Abstract
Excessive mechanical stress is thought to be a factor in the development of joint disorders through the expression of matrix metalloproteinases (MMPs) and related cytokines. Although studies revealed that mechanical stress on the synovium induces MMP expression, it is still not known which MMPs prolonged high level expression. The authors focused on MMP-3, which is one of the major factors in joint disorders such as rheumatism and temporomandibular joint disorders. They examined mRNA and protein levels of MMP-3, other MMPs and related cytokines after loading stress. Human synovial cells were seeded onto a collagen scaffold and different magnitudes of cyclic compressive load were applied for 1 h. Time-dependent mRNA and protein levels for catabolic genes were examined after loading. mRNA expressions of MMP-1, MMP-3, MMP-9, IL-6, IL-8 and IL-1β increased after excessive compression. In particular, only mRNA of MMP-3 was up-regulated and maintained at a high level for 24 h after excessive loading. The concentrations of MMP-3, IL-6 and IL-8 in culture media after loading increased with excessive compression. These results may account for the pathomechanism of MMP-3 induced by cyclic load on synovial cells in joint disorders.
Excessive mechanical stress on the articular joint is thought to erode the condylar cartilage and predispose the joint to osteoarthritis, and greatly contributes to the development of joint disorders . This pathomechanism is the same as for temporomandibular joint disorders (TMJD). In particular, in anterior disc displacement cases, the posterior part of the synovium in the temporomandibular joint (TMJ) is continuously subjected to direct tensile, compressive and shear mechanical stress during various functions, such as chewing and bruxism (grinding), resulting in inflammatory changes of synovitis, such as pain, discomfort and functional disorder .
There have been many reports on the relation between the expression of matrix metalloproteinases (MMPs) and the onset of joint disorders ; but this biomechanical and biological pathomechanism has not been clarified sufficiently. Previously, the authors established an in vitro three-dimensional (3D) culture system with cyclic compressive load. The results showed that the expressions of mRNAs and proteins for MMP-1, MMP-2, and MMP-3 increased with the application of cyclic compressive loads for 1 h per day for 5 and 15 consecutive days ; but it remains unknown how the expressions of these genes change after cyclic compression for 1 h. The authors hypothesized that MMP-3, which is one of the major factors in TMJD, prolonged high level expression after mechanical stress.
To test this hypothesis, the authors examined mRNA expression levels and the protein production of MMP-3, other MMPs and related cytokines after 1 h cyclic compressive loads.
Materials and methods
Human synovial cell culture
Surgical specimens of human synovial membranes were obtained during arthroscopic knee surgeries. Patients gave informed consent for the use of surgical specimens for experiments. The authors repeated the experiment three times. Experiments 1 and 2 were performed using cells from a female donor aged 21 years. In the third experiment, the cells from a male donor aged 40 years were used. There were no differences in the morphological characteristics and biological responses of mechanical stimulation between donors. The cell culture method was the same as in a previous study . Briefly, an excised synovium was digested with 0.2% collagenase in Dulbecco’s modified Eagle’s medium (DMEM). The liberated cells were resuspended in medium supplemented with 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were then cultured in a monolayer at 37 °C and in 5% CO 2 atmosphere. Cells from the fifth to seventh passage were used for experiments.
Collagen scaffold preparation
A porous collagen sponge was produced as described previously . Briefly, Atelocollagen ® (KOKEN, Tokyo, Japan) gel was freeze-dried, and then cross-linked and sterilized with formaldehyde and γ-ray (10 kGy) to produce a porous collagen sponge (AteloCell ® Atelocollagen sponge, MIGHTY; KOKEN). The pore size was designed to be 30–200 μm, and the pores were inter-connected. In this study, the porous collagen sponge, whose compressive modulus is close to that of native cartilage was 5 mm diameter and 3 mm thick to fit a 96-well cell culture plate ( Fig. 1 A ).
Cell seeding in collagen scaffold and 3D culture of cell-scaffold constructs
Collected cells (5.0 × 10 5 /scaffold) were resuspended in 1 × DMEM containing 10% FBS, 1% antibiotics, and 0.5% collagen solution (Atelocollagen ® gel, KOKEN). The cell–collagen solution mixture was then seeded onto a collagen scaffold in a 96-well cell culture plate by centrifugal force for 5 min at 500 × g and incubated at 37 °C for 1 h to produce a 3D cell-scaffold construct . After gelation, 3D cultured tissue was incubated for 3 days before cyclic compressive load.
Cyclic loading system and loading protocols
Intermittent dynamic loading was carried out using a custom-made apparatus, a cyclic loading bioreactor (CLS5J-Z ® ; Technoview, Osaka, Japan), described previously . In this study, 20 kPa and 40 kPa were applied at 0.5 Hz for 1 h. This apparatus, consists of cylindrical loading pistons connected to weights, a moving stage that raises and drops the loading pistons onto the constructs, and a linear actuator that controls the motion of the moving stage. The pistons (and weights) are raised by the moving stage and then allowed to fall onto the 3D constructs without actually being attached to the moving stage, during loading, so stimulation of each sample is subject to constant peak load, due to the weight on each piston. The weights on the top of each piston are exchangeable, so that the cyclic load bioreactor can apply a designated peak load to each 3D tissue in the culture wells. Specimens were cultured in 96-well culture plates for loading of uni-axial unconfined compression ( Fig. 1 B and C).
Histology
The constructs after cyclic loading for 1 h at 40 kPa were rinsed with PBS and fixed with 10% formalin. Paraffin sections were prepared for staining with haematoxylin and eosin ( n = 3). For histological sections, three constructs were used.
Time course of mRNA expression level
The constructs at pre-loading, and 0, 3, 6, 12, and 24 h post-loading were processed for extraction of total RNA using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Eighteen constructs were used this experiment at each time point by three constructs. After washing with 80% ethanol, RNA was treated with RNase-free DNase (DNase I; TAKARA Bio Inc., Shiga, Japan) to remove genomic DNA, and purified with phenol–chloroform. Single-strand cDNA was synthesized by reverse transcriptase (PrimeScript ® RT reagent Kit; TAKARA Bio Inc.). The cDNA was synthesized during 15 min incubation at 37 °C with reverse transcriptase and a random hexamer primer, followed by enzyme inactivation at 85 °C for 5 s. The mRNA expressions of the specimens were quantitatively determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) by Lightcycler ® DX400 (Roche Diagnostics Ltd., Lewes, UK) with associated enzyme and reagents (SYBR ® Premix Ex Taq ™ II; TAKARA Bio Inc.). Primer sequences for MMP-1, MMP-3, MMP-9, MMP-13, interleukin-6 (IL-6), IL-8, IL-1β, and tissue inhibitor of MP-1 (TIMP-1) were designed using Primer3 on the Web ( ) ( Table 1 ). PCR amplification of the cDNA samples was carried out by initial denaturing for 30 s at 94 °C, denaturing for 10 s at 94 °C, and annealing for 20 s at 60 °C for 40 cycles ( n = 9).
| Gene name | Primer set | Sequences | Size (bp) |
|---|---|---|---|
| GAPDH | Forward | 5-tctctgctcctcctgttcgac-3 | 101 |
| Reverse | 5-gttgactccgaccttcaccttc-3 | ||
| MMP-1 | Forward | 5-cccaaaagcgtgtgacagtaag-3 | 113 |
| Reverse | 5-cttccgggtagaagggatttg-3 | ||
| MMP-3 | Forward | 5-cgtgaggaaaatcgatgcag-3 | 134 |
| Reverse | 5-cttcagctatttgcttgggaaag-3 | ||
| MMP-9 | Forward | 5-agtccacccttgtgctcttc-3 | 122 |
| Reverse | 5-tttcgactctccacgcatc-3 | ||
| MMP-13 | Forward | 5-cttcccaaccgtattgatgc-3 | 149 |
| Reverse | 5-acttcttttggaagacccagttc-3 | ||
| IL-6 | Forward | 5-gtgtgaaagcagcaaagagg-3 | 140 |
| Reverse | 5-cctcaaactccaaaagaccag-3 | ||
| IL-8 | Forward | 5-tggcagccttcctgatttc-3 | 109 |
| Reverse | 5-gggtggaaaggtttggagtatg-3 | ||
| IL-1β | Forward | 5-tccagggacaggatatggag-3 | 133 |
| Reverse | 5-tctttcaacacgcaggacag-3 | ||
| TIMP-1 | Forward | 5-gcatcctgttgttgctgtgg-3 | 141 |
| Reverse | 5-aggtggtctggttgacttctgg-3 | ||
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