Cell culture
Human tendon cells were isolated from healthy human hamstring (semitendinosus) tendons (excess anterior cruciate ligament autograft material) as previously described [14]. Male (N = 2; age 28 ± 1.4) and female (N = 2; age 37 ± 5.7) donors provided written informed consent and the protocol was approved by University of British Columbia Clinical Research Ethics Board (certificate number H10-00220). Equal number of females and males were used in each experiment to exclude the effect of gender as a factor. To expand the tendon cell culture, the isolated tendon cells were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 2 mm L-glutamine, and 1x Antibiotic/Antimycotic solution in a humidified incubator containing 5% CO2 at 37°C. Then, 10% calf serum was replaced with 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) before and during incubation with oxLDL. Recombinant TGF-β protein (PeproTech, USA, #100 − 21) at concentration of 5ng/ml were added during the incubation with oxLDL.
Bioartificial tendon (BAT)
BATs were prepared as previously described [15] with some modifications. Human tendon cells were seeded in the mixture of Purecol EZ Gel (Sigma-Aldrich, USA, # 5074), 5× DMEM, and 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) with or without 50 µg/ml oxLDL and 5 ng/ml TGF-β. The mixture was pipetted between the anchor stems of untreated Tissue Train plate (Flexcell International Corp., USA, #TT-5001U) in each well. After setting the BATs in the incubator, 3ml 1x DMEM media with or without oxLDL and TGF-β was added to each well and the plate incubated for 72 hours. The BATs were then scanned using an image scanner and the images analyzed in ImageJ to measure the diameter of the BATs.
oxLDL preparation
Isolated Human Low Density Lipoprotein (LDL) (Lee Biosolutions, USA, #360 − 10) was oxidized as previously described [12]. LDL was diluted in DPBS containing 5 µM copper sulfate and incubated at 37°C for 24 hours. The oxidation was stopped by adding 1 mM EDTA. The oxLDL was washed and concentrated using Amicon Ultra centrifugal filter unit with the pore size of 10 kDa MWCO (Millipore, USA, #UFC901024). The concentration of oxLDL was measured by a bicinchoninic acid protein assay kit. The concentrated oxLDL was aliquoted and stored in -80°C.
Gene expression analysis
The changes in mRNA expression was analyzed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) as previously described [16]. ΔCt values were calculated as Ct Target – Ct GAPDH) and used for graphs and statistical analysis. The oligo primers that were used are listed in Table 1.
Table 1
The sequences of oligo DNA used as primer for qRT-PCR
| Gene | Forward primer | Reverse primer |
| COL1A1 | CACCAATCACCTGCGGTACAGAA | CAGATCACGTCATCGCACAAC |
| COL1A2 | AGAGTGGAGCAGTGGTTACTA | GATACAGGTTTCGCCAGTAGAG |
| COL3A1 | AATCAGGTAGACCCGGACGA | TTCGTCCATCGAAGCCTCTG |
| DCN | GTCACAGAGCAGCACCTACC | TTGTCCAGACCCAAATCAGAACA |
| ILK | CCCACGACATGCACTCAATA | GACCAGGACATTGGAAAGAGAA |
| ITGB1 | TGATCCTGTGTCCCATTGTAAG | TGACCTCGTTGTTCCCATTC |
| LOX | TACCCAGCCGACCAAGATA | TGGCATCAAGCAGGTCATAG |
| MMP1 | CAGAAAGAGACAGGAGACATGAG | GAAGAGTTATCCCTTGCCTATCC |
| MMP2 | AGAGAACCTCAGGGAGAGTAAG | CCTCGAACAGATGCCACAATA |
| MMP3 | GTGAGGACACCAGCATGAA | GACCACTGTCCTTTCTCCTAAC |
| MMP8 | GGGTGGGCTCTAAATCCATTAT | CCAATCTCTGCCTCTGTCTTC |
| MMP9 | GAACTTTGACAGCGACAAGAAG | CGGCACTGAGGAATGATCTAA |
| MMP10 | GGCCCTCTCTTCCATCATATTT | CCTGCTTGTACCTCATTTCCT |
| MMP13 | AGCATCTGGAGTAACCGTATTG | CCCGCACTTCTGGAAGTATT |
| MMP14 | GCCGACTAAGCAGAAGAAAGA | TGTCGGCTTGGAGTTAAAGG |
| TGFB1 | CCTGCCTGTCTGCACTATTC | TGCCCAAGGTGCTCAATAAA |
| GAPDH | TCTTTTGCGTCGCCAGCCGAG | TGACCAGGCGCCCAATACGAC |
Enzymatic assays
To evaluate the activity of LOX and MMPs in protein lysate of human tendon cells, fluorometric assay kits were used. According to the MMP activity assay kit (Abcam, USA, #ab112146), protein lysates were mixed with 4-aminophenylmercuric acetate (APMA) and incubated at 37°C for 3 hours to activate MMPs. Then, MMP green substrate solution was added and incubated for 30 minutes. The fluorescence intensity was measured at 490/525 nm (Ex/Em) using a microplate reader (Infinite F500, Tecan, Austria). Amplite™ Fluorimetric Lysyl Oxidase Assay kit (AAT Bioquest, USA, #15255) was used according to the manual to measure LOX activity in protein lysate. The recorded relative fluorescence units from the MMP and LOX assay kits were normalized to the total protein concentrations measured by BCA assay.
Protein quantification: The secretion of TGF-β1 was quantified in the conditioned media of human tendon cells using an ELISA kit (R&D System, USA, #DY246) according to the manual. The level of COL1A1 protein in cell lysate was measured using western blot as previously described [16]. The western blots of Col1A1 and Vinculin were detected by IRDye 680 and HRP-conjugated antibodies, then visualized using the G:BOX Chemi XT4 Gel Documentation System (Syngene, UK) and Odyssey CLx Imaging System (LI-COR, USA) with default settings, respectively.
Statistics
All data were analyzed by either paired t-test or one-way ANOVA followed by Dunnett's multiple comparison test. GraphPad prism version 7.03 was used to analyze data and generate graphs. The number of biological replicates is reported in the figure captions. P values of less than 0.05 were regarded as statistically significant.