ARTZ Dispo® (1% HA) and hyaluronidase were purchased from Seikagaku Corporation (Tokyo, Japan). Diclofenac sodium was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water for injection (WFI) was from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). Human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) patients and those from osteoarthritis (HFLS-OA) patients were purchased from Cell Applications, Inc. (San Diego, CA). Growth medium and a subculture reagent kit were from Cell Applications, Inc. α-MEM, α-MEM powder, penicillin/streptomycin and Dulbecco’s phosphate buffered saline (D-PBS) were from Life Technologies Corporation (Waltham, MA). FBS was from MP Biomedicals LLC. (Santa Ana, CA). IL-1β was from R&D Systems, Inc. (Minneapolis, MN). Bovine serum albumin (BSA) was from Sigma-Aldrich Co. LLC. (St. Louis, MO). Glucosamine hydrochloride, D-[6-3H (N)] ([3H]glucosamine) (37 MBq/mL) and Ultima-FloTMM (scintillation solution) were from PerkinElmer Co., Ltd. (Waltham, MA). OH pak SB-805 HQ and OH pak SB-807 HQ columns were from SHOWA DENKO K.K. (Tokyo, Japan). Select-HATM 500 k, Select-HATM 1000 k, and Select-HATM 2500 k were from Hyalose, LLC. (Oklahoma City, OK).
Preparation of SI-613
SI-613 was prepared by conjugating high-molecular-weight fermented HA (600,000 to 1200,000 Da) and DF via a 2-aminoethanol linker extended from the glucuronic acid moieties. The SI-613 active pharmaceutical ingredient was manufactured in accordance with good manufacturing practice (GMP) guidelines, and its solution was prepared in a laminar flow cabinet to maintain its sterility. SI-613 was dissolved in 5 mM phosphate-buffered saline (pH 6.0) at a concentration of 10 mg/mL and diluted appropriately with PBS before use. Although SI-613 has been used at 10 mg/mL in clinical trials, the maximum concentration of SI-613 was set at 1 mg/mL in this study because highly-concentrated SI-613 solution is very viscous and difficult to pipette.
Cells and cell culture
Three lots of human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) patients and three from osteoarthritis (HFLS-OA) (Cell Applications, Inc.) patients were cultured separately in basal medium containing 10% growth supplement and 1% penicillin/streptomycin (Cell Applications, Inc.). Cells were incubated at 37 °C under 5% CO2. The medium was changed every 2 days. After confluence, the cells were seeded at a density of 3.0 × 105 cells/2 mL/well in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 6-well plates.
Challenge to test materials
One day after cell seeding, the culture medium was replaced with 2 mL of α-MEM medium containing 10% FBS, 10 ng/mL recombinant human IL-1β/IL-1F2 (IL-1β) (R&D Systems, Inc.), 1% penicillin/streptomycin, 370 kBq/mL glucosamine hydrochloride D-[6-3H (N)] ([3H]glucosamine) and each test material: HA (ARTZ Dispo®), SI-613 (Seikagaku Corporation), diclofenac sodium (DF-Na) (Wako Pure Chemical Industries, Ltd.), or a mixture of DF-Na and HA (DF-Na + HA). For mRNA expression measurements, [3H] glucosamine was excluded from the medium. The cells were incubated at 37 °C for the indicated periods under 5% CO2.
Collection of the culture supernatant and cell lysate
Culture supernatant fraction for the measurement of high molecular weight HA (HMW-HA, MW > 2400 kDa) production was collected at each incubation time. Cell lysate for the measurement of RNA was collected at 48 h and stored frozen at -80 °C.
Fractionation of the culture supernatant and measurement of the radioactivity levels
Radiolabeled HA in the culture supernatant was separated using a size exclusion HPLC column (an OH pak SB-805 HQ column or an OH pak SB-807 HQ column) in an HPLC system (JASCO Corporation). HPLC was performed at 0.5 mL/min of mobile phase (5 mmol/L phosphate buffer, 0.82% NaCl: acetonitrile = 2: 1) at 35 °C. The injection volume of culture supernatant was set at 10 μL. Eluent was collected continuously as 0.5 min fractions from 9 min (OH pak SB-805 HQ column) or 14 min (OH pak SB-807 HQ column) after starting the elution using a fraction collector (JASCO Corporation). Liquid scintillator (Ultima-FloTMM, PerkinElmer Co., Ltd.) was added to each HPLC fraction and mixed well. The disintegrations per minute (dpm) of each fraction were measured in a liquid scintillation counter (PerkinElmer Co., Ltd.). Each sample was analyzed in a single HPLC run.
Evaluation of the molecular weight of HA in each fraction using HA standard solutions
Select-HA™ 500 k, Select-HA™ 1000 k and Select-HA™ 2500 k, for which weight-average molecular weights were 528 kDa, 1076 kDa, and 2420 kDa respectively, were used for molecular weight calibration. Each standard was subjected to size exclusion HPLC analysis with online monitoring of UV absorption at 210 nm. The peak top fraction of each HA standard was calculated by considering the lag between the UV monitor and the fraction collector.
The culture supernatant of the 0.01% HA-treated group (90 μL) was mixed with 10 μL of 100 turbidity reducing units (TRU)/mL hyaluronidase or WFI and incubated overnight at 37 °C, except for the culture supernatant of the 0.01% SI-613-treated group, which was digested by hyaluronidase treatment (30 μL hyaluronidase solution at 60 °C for 3 h). This more aggressive treatment was required because HA in the culture supernatant of the 0.01% SI-613-treated group was more difficult to digest.
Immediately after collecting the culture supernatant, each well was washed with Dulbecco’s phosphate-buffered saline (D-PBS) (GIBCO, Life Technologies Corporation). Trypsin/EDTA solution (Cell Applications, Inc.) was added to the wells and the cells were incubated at 37 °C under 5% CO2. The cells were detached from the wells by tapping the plate, and 0.5 mL of trypsin-neutralizing solution (Cell Applications, Inc.) was added. The live cells were counted using a hematocytometer after staining with trypan blue.
RNA extraction and cDNA sample preparation
RNA was extracted from the cell lysates according to the instruction manual for the RNeasy plus mini kit (QIAGEN). RNA concentrations were measured by ultramicro spectrophotometry (Thermo Fisher Scientific, Inc.). The RNA was stored frozen at -80 °C. cDNA samples were prepared using the Super Script III First-Strand Synthesis System (Invitrogen, Life Technologies Corporation).
Real-time quantitative PCR
The mRNA expression levels of the target genes (HAS1, HAS2, HAS3, HYAL1, HYAL2, and HYAL3) and GAPDH were measured in duplicate using real-time quantitative polymerase chain reaction (PCR; RT-qPCR). RT-qPCR was performed using TaqMan Gene Expression Assay (TaqMan) (Applied Biosystems) and Premix Ex Taq (Perfect Real Time, Takara Pac LTD.) according to the instruction manuals. The following sets of TaqMan probes and primers were used to measure the expression levels of the target genes: HAS1 (ID: Hs00987418_m1), HAS2 (ID: Hs00193435_m1), HAS3 (ID: Hs00193436_m1), HYAL1 (ID: Hs00201046_m1), HYAL2 (ID: Hs01117343_g1), HYAL3 (ID: Hs00185910_m1), and GAPDH (ID: Hs03929097_g1). Reagents were aliquoted into each well at the following volumes: 1 μL of TaqMan, 12.5 μL of Premix Ex Taq, 2 μL of ROX II (× 40), and 7.5 μL distilled water. Next, 2 μL of the cDNA sample was added. The PCR conditions were as follows: first denaturation at 95 °C for 30 s., followed by a 40-cycle sequence of denaturation (95 °C, 5 s.), annealing (60 °C, 30 s.), and extension (72 °C, 20 s.). The cycle threshold (Ct) value was measured for each gene using a real-time PCR system (MX3000P; Stratagene Corporation). The Ct value was analyzed using the comparative Ct method (ΔΔCt method) to calculate the target mRNA expression level, followed by normalization to GAPDH level. The relative mRNA expression levels were presented as the times-fold increase compared to that of the control.