Osteoarthritis is a common cause of disability in humans and other animals. To study molecular mechanisms responsible for cartilage degradation in osteoarthritis, the catabolic cytokine IL-1, originally termed catabolin, has been used since 1980 and is commonly used at the present time.1–11 Studies on either isoform of IL-1 (IL-1α or IL-1β) have contributed greatly to the understanding of cartilage degradation. However, results of studies measuring catabolic mediators in naturally occurring osteoarthritic samples suggest that MMP-13, rather than IL-1, is emerging as the primary catabolic molecule responsible for matrix degradation.
Results of most microarray studies12–16 on normal (unaffected) and osteoarthritic human articular cartilage or primary chondrocytes suggest that MMP-13, but neither IL-1α nor IL-1β, is increased with osteoarthritis. In 1 study17 of early osteoarthritis, mRNA expression for IL-1 and MMP-13 genes was increased. Similarly, by use of immunohistochemistry, chondrocytes in the superficial zone of osteoarthritic cartilage have been found to have more MMP-13 and IL-1 than the other zones, with MMP-13 immunostaining present to a greater extent than IL-1.18 Increased protein expression of MMP-13 has been observed in subchondral bone resorption pits in osteoarthritic cartilage,19 and when administered intra-articularly, MMP-13 resulted in cartilage damage as indicated by the increased presence of collagen fragments.20 Together, these studies suggest that treatment with MMP-13 may be an effective method to induce articular cartilage degeneration for in vitro studies.
The role of MMP-13 and other MMPs in cartilage degradation and osteoarthritis has recently been reviewed.21 Matrix metalloproteinase-13 is also known as collagenase-3 and is best known for its ability to efficiently cleave type II collagen into characteristic three-fourth and one-fourth fragments.22 Matrix metalloproteinase activity can be regulated at the level of transcription, mRNA stability, and protein activation.21 Matrix metalloproteinase-13 is secreted as an inactive proenzyme, and proteolytic cleavage of pro-MMP-13 to the active form is enhanced by exposure to inflammatory cytokines such as IL-1 and tumor necrosis factor, by products of cartilage degradation including fibronectin fragments23 and collagen,24 and by mechanical injury.25
Despite the documented importance of MMP-13 in the development of osteoarthritis, investigations on cartilage with MMP-13 are limited to studies26–28 of proteoglycan cleavage mechanics and characterization of MMP-13 cell-surface binding and internalization. The objective of the study reported here was to determine the effects of MMP-13 on cartilage matrix molecule gene expression and to compare the outcomes with those achieved following treatment with IL-1. Our hypothesis was that MMP-13 would induce a similar cartilage matrix degradation pattern to IL-1 and that it would therefore be an acceptable method for in vitro studies of cartilage degradation. To further model the native joint environment, a coculture system in which cartilage explants are grown in the presence of synoviocytes was used.
Materials and Methods
Cartilage and synoviocyte coculture—Cartilage and synovium were harvested from 4 horses, aged 3 to 5 years, which were euthanatized by an overdose of pentobarbitol for reasons unrelated to the musculoskeletal system. The Institutional Animal Care and Use Committee of Cornell University approved all procedures.
Full-thickness cartilage was removed from the lateral trochlear ridges of the distal aspects of the femora, and synovium was harvested from the femoropatellar joints. Careful dissection was performed to minimize inclusion of fat and fibrous joint capsule in the synovium specimens. Cartilage was cut into approximately 5 × 5-mm full-thickness pieces and stored overnight in complete DMEMa containing 25mM HEPES, ascorbic acid (50 μg/mL), α-ketoglutaric acid (30 μg/mL), L-glutamine (300 μg/mL), sodium penicillin (100 U/mL), and streptomycin sulfate (100 μg/mL) with 5% FBS at 37°C, 5% CO2, and 90% humidity.
Synoviocytes were isolated from the synovium by digestion in complete DMEM containing 0.015% collagenase type 2b and 0.0015% DNase (0.1 g of synovium/mL of medium) for 2 hours at 350 × g and 37°C. The resulting cell slurry was filtered through 44-μm-diameter nylon mesh, and cells were pelleted by centrifugation for 10 minutes at 350 × g and 25°C. The cell pellet was resuspended in complete DMEM, and a cell count and test for viability with trypan blue dye exclusion were performed. Synoviocyte mono-layer cultures were established with 0.32 × 106 viable synoviocytes/cm2 in the bottom of split-well plates specifically designed for coculture.c No further characterizations or separation of synoviocyte subtypes was performed because the goal of the model system was to mimic the native articular environment where all synovial cells would be present. Synoviocytes were allowed to adhere overnight, after which cocultures of synoviocytes and cartilage explants were established by suspending 5 cartilage explants in the medium of each well via low–protein-binding polyester membrane inserts (pore size, 3 μm). Media were then changed to serum-free DMEM, and treatments were initiated.
Cocultures of cartilage explants and synoviocytes were treated with human recombinant MMP-13d (1, 25, or 100 ng/mL of medium) or human recombinant IL-1αe (0.01, 0.1, 1.0, or 10 ng/mL of medium) for 96 hours with medium exchange at 48 hours. Concentrations of MMP-13 and IL-1α tested were chosen on the basis of results of preliminary experiments with the coculture system. Activation of MMP-13 was accomplished through trypsin-induced autoproteolysis to the active form with 10 μg of trypsin/μg of MMP-13 in a total volume of 1 mL for 10 minutes at 25°C. The trypsin was inactivated by addition of FBS to a final concentration of 10%. Active MMP-13 was added to the respective treatment groups, and FBS was added to all groups to achieve a final concentration of 2.5% FBS. Control groups consisted of FBS-inactivated, trypsin-treated control and nontreated control cultures. Treatments were performed in triplicate, and the experiment was repeated 4 times.
Mean ± SD (n = 4 horses) GAG content (μg/mL) in media of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL) for 48 or 96 hours. *Significant (P < 0.05) difference from nontreated (NT) controls.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) GAG content (μg/mL) in media of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL) for 48 or 96 hours. *Significant (P < 0.05) difference from nontreated (NT) controls.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) GAG content (μg/mL) in media of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL) for 48 or 96 hours. *Significant (P < 0.05) difference from nontreated (NT) controls.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
At medium exchange (48 hours) and culture harvest (96 hours), media were collected and 10% (vol/vol) protease inhibitorsf were added to inhibit endogenous enzyme activity. Media were centrifuged at 350 × g for 10 minutes to remove particulate matter and stored at −80°C until analyzed for GAG content. Cartilage explants and synoviocytes were harvested after 96 hours of treatment. Explants were rinsed in protease inhibitors, wiped dry, weighed, and pulverized in a freezer mill and stored at −80°C until used for GAG analysis and RNA isolation.
GAG content in medium and cartilage—Total GAG content of medium and cartilage was assayed by routine 1,9-dimethyl-methylene blue dye binding microwell spectrophotometric assay.29 Medium was digested (1:1 ratio [vol/vol]) in 0.05% papain. Pulverized cartilage (100 mg) was lyophilized, weighed, and digested (10% [dry wt/vol]) in 0.05% papain. The optical density at 595 nm was determined on a multiple detection plate reader.g Mixed isomer shark chondroitin sulfate was used to construct the standard curve. Cartilage GAG was expressed per micrograms of DNA, which was assessed fluorometrically.30 Calf thymus DNA was used to construct a standard curve.
RNA isolation and gene expression by quantitative PCR—Total RNA was isolated from pulverized cartilage (150 mg) by use of a monophasic solution of phenol and guanidine isothiocyanatea according to the manufacturer's directions for RNA extraction from whole tissue, with further purification by use of spin columns.h Similarly, RNA was isolated from synoviocytes according to the manufacturer's directions for cells in monolayer culture. Total RNA was reverse transcribed and amplified by use of a 1-step quantitative reverse transcriptase–PCR technique and sequence detection system.i For synoviocytes, MMP-3 and MMP-13 mRNA expression were measured. For cartilage, COL2A1, aggrecan, MMP-3, and MMP-13 mRNA expression were assessed. All results were adjusted to 18S mRNA expression. Primers and dual-labeled fluorescent probe were designed by use of designated software.j All probes and primers were designed by use of equine-specific sequences either published in GenBank, provided by another investigator,31 or sequenced in our laboratory.
Statistical analysis—Results from replicate samples were averaged and expressed as the mean ± SD of 4 independent experiments, which were performed on separate occasions by use of tissues obtained from different horses. Quantitative PCR, 1,9-dimethyl-methylene blue dye binding, and DNA data were analyzed by use of an ANOVA to compare values between treatments; all treatments (with or without IL-1α or MMP-13) were included in the model. For ANOVAs with a significant F test value, a Tukey post hoc test was performed. Values of P < 0.05 were considered significant.
Results
Control cultures—No significant differences were found between nontreated control and trypsin-treated control cultures for any outcome parameter evaluated. For graphic display, only nontreated control cultures are presented.
GAG analysis—Treatment of cartilage-synoviocyte cocultures with MMP-13 or IL-1α stimulated cartilage degradation with concomitant reparative responses. Significant GAG accumulation was found in media at 48 hours in cultures treated with MMP-13 at ≥ 25 ng/mL or with IL-1α at ≥ 1.0 ng/mL (Figure 1). At 96 hours, GAG content in media again increased in cultures treated with MMP-13 at ≥ 25 ng/mL, but the highest concentration of IL-1α (10 ng/mL) was required to observe a significant increase GAG accumulation. No changes were found in cartilage in GAG content regardless of MMP-13 or IL-1α treatment concentration (Figure 2). Increased media GAG content without a decrease in cartilage GAG content has been observed in a previous study32 when a similar coculture design was used. In the present study, cartilage GAG content results remained unaltered when analyzed on the basis of per milliliter of medium, per milligram of cartilage wet weight, per milligram of cartilage dry weight, or per microgram of DNA. No significant (P = 0.25) differences were found in DNA content as a result of treatment (data not shown), allowing for adjustment of GAG to DNA content.
Mean ± SD (n = 4 horses) adjusted GAG content in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (at 1, 25, or 100 ng/ mL). See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) adjusted GAG content in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (at 1, 25, or 100 ng/ mL). See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) adjusted GAG content in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (at 1, 25, or 100 ng/ mL). See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) aggrecan mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL).A,B Different letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) aggrecan mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL).A,B Different letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) aggrecan mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL).A,B Different letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) COL2A1 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) COL2A1 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) COL2A1 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 105 [n = 4 horses]) MMP-3 mRNA expression relative to 18S mRNA expression in cartilage and synoviocytes treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). a-c,A-CDifferent letters above bars indicate values that differ significantly (P < 0.05) for cartilage (lowercase letters) and synoviocytes (uppercase letters). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 105 [n = 4 horses]) MMP-3 mRNA expression relative to 18S mRNA expression in cartilage and synoviocytes treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). a-c,A-CDifferent letters above bars indicate values that differ significantly (P < 0.05) for cartilage (lowercase letters) and synoviocytes (uppercase letters). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 105 [n = 4 horses]) MMP-3 mRNA expression relative to 18S mRNA expression in cartilage and synoviocytes treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). a-c,A-CDifferent letters above bars indicate values that differ significantly (P < 0.05) for cartilage (lowercase letters) and synoviocytes (uppercase letters). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 104 [n = 4 horses]) MMP-13 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). A-CDifferent letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 104 [n = 4 horses]) MMP-13 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). A-CDifferent letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (X 104 [n = 4 horses]) MMP-13 mRNA expression relative to 18S mRNA expression in cartilage of cocultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/mL) or MMP-13 (1, 25, or 100 ng/mL). A-CDifferent letters above bars indicate values that differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) MMP-13 mRNA expression relative to 18S mRNA expression in synoviocyte cultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/ mL) or MMP-13 (1, 25, or 100 ng/mL). See Figures 1 and 6 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) MMP-13 mRNA expression relative to 18S mRNA expression in synoviocyte cultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/ mL) or MMP-13 (1, 25, or 100 ng/mL). See Figures 1 and 6 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Mean ± SD (n = 4 horses) MMP-13 mRNA expression relative to 18S mRNA expression in synoviocyte cultures treated with various concentrations of IL-1α (0.01, 0.1, 1.0, or 10 ng/ mL) or MMP-13 (1, 25, or 100 ng/mL). See Figures 1 and 6 for remainder of key.
Citation: American Journal of Veterinary Research 68, 4; 10.2460/ajvr.68.4.379
Gene expression in cartilage—Aggrecan mRNA expression was significantly increased in cultures treated with MMP-13 at ≥ 25 ng/mL, compared with control cultures and cultures treated with the lowest tested concentrations of MMP-13 (1 ng/mL) and IL-1α (0.01 ng/mL; Figure 3). However, aggrecan mRNA expression was not significantly different between cultures treated with MMP-13 at ≥ 25 ng/mL or IL-1α at ≥ 0.1 ng/mL.
The COL2A1 gene expression was significantly increased in cultures treated with MMP-13 at 100 ng/mL, compared with all other treatment groups (Figure 4). There was no significant effect of IL-1α on COL2A1 mRNA expression.
For the catabolic mediator, MMP-3, mRNA expression was increased in cultures treated with MMP-13 at ≥ 25 ng/mL or IL-1α at ≥ 1.0 ng/mL, compared with lower concentrations of either cytokine or control cultures (Figure 5). Treatment with IL-1α resulted in a dose-response increase in MMP-3 mRNA expression, with IL-1α at ≥ 10 ng/mL stimulating MMP-3 mRNA expression to a greater extent than all other treatments. A similar dose response to IL-1α was observed for MMP-13 mRNA expression, in which IL-1α at ≥ 1.0 ng/mL stimulated mRNA expression to a greater extent than all other treatment groups and control cultures (Figure 6). No significant effect of MMP-13 treatment on MMP-13 mRNA expression was found.
Gene expression in synoviocytes—Matrix metal-loproteinase-3 mRNA expression in synoviocytes was greater in all groups, compared with MMP-3 mRNA expression in cartilage. Similar to cartilage, MMP-13 at ≥ 25 ng/mL and IL-1α at ≥ 0.1 ng/mL stimulated a significant increase in MMP-3 mRNA expression in synoviocytes, with IL-1α at 10 ng/mL resulting in the greatest increase (Figure 5). Conversely, MMP-13 mRNA expression in synoviocytes was lower in all treatment groups, compared with cartilage samples. A significant increase was found in MMP-13 mRNA expression in synoviocytes treated with IL-1α at ≥ 1.0 ng/mL and a significant decrease in MMP-13 mRNA expression in synoviocytes treated with MMP-13 at all concentrations tested (Figure 7).
Discussion
Results of the present study indicated that in cocultures of cartilage explants and synoviocytes, either-IL-1α (1 ng/mL) or MMP-13 (25 ng/mL) can be used to stimulate cartilage degradation. Both substances induced significant accumulation of GAG in the medium, but parallel GAG loss was not detected in cartilage explants. An increase in medium GAG content without cartilage GAG loss has been observed in another co-culture study32 and appears to be unique to this type of culture system in which synoviocytes are present. The molecular mechanism for this result remains to be elucidated, but results of a previous study32 suggest that a mediator is released from the synoviocytes that preferentially protects GAG synthesis.
An increase in GAG synthesis might suggest an early reparative effort by the cartilage in response to treatment with IL-1 or MMP-13. In our study, this concept is supported by increased aggrecan mRNA expression in response to MMP-13 or IL-1α treatment. We did not investigate GAG synthetic rate through the use of 35S-sulfate incorporation because all of the cartilage was required for mRNA isolation, but it would be of interest. Collagen type IIB mRNA expression was also upregulated by MMP-13 treatment at 100 ng/mL, which would also suggest a reparative response. Simultaneous degradative and synthetic activities in articular cartilage have been observed in several other studies.13,17,32–35
It was anticipated that treatment with MMP-13 would stimulate cartilage degradation in our study. As mentioned, loss of GAG into the medium was observed in response to MMP-13 treatment. Application of MMP-13 also resulted in increased MMP-3 mRNA expression in cartilage and synoviocytes. These effects were also observed in IL-1α–treated cultures. In contrast, MMP-13 treatment had an autoinhibitory effect on MMP-13 mRNA expression in synoviocytes but had no significant effect on MMP-13 mRNA expression in cartilage. The inhibition of MMP-13 mRNA expression in synoviocytes was observed at the lowest concentration of MMP-13 (1 μg/mL) tested and was the only response observed at an MMP-13 concentration of 1 μg/mL.
Feedback inhibition of mRNA expression subsequent to exogenous application of the protein from the same gene is not surprising, but the lack of negative regulation of MMP-13 mRNA in cartilage, when feedback inhibition was observed in synoviocytes, was unexpected. This result is likely the result of the low amounts of MMP-13 mRNA expression in cartilage, compared with synoviocytes. The relatively high MMP-13 and MMP-3 mRNA expression in synoviocytes, compared with cartilage, might suggest that treatments aimed at diminishing MMP-13 and MMP-3 production should be preferentially targeted toward synoviocytes rather than chondrocytes.
In our study, few differences were found between the effects of MMP-13 and IL-1α on matrix molecule gene expression. The lack of overt differences was expected because IL-1α upregulates MMP-13 mRNA expression in articular cartilage; therefore, the effects of the 2 treatments should overlap to some extent. However, IL-1α and MMP-13 each likely stimulate distinct and unique cellular pathways. The goal of our study was not to determine the mechanism of actions of IL-1α or MMP-13, but rather to determine whether MMP-13 could be used to stimulate cartilage degradation in vitro and how the effects might differ from IL-1α on the pattern of matrix molecule gene expression.
The capacity of recombinant MMP-13 to stimulate cartilage catabolism for studies of cartilage degradation is supported by results of our study. Given increasing evidence that MMP-13 is upregulated more commonly than IL-1 in naturally occurring osteoarthritis, the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants might better mimic the native articular environment and may lead to more clinically translatable results than studies with IL-1 in cartilage or synovium cultures.
ABBREVIATIONS
| IL | Interleukin |
| MMP | Matrix metalloproteinase |
| DMEM | Dulbecco modified Eagle medium |
| FBS | Fetal bovine serum |
| GAG | Glycosaminoglycan |
| COL | Collagen type |
| 18S | 18S ribosomal subunit |
Trizol, Invitrogen, Carlsbad, Calif.
Worthington Biochemicals, Lakewood, NJ.
Costar Transwells, Cole-Parmer Instrument Co, Vernon Hills, Ill.
Chondrex Inc, Redmond, Wash.
R&D, Minneapolis, Minn.
Hoffmann-La Roche Ltd, Basel, Switzerland.
Tecan SPECTRAFlour, Tecan Systems Inc, San Jose, Calif.
RNeasy, QIAGEN, Valencia, Calif.
Applied Biosystems, Foster City, Calif.
Primer Express Software, version 2.0b8a, Applied Biosystems, Foster City, Calif.
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