Abstract
This study investigated the effects of in vitro chondrogenic differentiated mesenchymal stem cells (MSCs) on cartilage and subchondral cancellous bone in temporomandibular joint osteoarthritis (TMJOA). Four weeks after induction of osteoarthritis (OA), the joints received hylartin solution, non-chondrogenic MSCs or in vitro chondrogenic differentiated MSCs. The changes in cartilage and subchondral cancellous bone were evaluated by histology, reverse transcription polymerase chain reaction and micro-computed tomography (CT). Implanted cells were tracked using Adeno-LacZ labelling. The differentiated MSC-treated group had better histology than the MSC-treated group at 4 and 12 weeks, but no difference at 24 weeks. Increased mRNA expression of collegan II, aggeran, Sox9 and decreased matrix metalloproteinase 13 (MMP13) were observed in differentiated MSC-treated groups compared to the undifferentiated MSC-treated group at 4 weeks. The differentiated MSC-treated group had decreased bone volume fraction, trabecular thickness and bone surface density, and increased trabecular spacing in the subchondral cancellous bone than the undifferentiated MSC-treated group. Transplanted cells were observed at cartilage, subchondral bone, and the synovial membrane lining at 4 weeks. Intra-articular injection of MSCs could delay the progression of TMJOA, and in vitro chondrogenic induction of MSCs could enhance the therapeutic effects. This provides new insights into the role of MSCs in cell-based therapies for TMJOA.
Osteoarthritis (OA) is a degenerative joint disease, its main characteristics are progressive cartilage degeneration and subchondral bone sclerosis. Many attempts have been made to treat the lesions caused by OA and cell-based therapy has gained increasing attention and popularity. Mesenchymal stem cells (MSCs) can be easily obtained from bone marrow and have the capacity for differentiation into multiple cell lineages such as cartilage and bone. Murphy et al. used a scaffold-free approach to deliver MSCs as a suspension by direct intra-articular injection in the knee of goats.
Although positive outcomes were found, it did not yield satisfactory repair of articular cartilage. One possible reason is that in osteoarthritic joints, there is insufficient local stimulation to the delivered MSCs by cytokines which are necessary to drive chondrogenic differentiation in vivo . Since in vitro preconditioned MSCs have greater chondrogenic potential in promoting cartilage repair, they may be considered a better candidate for OA treatment.
The changes of subchondral cancellous bone are also recognized as a typical OA characteristic. Considerable attention has been paid to the role of the subchondral bone in OA. It is not known if MSC-based therapy has any effect on the underlying subchondral bone, for example the micro-architectural properties, which is of great importance in understanding the relationship between subchondral bone and the cartilage damage in OA.
The authors’ previous study and other studies have shown that OA-like characteristics could be induced by a partial resection of articular disc in the temporomandibular joint (TMJ).
In this study, the authors hypothesized that intra-articular injection of in vitro chondrogenic differentiated autologous MSCs could enhance cartilage and subchondral cancellous bone repair more significantly than non-chondrogenic MSCs. To test that, the changes in cartilage and subchondral cancellous bone of TMJ were evaluated by histology, reverse transcription polymerase chain reaction (RT-PCR) and micro-computed tomography (CT).
Materials and methods
A total of 46 skeletally mature New Zealand white rabbits (6 months old, 2.5–3.2 kg) were used in this study. They were given tap water and food ad libitum during the experiment. They were kept in separate cages and allowed to move freely. The study was authorized by the Animal Ethics Committee of the University.
Isolation and culture of MSCs
Bone marrow was aspirated from the tibia by an 18-gauge needle in a 5 ml syringe containing 0.1 ml of heparin (3000 U/ml), and was transferred to a 50 ml centrifuge tube and mixed with twice the volume of phosphate buffer solution (PBS). The diluted marrow mixture was centrifuged at 200 × g for 15 min. After centrifugation, the cells were cultured in 10 ml complete medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) containing 10% foetal bovine serum (FBS) and antibiotics (penicillin G, 100 U/ml; streptomycin, 0.1 mg/ml; amphotericin B, 0.25 mg/ml; Gibco) at 37 °C and 5% CO 2 and seeded at a concentration of 1.5–1.6 × 10 6 cells/ml. When the adherent cells reached subconfluence they were freed from the dish with 0.25% trypsin and subcultured for implantation.
In vitro chondrogenic differentiation of MSCs
The expanded autologous MSCs were cultured in different media for implantation. Non-chondrogenic MSCs were cultured only in basal medium (DMEM, 10% FBS, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, 0.25 mg/ml amphotericin B). For chondrogenic differentiation, confluent monolayers of MSCs were trypsinized, counted and 2 × 10 5 cells were centrifuged at 500 × g in 15 ml polypropylene conical tubes to generate pellets. FBS containing medium was then replaced with a defined medium, consisting DMEM with ITS + Premix (BD Sciences, San Diego, CA, USA), containing 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.35 μg/ml linoleic acid, and 1.25 μg/ml bovine serum albumin (BSA). Medium was also supplemented with 10 ng/ml of transforming growth factor (TGF)-β1, 1 mM pyruvate, 37.5 μg/ml of ascorbate-2-phosphate, and 10 −7 M of dexamethasone. The pellets were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 for 2 weeks with a change of medium every 2–3 days. Pellets were fixed in 10% buffered formalin for 2 h and embedded in paraffin, and 5 μm sections were cut for histology (sanfranin-O and toluidine blue staining) and histoimmunochemistry analysis (collagen type II).
Other pellets were cut into small pieces and digested with 0.1% collagenase in DMEM with 10% FBS for 12 h at 37 °C with gentle stirring. Isolated chondrogenic inductive cells were cultured in monolayer manner in DMEM (penicillin G 100 U/ml, streptomycin 100 μg/ml, HEPES 2.4 mg/ml, NaHCO 3 3.7 mg/ml, and 10% FBS), and 10 ng/ml of TGF-β1 and 10 −7 M of dexamethasone were added to maintain chondrogenic differentiation. After 3 days of culturing, the nonadherent cells were removed by changing the medium. When cells began to reach near-confluent stage, they were trypsinized and counted, and suspended in hylartin solution at a density of 2 × 10 6 cells/ml for intra-articular injection.
Animal model
Sixty animals were anaesthetized by intravenous injection of sodium pentobarbital 20 mg/kg and sodium ketamine hydrochloride 4 mg/kg. In brief, the right TMJ was exposed over the zygomatico-squamosal suture. One-third of the disc in the anterior and lateral regions of the joint was resected with a scalpel. The articular capsule and skin were closed independently in layers with 5-0 nylon sutures. The contralateral joint received identical surgery. All rabbits were returned to their cages after the operation and were allowed to move freely.
Study design
Both the non-chondrogenic and chondrogenic differentiated MSCs were suspended in the hylartin solution at a density of 2 × 10 6 cells/ml for autologous injection. Hylartin solution consisted of 0.1 ml sodium hyaluronan (Hylartin-V; Pharmacia & Upjohn, Peapack, NJ) at a concentration of 4 mg/ml.
Four weeks after OA induction, the location of the joint was identified and a syringe with a 26 G needle was inserted into the upper joint cavity. The TMJs in the animals from group A received 0.1 ml hylartin solution (vehicle group, n = 12), the animals in group B (non-chondrogenic MSCs group, n = 12) received a single injection of 0.2 × 10 6 non-chondrogenic autologous MSCs in the operated joint (0.1 ml) as a suspension in the vehicle, and the animals in group C ( in vitro chondrogenic differentiated MSCs group, n = 12) received identical volume of vehicle (0.1 ml) suspended with 0.2 × 10 6 in vitro chondrogenic differentiated autologous MSCs. The animals in group D were used as normal controls ( n = 6) and their TMJs remained intact. Four animals with OA induction were used for labelling of implanted autologous MSCs ( n = 4).
Six animals in groups A, B, and C, and two animals in group D were killed at 4, 12, and 24 weeks after injection, respectively. The animals for autologous MSCs labelling were killed at 4 weeks ( Table 1 ).
| Groups | Number of animals | Total | ||
|---|---|---|---|---|
| 4 weeks | 12 weeks | 24 weeks | ||
| A. Vehicle group | 6 | 6 | 6 | 18 |
| B. Non-chondrogenic MSCs group | 6 | 6 | 6 | 18 |
| C. In vitro chondrogenic differentiated MSCs group | 6 | 6 | 6 | 18 |
| D. Normal control | 2 | 2 | 2 | 6 |
| In vivo cell tracing | 4 | 4 | ||
| Total | 24 | 20 | 20 | 64 |
Histology and immunohistochemistry
The samples were fixed, decalcified and embedded in paraffin. 5 μm thick sections were cut and stained with toluidine blue. For each specimen, five randomly selected sections in each joint were evaluated for severity of arthritis by two independent blinded observers using the modified criteria as shown in Table 2 .
| Parameters | Grade |
|---|---|
| Matrix staining | |
| Normal | 0 |
| Mild increase or decrease | 1 |
| Moderate increase or decrease | 2 |
| Severe increase or decrease | 3 |
| No staining | 4 |
| Arrangement of chondrocytes | |
| Normal | 0 |
| Appearance of clustering | 1 |
| Mild hypocellularity | 2 |
| Moderate hypocellularity | 3 |
| Severe hypocellularity | 4 |
| No chondrocytes | 5 |
| Surface regularity | |
| Normal | 0 |
| Mild surface irregularities | 1 |
| Moderate surface irregularities | 2 |
| Severe surface irregularities | 3 |
| Thickness of fibrocartilage | |
| Normal | 0 |
| Mild increase or decrease | 1 |
| Moderate increase or decrease | 2 |
| Severe increase or decrease | 3 |
| No fibrocartilage | 4 |
| Total | 16 |
To detect expression of type II collagen, immunohistochemistry was performed using the avidine–biotin immunoperoxidase method with an antibody raised against type II collagen (Santa Cruz Biotechnology, Inc., CA, USA).
Total mRNA extraction and real-time RT-PCR
Total RNA of the mandibular cartilage was extracted at 4 weeks using Transcriptor First Strand cDNA Synthesis Kit (Roche Molecular Biochemicals, Mannheim, Germany) and analyzed by real-time RT-PCR. Amplimer pairs for extracellular matrix proteins Col2a1 (collagen type II), aggrecan, SRY-box containing gene 9 (Sox9), matrix metalloproteinase 13 (MMP13), and GAPDH were 5′-AGAAGAACTGGTGGAGCAGCAAGA-3′ and 5′-TGCTGTCTCCATAGCTGAAGTGGA-3′, 5′-ACACCAACGAGACCTATGACGTGT-3′ and 5′-ACTTCTCTGGCGACGTTGCGTAAA-3′, 5′-AGAAGGAGAGCGAAGAGGACAAGT-3′ and 5′-TTGTTCTTGCTGGAGCCGTTGA-3′, 5′-AACGAGGATGATGATTTGGTCCG-3′ and 5′-TTGGCCAGGAGGAAAAGCGTGAG-3′, 5′-TCACCATCT TCCAGGAGCGA-3′ and 5′-CACAATGCCGAAGTG GTC GT-3′, respectively. Expression of collagen type II, aggrecan, Sox9 and MMP13 were normalized as a function of GAPDH expression.
Micro-CT examination of subchondral cancellous bone
Subchondral cancellous bone was defined as the cancellous bone region 0.5 mm beneath the calcified cartilage–bone junction. The region of interest was selected as the cancellous bone region 0.5 mm beneath the calcified cartilage–bone junction and 3 mm distal. The subchondral cancellous bone was scanned by a micro-CT 80 scanner (Scanco Medical, Bassersdorf, Switzerland) with a resolution of 2048 × 2048 pixels and at a fixed threshold of 80 according to the manufacturer’s protocol. The parameters for analysis included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp).
Labelling of implanted autologous MSCs
The adenoviral vector containing the reporter gene LacZ (Adeno-LacZ) was used for implanted cell tracing. On the day of transduction, the in vitro chondrogenic differentiated MSCs were pre-incubated with 5 ml DMEM and then infected with Adeno-LacZ at multiplicities of infection at 800 ifu per cell for 4 h. 10 ml of fresh culture medium were added to the cells and subjected to 48 h incubation for the experiment.
Four rabbits with OA induction were used for tracing the implanted cells and were killed 4 weeks after intra-articular injection. The samples were fixed and stained in X-gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) substrate 1 mg/ml, 1 mmol/l MgCl 2 , 10 mmol/l K 4 Fe(CN) 6 , and 10 mmol/l K 3 Fe(CN) 6 in PBS for 24 h. After X-gal staining, the samples were decalcified and embedded. The sections were counterstained with haematoxylin.
Statistical analysis
One-way analyses of variance (ANOVA) were performed followed by post hoc Bonferroni test to determine which specific differences were significant within the three groups (SPSS Software; SPSS Inc., Chicago, IL, USA). The data were shown as the means ± SD. P < 0.05 was considered as statistically significant.
Results
In vitro chondrogenic differentiation of MSCs
Safranin-O and toluidine blue staining, and immunohistochemical staining of type II collagen were positive in chondrogenic differentiated MSCs ( Fig. 1 A–C ) but were negative in non-chondrogenic MSCs.
Histology and immunohistochemistry
At 4 weeks ( Fig. 2 ), the fibrocartilage in the vehicle group ( Fig. 2 A and D) was thickened, but the number of chondrocytes was mildly or moderately reduced. Clusters of chondrocytes were more apparent in the undifferentiated MSC-treated group ( Fig. 2 B and E) than in the differentiated MSC-treated group ( Fig. 2 C and F) and only mild surface irregularity was found in the differentiated MSC-treated group compared to the undifferentiated MSC-treated group.
