and cartilage repair to postpone cartilage degeneration.

This study was designed to compare the number and function of mesenchymal progenitor cells in articular cartilage collected from human fetuses, healthy adults (aged 28–45 years), and elderly adults (aged 60–75 years) and cultured in vitro. We detected multipotent mesenchymal progenitor cells, defined as CD105+/CD166+ cells, in human articular cartilage of all ages. However, mesenchymal progenitor cells accounted for 94.69%±2.31%, 4.85%±2.62%, and 6.33%±3.05% of cells in articular cartilage obtained from fetuses, adults, and elderly patients, respectively ( P<.001). Furthermore, fetal mesenchymal progenitor cells had the highest rates of proliferation measured by cell doubling times and chondrogenic differentiation as compared to those from adult and elderly patients. In contrast, alkaline phosphatase levels, which are indicative of osteogenic differentiation, did not show significant reduction with aging. However, spontaneous osteogenic differentiation was detected only in mesenchymal progenitor cells from elderly patients (with lower Markin scales). The lower chondrogenic and spontaneous osteogenic differentiation of mesenchymal progenitor cells derived from elderly patients may be associated with the development of primary osteoarthritis. These results suggest that measuring cartilage mesenchymal progenitor cells may not only identify underlying mechanisms but also offer new diagnostic and therapeutic potential for patients with osteoarthritis.

Drs Chang, Yang, Li, Chen, and Dai are from the Department of Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing, and Dr Chang is also from the Department of Orthopedics, Beijing Army General Hospital of PLA, Beijing, China.

Drs Chang, Yang, Li, Chen, and Dai have no relevant financial relationships to disclose. This study was supported by The National Natural Science Foundation of China (no. 30901576 and 30672200).

Correspondence should be addressed to: Gang Dai, MD, PhD, Department of Joint Surgery, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China (daigang60@163.com).

Posted Online: August 08, 2011

Osteoarthritis results from an imbalance between damage to and repair of articular cartilage. Chondrocytes are thought to be terminal cells with limited capacity for proliferation, primarily because of poor blood circulation and a limited number of available stem cells. ¹ The articular cartilage is, therefore, often considered to be at high risk for age-related diseases. ^2,3 However, several recent studies have determined that chondrocytes from healthy primary osteoarthritis patients express markers specific for stem cells. ^4,5 These cells are called mesenchymal progenitor cells. Why, then, can’t the existing mesenchymal progenitor cells prevent or postpone cartilage degeneration? Previous studies have shown that aging stem cells from the bone marrow reduce the rate of generation and have a lower capacity of proliferation and differentiation. ^6,7 However, no report exists on the correlation between age and the rate of proliferation of cartilage mesenchymal progenitor cells.

We hypothesized that the ability of cartilage mesenchymal progenitor cells to proliferate and differentiate declines with age, leading to age-related decrease in chondrocyte numbers and function. This age-related decline in chondrocyte number and function directly contributes to the development of primary osteoarthritis. This study compared the in vitro functions of mesenchymal progenitor cells, defined as CD166+/CD105+ cells, isolated from cartilage and surgically removed from patients of different ages. The results may offer new insights into the pathogenesis of osteoarthritis and lead to the development of new preventive and therapeutic agents to improve the quality of life for patients with osteoarthritis.

Materials and Methods

Collection of Articular Cartilage

Fetal cartilage samples were obtained from fetuses aged 20 to 24 weeks who had died of congenital heart abnormalities. Samples of knee joint cartilage were taken from adult and elderly patients who had either died of other diseases or had undergone limb amputation. All cartilage was without visible joint disease (Table 1). Articular cartilage was dissected from the femoral condyle with perichondrium and subchondral bone excised and washed 3 times with phosphate buffered saline to remove blood and soft tissue on the surface. All samples were graded according to a modified Mankin scale (0–2). ⁸ Despite our efforts in choosing cartilage with minimal evidence of degeneration, cells collected for the study may have had age-associated degenerative changes in cartilage, which may have impacted the study. Samples were obtained after an informed consent form was signed by the patients and/or guardians. The study was approved by the hospital ethics committee.

: Cartilage Resource and Modified Mankin Scale8

Culture of Cartilage Cells

Articular cartilage cells were isolated as previously described. ⁹ Briefly, an articular cartilage was cut into 1-mm ³ pieces and incubated with phosphate buffered saline containing 0.1% trypsin, 0.1% hyaluronidase, and 0.2% type II collagenase at 37°C for 1 hour and 3 hours, respectively. Chondrocytes were harvested by centrifugation at 1000 rpm for 5 minutes and seeded into a cell culture flask at a plating density of 3×104 cells/cm ² in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) medium with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cell culture was maintained at 37°C with air that contained 5% (v/v) CO ₂. The medium was changed 72 hours after initial seeding, and chondrocytes continued to be cultured until 80% confluent. Cells were then detached with trypsin (0.25%) and reseeded at control densities for functional assays.

Detection of CD105+/CD166+ Cells in Chondrocytes From Articular Cartilage

Cells from the primary culture and after the second and fourth passages were detached with trypsin and resuspended to a final concentration of 105 cells/50 µl in phosphate buffered saline supplemented with 1% bovine serum albumin and incubated with phycoerythrin-conjugated anti-CD105 (monoclonal antibody SN6) and fluorescein isothiocyanate (FITC)-conjugated anti-CD166 antibodies for 45 minutes at 4°C. Cells stained with a mouse isotype IgG were used as negative control. After antibody binding, cells were washed 3 times with phosphate buffered saline containing 1% bovine serum albumin to remove unbound antibodies and fixed in 4% paraformaldehyde in phosphate buffered saline for 15 minutes at room temperature. They were subjected to fluorescence-activated cell sorting using a FACScan and analyzed with the CellQuest program (Becton Dickinson, San Jose, California).

For flow cytometry analysis, cells were first gated on the forward and side scatters to exclude debris and cell aggregates and then detected for specific antibody binding. The percentage of dual-positive cells was calculated after subtracting non-specific binding of mouse isotype IgG as described previously. ⁴

Isolation of Articular Cartilage Mesenchymal Progenitor Cells

An Anti-FITC MultiSort Kit and a miniMACS separation system were used to purify mesenchymal progenitor cells according to instructions from the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, chondrocytes from the fourth passage in culture were harvested and suspended in phosphate buffered saline. They were incubated with an FITC-CD166 antibody (1:50 dilution) for 45 minutes in the dark at 4°C. The stained cells were washed twice and then resuspended in 80 µl of 1× phosphate buffered saline containing 1% bovine serum albumin and 0.1% NaN3. They were then incubated with 10 µl of an anti-FITC antibody coupled to magnetic beads for 30 minutes at 4°C. After washing, CD166+ cells were selected with a magnetic apparatus (Miltenyi Biotec). The selected CD166+ cells were further selected with CD105 micromagnetic beads (Miltenyi Biotec) to eventually obtain cells that are positive for CD166 and CD105. The sorted cells were cultured at a density of 5×104 cells/cm ².

Laser Scanning Confocal Fluorescence Microscopy

Magnetic bead-purified mesenchymal progenitor cells were either prepared as smears for histological examination or seeded on a 10×10-mm cover glass to be cultured in DMEM/F-12 culture medium for 3 days. Cells prepared in both techniques were fixed in 4% polyoxymethylene (30 minutes at room temperature). The fixed cells were first blocked with a buffer containing 5% fetal bovine serum and 2% bovine serum albumin for 30 minutes and then stained for phycoerythrin-conjugated CD105 (1:50 dilution) and FITC-conjugated CD166 (1:50 dilution) antibodies (phycoerythrin or FITC mouse IgG as controls) for 30 to 45 minutes at room temperature. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to mark the nucleus. Fluorescently labeled cells were visualized and imaged under a confocal laser scanning microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany).

Cell Doubling Time

Cell doubling time (Td) was used to calculate the ability to proliferate. Mesenchymal progenitor cells in culture (second passage) were detached with trypsin (0.25%), suspended in DMEM/F-12 medium containing 10% fetal bovine serum, and plated into a 96-well plate (2×103 cells/well). Cells in 3 wells from each age group were processed in a standard MTT procedure ¹⁰ and examined every 24 hours (optical density, 570 nm). A growth curve was plotted from these optical density values to calculate mesenchymal progenitor cells-cell doubling time in each age group based on the following formula: Td=t*lg2/(lgNt-lgN0), where t is time in culture (hours), N0 is the seeding density of cells, and Nt is the cell density after t hours in culture.

Detection of Articular Cartilage Mesenchymal Progenitor Cells Differentiation

Chondrogenic Induction. Mesenchymal progenitor cells were cultured with chondrogenic medium (DMEM/F-12 supplemented with 1% ITS Liquid Media Supplement (Sigma-Aldrich Co, St Louis, Missouri), 1 mM sodium pyruvate, 37.5 g/mL ascorbate 2-phosphate, 10-8 M dexamethasone, and 10 ng/mL recombinant human transforming growth factor-ß1) for 2 weeks. ⁴ Control cells were cultured in DMEM/F-12 containing 10% fetal bovine serum.

After 2 weeks in culture, total RNA was isolated using TRIzol reagent (Tiangen Biotech Co Ltd, Beijing, China) and used for semiquantitative reverse transcription-polymerase chain reaction to detect the expression of type II collagen and aggrecan gene. Reverse transcription was first performed with 1 µg of total RNA from each sample using oligo(dT)18 primer and 200 units of SuperScript II RT (Life Technologies Inc, Gaithersburg, Maryland) for cDNA synthesis. DNA (in triplicate) was then amplified in 20 µL solution that contained 2 µl diluted template, 10 pmol primer pairs for type II collagen, and aggrecan and control glyceraldehyde 3-phosphate dehydrogenase (Table 2), respectively, and 10 µl Taq PCR Master Mix (TianGen Biotech Co Ltd). The amplification was induced first at 94°C for 5 minutes, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 57°C, and 30 seconds at 72°C. The reaction was completed by a final incubation at 72°C for 10 minutes. Gene expression was expressed as 2- ^??(Ct), where Ct is the cycle threshold, ?(Ct) is the Ct of the tested gene–Ct of glyceraldehyde 3-phosphate dehydrogenase, and ??(Ct) is the ?(Ct) of sample 1–?(Ct) of sample 2. ¹¹

: Primers Used for Real-time Polymerase Chain Reaction

Osteogenic Induction. Mesenchymal progenitor cells (1×104/cm ²) were first cultured in DMEM/F-12 medium for 24 hours and then switched to the osteogenic medium (50 µmol/L ascorbic acid, 10 µM ß-sodium glycerophosphate, and 0.1 µM dexamethasone) ¹² for 14 days. Control cells were cultured in DMEM/F-12 containing 10% fetal bovine serum. Intracellular alkaline phosphatase was measured with a commercial kit (LabAssay; Wako Pure Chemical Industries, Ltd, Osaka, Japan) at 405 nm and calculated as instructed by the manufacturer.

Statistical Analysis

All values are presented as mean±standard error of mean from repeated experiments. The quantitative data were analyzed using SPSS 13.0 software (SPSS, Inc, Chicago, Illinois). The Kruskal-Wallis test (non-parametric) was used to compare among multiple groups. The independent samples t test was used to assess difference between 2 groups of variables. A P value <.05 was considered statistically significant.

Results

CD105+/CD166+ Cells in Articular Cartilage

The percentage distributions of CD105+/CD166+, CD34+, and CD45+ cells purified from articular cartilage were similar to mesenchymal progenitor cells from the bone marrow (Figure 1), consistent with a previous report on mesenchymal progenitor cells. ⁴ In primary culture, articular cartilage from fetuses had the highest percentage of CD166+/CD105+ cells as compared to those from adult and elderly patients ( P<.001). There was no statistical difference in the counts of CD166+/CD105+ cells between articular cartilage obtained from adult and elderly patients. However, the percent of CD166+/CD105+ cells from adult and elderly patients significantly increased in culture, whereas those from fetal tissue (which counted for >90%) did not show significant changes (Table 3; Figure 2).

CD166-fluorescein isothiocyanate/CD105-phycoerythrin positive cells after immunomagnetic purification was identified by fluorescence-activated cell analysis (A). The same technology was also used to detect positivity for CD34 (0.50%) and CD45 (0.69%) (B). Laser scanning confocal fluorescence microscope detected mesenchymal progenitor cells after labeling with phycoerythrin-CD105 and fluorescein isothiocyanate-CD166 antibodies. DAPI (4',6-diamidino-2-phenylindole) was used to highlight the nucleus. Geometrical mean fluorescence was used to measure the antibody binding in fluorescence intently (X axis). Representative of 5 independent experiments (C).

: CD105+/CD166+ Mesenchymal Progenitor Cells in Articular Cartilage in Different Age Groups

Fluorescence-activated cell-sorting analysis of chondrocytes was presented on forward/side scatter plot for primary culture (A) and after 4 passages (B). The scatter plot was set on a linear scale to indicate particle size and was used to exclude cellular debris and aggregates. The CD166-fluorescein isothiocyanate/CD105-phycoerythrin staining was detected in the primary culture (C) and after 4 passages (D) in geometrical mean fluorescence.

Growth and Proliferation of Mesenchymal Progenitor Cells

The mesenchymal progenitor cells from fetal articular cartilage were short, spindle-shaped cells that grew rapidly, whereas most of the mesenchymal progenitor cells from articular cartilage of adult and elderly patients were longer spindle-shaped cells (Figure 3). The proliferation capacity was lower for mesenchymal progenitor cells from adult and elderly patients as compared to those from fetal samples, with the cell doubling times of 25.68±7.71 hours, 45.35±15.41 hours, and 55.69±16.52 hours for fetal-, adult-, and elderly-derived mesenchymal progenitor cells, respectively (Figure 4).

Morphological characterization of chondrocytes (A–C) and CD166+/CD105+ mesenchymal progenitor cells (D–F) from fetal, adult, and elderly patients, respectively, after 2 passages in culture.

Cell growth (1 passage in culture) was measured for mesenchymal progenitor cells from different age groups, indicating a higher grown rate for fetal mesenchymal progenitor cells (A). Cell doubling time was calculated for mesenchymal progenitor cells from different groups of patients, showing a lowest double time (Kruskal-Wallis test, P=.001) (B).

Chondrogenic Differentiation

mRNA for aggrecan and type II collagen were the highest in chondrogenic-induced fetal mesenchymal progenitor cells as compared to those from adult and elderly patients ( P<.05) (Figure 5). These results indicate that CD105+/CD166+ mesenchymal progenitor cells in the resting state differentiated into chondrocytes and upregulated the expression of aggrecan and type II collagen mRNA in the presence of growth factors.

mRNA for aggrecan and type II collagen was detected by real-time polymerase chain reaction with the unit of Y-axis as the relative expression level measured in quantitative polymerase chain reaction. The expression levels of aggrecan and type II collagen mRNA in fetal (group A), adult (group B), and elderly (group C) mesenchymal progenitor cells were 7.79 and 9.34 times the control, 5.82 and 7.02 times the control, and 2.24 and 2.93 times the control, respectively. The chondrogenic potential declines with age ( P<.01), and chondrogenic differentiation was also significantly reduced in aged cartilages.

Osteogenic Differentiation

Levels of alkaline phosphatase in mesenchymal progenitor cells from all age groups increased after osteogenic induction, indicating that cartilage mesenchymal progenitor cells from all ages maintained a normal osteogenic differentiation capacity. However, a 2-week induction resulted in a higher level of alkaline phosphatase in mesenchymal progenitor cells from adult patients as compared to those from fetal and elderly patients, but the difference did not reach statistical significance (Figure 6). Without induction, alkaline phosphatase of mesenchymal progenitor cells from elderly patients was significantly higher than that of the other 2 groups ( P=.007), suggesting that mesenchymal progenitor cells from elderly patients’ cartilage might have undergone spontaneous osteogenic differentiation.

The alkaline phosphatase (ALP) levels after osteogenic induction of mesenchymal progenitor cells show no significant difference among the 3 groups (P=.215), but show a significant difference among different patients without induction (controls).

Discussion

Chondrocytes are thought to be terminal cells with low capacity for reproduction or self-renewal. Blood circulation in articular cartilage is also poor, potentially resulting in limited supply of progenitor cells critical for tissue renewal. ^2,3 These factors contribute in part to a high risk for age-related disease in articular cartilage. However, recent studies have found that osteoarthritis chondrocyte express stem cell markers. ^4,5 A key question is whether these mesenchymal progenitor cells undergo functional changes that result in progressively reduced capacity for self-renewal and differentiation. We have provided experimental evidence that human cartilage of all ages contained CD105+/CD166+ mesenchymal progenitor cells, ^13,14 which are known to differentiate into mature cells of chondrogenic as well as adipogenic and osteogenic lineages. ^4,15 As expected, mesenchymal progenitor cells were found to have a higher quantity in fetal cartilage, consistent with a previous report. ⁴ Interestingly, mesenchymal progenitor cells in cartilage from elderly patients were similar in quantity to those from adult patients, indicating a minimal decline in mesenchymal progenitor cells between the 2 age groups.

Despite their universal presence, we found that fetal mesenchymal progenitor cells had a higher rate of proliferation and chondrogenic capacity as compared to aged cartilage (Figures 4, 5), similar to those found in bone marrow mesenchymal progenitor cells. ^6,16 This low rate of proliferation and slow induction is consistent with finding that aged cartilage had a lower percentage of mesenchymal progenitor cells that had a reduced chondrogenic capacity, as shown in Figure 5. A role of aging in mesenchymal stem cell differentiation has been debated. ^7,17 Our observations strongly suggest a mechanism that supports for age-related development of cartilage degeneration and osteoarthritis.

Cell growth (1 passage in culture) was measured for mesenchymal progenitor cells from different age groups, indicating a higher grown rate for fetal mesenchymal progenitor cells (A). Cell doubling time was calculated for mesenchymal progenitor cells from different groups of patients, showing a lowest double time (Kruskal-Wallis test, P=.001) (B).

mRNA for aggrecan and type II collagen was detected by real-time polymerase chain reaction with the unit of Y-axis as the relative expression level measured in quantitative polymerase chain reaction. The expression levels of aggrecan and type II collagen mRNA in fetal (group A), adult (group B), and elderly (group C) mesenchymal progenitor cells were 7.79 and 9.34 times the control, 5.82 and 7.02 times the control, and 2.24 and 2.93 times the control, respectively. The chondrogenic potential declines with age ( P<.01), and chondrogenic differentiation was also significantly reduced in aged cartilages.

Furthermore, spontaneous osteogenic differentiation in mesenchymal progenitor cells derived from elderly patients may be related to primary osteoarthritis. For example, mesenchymal progenitor cells cultured in a transforming growth factor-ß-containing chondrogenic medium display signs consistent with chondrocyte hypertrophy. ¹⁸ Osteoarthritis chondrocytes in culture show significant hypertrophy after transforming growth factor-ß induction, ¹⁹ which precedes cartilage apoptosis, vessel invasion, and calcification during cartilage development. Xiao et al ²⁰ compared the gene-expression pattern in the bone marrow-mesenchymal stem cells of geriatric (>2 years), osteoporotic and nonosteoporotic adult (7 months), and juvenile (7 weeks) rats and detected the highest high expression of osteoblast-related genes in geriatric rats. Together, these findings imply that mesenchymal progenitor cells from elderly animals could promote the development of osteoarthritis by differentiating into bone cells.

Although we have observed significant differences in mesenchymal progenitor cell function in patients from different age groups, the sample size for this study is too small to establish a linear relationship between age and mesenchymal progenitor cell functionalities, due to difficulties obtaining cartilage tissue. It may be more informative to study cartilage mesenchymal progenitor cells at epiphyses fusion age, when cartilage evolves from the growth phase to the stationary phase. This concern is partially addressed by examining fetal cartilage, which represents the growth phase, as well as adult and elderly cartilage, which represent the stationary and degenerative phases. Nevertheless, more studies on the subject are called for, including larger randomized controlled trials to study how mesenchymal progenitor cells associate with osteoarthritis development and to explore the potential of using mesenchymal progenitor cells as a therapeutic alternative to the standard care of patients with osteoarthritis.

For further information: http://www.orthosupersite.com/view.aspx?rid=86265

FOOT AND ANKLE

ORTHOPEDICS August 2011;34(8):356.

Comparison of MRI and Arthroscopy in Modified MOCART Scoring System After Autologous Chondrocyte Implantation for Osteochondral Lesion of the Talus

by Kyung Tai Lee, MD; Yun Sun Choi, MD; Young Koo Lee, MD; Seung Do Cha, MD; Hyung Mo Koo, MD

DOI: 10.3928/01477447-20110627-10

Abstract

Magnetic resonance imaging (MRI) and arthroscopy have frequently been used to evaluate articular cartilage. Many studies have compared the accuracy of MRI to that of arthroscopy. However, there have been no previous comparison studies between MRI and arthroscopy in the evaluation of repaired cartilage after autologous chondrocyte implantation using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring system. The purpose of this study was to compare the results between MRI and arthroscopy after autologous chondrocyte implantation of an osteochondral lesion of the talus using a modified MOCART scoring system.

Our study investigated 27 consecutive cases in 26 patients who underwent follow-up MRI and second-look arthroscopy 1 year following autologous chondrocyte implantation based on their osteochondral lesion of the talus diagnosis. According to the comparison results of those 5 categories, the agreement between MRI and arthroscopy evaluation results was statistically significant with good reliability in the categories of the degree of defect repair and defect filling, the quality of repaired tissue surface, and synovitis. However, the integration with the border zone and the adhesion category showed poor to moderate reliability. There has been no well-established correlation method between arthroscopy and MRI after autologous chondrocyte implantation of an osteochondral lesion of the talus.

Dr Lee (Kyung Tai) is from the Foot and Ankle Clinic, KT Lee’s Orthopedic Hospital, Dr Choi is from the Department of Radiology, Eulji Hospital, Eulji University School of Medicine, Seoul, Drs Lee (Young Koo) and Koo are from the Department of Orthopedic Surgery, Soonchunhyang University, Bucheon Hospital, Gyeonggi-Do, and Dr Cha is from the Department of Orthopedic Surgery, Kwandong University Hwajung dong, Dukyang-Gu, Koyang-Si, Gyeonggi-Do, Republic of Korea.

Drs Lee (Kyung Tai), Choi, Lee (Young Koo), Cha, and Koo have no relevant financial relationships to disclose.

Correspondence should be addressed to: Young Koo Lee, MD, Department of Orthopedic Surgery, Soonchunhyang University 4 Jung-Dong, Wonmi-Gu, Bucheon-Si, Gyeonggi-Do, 420-767, Republic of Korea (brain0808@hanmail.net).

Posted Online: August 08, 2011

Magnetic resonance imaging (MRI) and arthroscopy frequently have been used to evaluate articular cartilage. ^1,2 Many studies have compared the accuracy of MRI to that of arthroscopy. With regard to the knee joint, O’Connor et al ³ reported the accuracy of MRI compared to arthroscopy for osteochondritis dissecans was 85%. For the ankle joint, Mintz et al ² reported the accuracy of MRI of osteochondral lesions of the talus was 83% and Lee et al ¹ reported an accuracy of 81%. However, none of the prior studies were based on the evaluation results of repaired cartilage after autologous chondrocyte implantation of an osteochondral lesion of the talus.

In addition, there have been no previous comparison studies between MRI and arthroscopy in the evaluation of repaired cartilage after autologous chondrocyte implantation using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring system. The MOCART scoring system is an evaluation method for repaired cartilage with low interobserver variability. ⁴ It is a useful method for long-term follow-up as it is based on a scoring system. ⁵ However, previous MOCART-based studies have dealt only with the knee joint; none of the MOCART studies have examined the ankle joint. The purpose of this study was to compare the results between MRI and arthroscopy after autologous chondrocyte implantation of an osteochondral lesion of the talus using a modified MOCART scoring system.

Materials and Methods

This study was performed from September 2005 to March 2008 and included 27 cases in 26 consecutive patients who received a follow-up MRI and second-look arthroscopy 1 year after autologous chondrocyte implantation caused by an osteochondral lesion of the talus. Average patient age was 33.9 years (range, 16–56 years), and the study population comprised 19 men and 7 women. A total of 13 right and 14 left ankles were examined. The study was approved by our Institutional Research Board, and all study participants provided informed consent prior to study enrollment.

All patients were diagnosed based on MRI and physical examinations, and the diagnosis was confirmed during surgery using arthroscopy. Approximately 1 year following their initial surgery, patients underwent MRI and second-look arthroscopy. A modified MOCART scoring system was used to classify MRI and arthroscopy evaluation of the osteochondral lesion of the talus. Five categories of the MOCART scoring system that could be applied to both MRI and arthroscopy were used with some modification to compare the results of the 1-year follow-up MRI and second-look arthroscopy (Table 1).

Modified MOCART Scoring System for the Evaluation of Autologous Chondrocyte Implantation

Surgical Technique

During the first surgical stage, a cartilage biopsy was performed after a local anesthesia block was administered to the foot and ankle. Autologous chondrocyte implantation, the second-stage surgery, was performed under spinal anesthesia, with patients placed in a frog-leg position. An incision was made along the midline of the medial malleolous, which exposed approximately 10 cm of the posterior tibial tendon and anterior ankle joint. Oblique medial malleolar osteotomy was performed under fluoroscopy.

The osteochondral lesion of the talus then was exposed by retracting the distal malleolar fragment. The lesion was removed, and a 2.5-mm drill was used to prevent delamination of the gel matrix. The area was injected with Chondron (Sewoncellontech Corp, Seoul, Korea), which hardened within 5 minutes given its properties to convert the fibrin-mixed chondrocyte liquid mixture into the gel matrix within the lesion. Through a pre-drilled hole, the osteotomy site was fixed using a 4.5-mm diameter cannulated screw and a K-wire without a rotational deformation. The ankle was immobilized at 90° using a plaster splint, and nonweight-bearing measures were initiated.

The third-stage surgery was performed under spinal anesthesia. Patients were placed in a kneeling position to retract the ankle using an 8-lb weight to check the joint condition through the anteromedial and anterolateral portals. Patients then were placed in a supine position with their legs on the table, and the previous incision scar was incised to remove the screw.

Imaging Technique

Magnetic resonance imaging was performed 1 year postoperatively using a 1.5-Tesla unit (Twinspeed; General Electric Health Care, Milwaukee, Wisconsin) and an extremity coil. Sagittal inversion recovery images were obtained with a 16-cm field of view, repetition time of 5000 milliseconds, echo time of 16 ms, inversion time of 150 milliseconds, echo train length 8, and slice thickness of 3.5 mm with no interslice gap. Sagittal, axial, and coronal intermediate-weighted fast-spin echo images were obtained with a repetition time of 4000 milliseconds, effective echo time of 25 to 26 milliseconds, echo train length 8, slice thickness of 3 mm with no inter-slice gap, and field view of 11 to 15 cm. A matrix of 512×256 was obtained with a number of excitations of 1 to 2. Coronal fat-suppressed 3-dimensional spoiled gradient-recalled images were added with a 12-cm field of view, repetition time of 40 milliseconds, echo time of 6 milliseconds, flip angle of 40°, slice thickness of 1.5 mm with no interslice gap, and matrix of 256×192. An experienced musculoskeletal radiologist assessed all MRIs and was unaware of the second-look arthroscopic findings.

Statistical Analysis

Statistical analysis was performed using the modified MOCART scoring system under the hypothesis that there is no difference between MRI and arthroscopy and under the alternative hypothesis that there is a difference between MRI and arthroscopy, to determine any difference between MRI and arthroscopy. To assess agreement and reliability, the intraclass correlation coefficients (ICC) were obtained from random effects 1-way analysis of variance. Intraclass correlation coefficient values close to zero indicated no interrater reliability, and ICC values close to 1 indicated perfect reliability. All analyses were performed using SPSS version 12.0 (SPSS Institute, Chicago, Illinois), and all significance tests were 2-tailed. For all tests, a P value of <.05 was considered statistically significant.

Results

Among the 5 categories of the MO-CART scoring system that are applicable to both second-look arthroscopy and MRI, the degree of defect repair and filling of the defect category (Table 2) showed congruent results in 16 of 27 cases. Of the 14 cases in which arthroscopy showed complete manifestations, MRI showed complete manifestations in 12 cases (Figure 1) and >50% of the adjacent cartilage filling manifestations in 2 cases. However, in 5 cases in which arthroscopy showed <50% of the adjacent cartilage filling, MRI showed >50%, indicating complete disagreement. Therefore, the degree of defect repair and filling of the defect category showed a relatively high correlation, resulting in an ICC value of 0.7222 ( P<.0001).

Degree of Defect Repair and Filling Category

Coronal (A) and sagittal (B) intermediate-weighted fast-spin echo MRIs of a 16-year-old girl 1 year after autologous chondrocyte implantation of the medial talar dome show complete filling of the defect of the osteochondral lesion (white arrows) and complete integration. The repaired tissue surface is relatively smooth and synovitis (black arrows) is seen. Arthroscopic photograph shows complete defect filling with a smooth surface over the talus. In this case, MRI demonstrates a good correlation with arthroscopy (C).

In the integration into the border zone category (Table 3), arthroscopy showed complete manifestations in 18 cases; MRI results were in agreement in 17 cases, with 1 case displaying a demarcating border. Nevertheless, of the 5 cases in which arthroscopy showed visible integration defects, MRI displayed incongruent results in 3 cases (Figure 2). Therefore, in this category, there was a tendency to disagree, with an ICC value of 0.48 ( P=.0034).

Integration Into the Border Zone

Sagittal (A) and coronal (B) intermediate-weighted fast-spin echo and coronal fat-suppressed 3D-spoiled gradient-recalled MRIs (C) of a 39-year-old woman show complete filling of the defect (arrows) of the osteochondral lesion of talus and complete integration. The repaired tissue shows damaged surface with fibrillation. Arthroscopic photograph shows complete filling of the defect with fibrillation of the surface but incomplete integration (arrow), which reveals disagreement with the MRI findings (D).

For the surface of the repaired tissue category (Table 4), the results were in agreement in 24 of 27 cases (Figure 3), with an ICC value of 0.8523 ( P<.0001). In the adhesion category (Table 5), MRI failed to detect any adhesions, whereas arthroscopy showed adhesion in 2 cases. Therefore, there was complete disagreement in the adhesion category, with an ICC of 0.00 ( P=.50). Finally, with regard to the synovitis category (Table 6), MRI and arthroscopy agreed in 24 of 27 cases, showing a fairly high degree of agreement and an ICC value of 0.7797 ( P<.0001).

Surface of the Repaired Tissue

Sagittal (A) and coronal (B) intermediate-weighted fast-spin echo MRIs of a 28-year-old man show complete filling of the defect of the osteochondral lesion of the medial talar dome. The repaired tissue shows damaged surface (arrows) with ulcer and fibrillation. Arthroscopic photograph shows complete defect filling and the surface of repaired tissue is damaged. In this case, MRI demonstrates a good correlation with arthroscopy (C).

Adhesion

Synovitis

Among the 5 categories, arthroscopy and MRI showed a high degree of agreement, producing ICC values with good reliability in the categories of degree of defect repair and filling, surface of the repair tissue, and synovitis. However, moderate to poor reliability was noted with respect to the categories of integration into the border zone and adhesion, suggesting that there might be some limitations to using these categories. In addition, the adhesion category showed complete disagreement, with an ICC value of 0.00 (Table 7).

Statistical Analysis Results

Discussion

Articular cartilage represents the most easily injured portion of a joint for which there is still no completely established treatment. There are various treatment methods for articular cartilage injury, the first being a primary repair technique in patients with acute symptoms ⁶ and attempted lavage and debridement in patients with chronic symptoms. ^7,8 Marrow-inducing repair techniques are widely used, although they have low biomechanical properties as cartilage is formed by fibrous cartilage. ⁹Restorative techniques recently have attracted attention as the cartilage is formed by hyaline cartilage restored to the mechanical properties of an uninjured normal cartilage. Our study technique also was used in patients with articular cartilage injury who underwent autologous chondrocyte implantation using restorative techniques.

Regarding patients who underwent various types of surgery, we compared the MRI and arthroscopy results. ^3,9 Although Mintz et al ² reported that the accuracy of MRI for osteochondral lesions of the talus was 83%, this report was not used in the postautologous chondrocyte implantation outcome. To address the increased need for such an outcome, Marlovits et al ¹⁰ reported the MOCART scoring system in 2004. The MOCART scoring system gradually developed for high-resolution MRI with the intent of defining pertinent variables for an objective description of repaired tissue, given the current technological limitations. Since Marlovits’ study, multiple articles have been published reporting the use of the MOCART scoring system, suggesting it is a useful method for long-term follow-up using point scales. ⁵

Recent articles also have highlighted the accuracy of MOCART for evaluating repaired knee cartilage after surgery for osteochondritis dissecans producing assessments with low interobserver variability. ⁴However, previous articles primarily addressed knee evaluations, prompting the need for evaluation studies on osteochondral lesions of the talus. In our study, the MOCART scoring system was modified (Table 1) by excluding the status of the subchondral lamina injured by drilling from the original MO-CART scoring system. In the arthroscopic fields, not only the structure of repaired tissue but also their signal characteristics, as shown in Table 1, could not be evaluated.

In our study, the categories for the degree of the defect repair and filling, surface of the repair tissue, and synovitis all showed fairly high ICC results. However, the categories of integration and adhesion showed low ICC. Although Mintz et al ² reported that the accuracy of MRI for osteochondral lesions of the talus was 83%, the report was not based on the results after autologous chondrocyte implantation. Therefore, an evaluation using the MO-CART scoring system was performed to verify accuracy.

According to the results of our study, the integration and adhesion category showed a low ICC, which was an unexpected result. A possible reason for this finding may be because the ankle cartilage is one-third thinner than knee joint cartilage ¹¹ and the articular surfaces of the ankle are naturally closely packed and congruous, unlike the knee where the joint surfaces are not congruent and there is no clear separation of the articular surfaces of the tibiotalar joint. As a result, ankle cartilage is hardly ever evaluated using MRI, in contrast to knee cartilage. It may be necessary to consider new isotropic MRI techniques at a high field (3T) MRI with a dedicated multichannel coil that potentially improves evaluation of repair tissue in the anatomically challenging ankle joint. ¹²

Furthermore, the adhesion category in our study showed complete disagreement, possibly due to one of the postautologous chondrocyte implantation complications where routine MRI may be limited in distinguishing adhesion from a closely packed joint with thin cartilage. In addition, compared to the results reported by Mintz et al, ² the MOCART scoring system is much more subdivided than in their study. Consequently, there will be some limitations in correlating arthroscopy and MRI after autologous chondrocyte implantation using those subtypes, which would be better used as an alternative method for following long-term clinical outcomes.

This study has limitations in that we studied relatively few cases and did not include all subtypes of the MOCART scoring system. Our results suggest further studies with more cases are needed. In addition, if modifications of the procedure or advancements in techniques allow for the study of the rest of the MOCART scoring system subtypes not included in this study because of the surgical procedure performed, more accurate data may be obtained in the future.

Conclusion

According to this comparison study between arthroscopy and MRI using the modified MOCART scoring system after autologous chondrocyte implantation in osteochondral lesion of the talus cases, the categories of degree of defect repair and filling, surface of the repair tissue, and synovitis all showed high ICC results, whereas the categories of integration into the border zone and adhesion showed low ICC values. There has been no well-established correlation method between arthroscopy and MRI after autologous chondrocyte implantation for osteochondral lesions of the talus. Although MRI allows for noninvasive evaluation of the repair tissue, limitations remain in using the MOCART scoring system to evaluate repaired cartilage after autologous chondrocyte implantation for osteochondral lesions of the talus.

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