Table 2

Biomarkers investigated and laboratory assay protocols

Author (year)Biomarker typeBiomarker sourceSample collection and storageAssay typeAssay analysis
Abrams et al (2014)16Proteins: FAC (OD), IL-1β, IL-1RA, IL-6, Eotaxin, IFN-γ, IP-10, MCP-1, MIP-1β, PDGF-BB, RANTES, TNFα, VEGFSynovial fluidCollection of synovial fluid done at time of hip arthroscopy or arthroplasty by injecting 10 mL of sterile normal saline intra-articularly and aspirating back the fluid.
Lavasate placed into 2 mL tubes containing 130 µuL of protease inhibitor (Roche Diagnostics, Indianapolis, Indiana, USA) dissolved in phosphate-buffered saline solution (0.045 tablet/mL sample) at pH 7.4 and stored at −80°C.
ELISAHuman multiplex inflammatory cytokine panel and the BioPlex 200 System (Bio-Rad Laboratories, Hercules, California, USA) used to determine biomarker concentration in a 96-well plate.
Bedi et al (2013)10Proteins: COMP, CRPPlasma~3 mL of blood was collected from an antecubital vein into a K2-EDTA tube, spun down at 1000 g for 10 mins and plasma was removed and stored at −80°C.ELISAPlasma analysed in duplicates using ELISA for COMP and ultrasensitive CRP in a SpectraMax plate reader.
Chinzei et al (2016)11mRNA: IL-1β, IL-8, MMP-3, COL1A1, COL2A1, ACAN, ADAMTS-4, MMP-13Tissue: synovium, labrum, articular cartilageTissue samples collected from anterolateral femoral head–neck junction.Transcript analysisTotal RNA extracted from all tissue samples using TRIzol Reagent (Invitrogen (ThermoFisher Scientific)) and RNeasy spin columns (Qiagen, Valencia, California, USA).
cDNA was then synthesised using SuperScript II reverse transcriptase (Invitrogen).
Finally, qPCR was performed using 20 mL of reaction mixture containing SYBR Green PCR Master Mix (Applied Biosystems (ThermoFisher Scientific)) and primers on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
Gene expression levels were compared between FAI and OA groups using the comparative Ct cycle method.
Elias-Jones et al (2015)18Proteins: CD68, CD3, CD4, CD34, VEGF, CD206, IL-13, mast cell tryptaseTissue: labrumLabrum samples obtained at time of arthroscopy or arthroplasty.
Tissue samples immediately fixed in 10% (vol/vol) formalin for 4–6 hours and then embedded in paraffin.
Sections were cut to 5 mm thickness using a Leica-LM microtome (Leica Microsystems) and placed onto Superfrost Ultra Plus glass slides (Gerhard Menzel).
Immunohistological analysisAfter the removal of parrafin from the slides, the sections were stained with H&E and toluidine blue.
Then, the sections were stained with primary monoclonal antibodies directed against the biomarkers of interest.
Next, endogenous peroxidase activity was quenched, and non-specific antibody binding was blocked.
Afterwards, antigen retrieval was performed, and the sections were incubated with the primary antibody.
After two washes, the slides were incubated with an ImmPRESS Reagent kit (Vector Laboratories Ltd) per manufacturer instructions.
The slides were washed and incubated with Vector ImmPACT DAB chromagen solution.
Finally, the sections were counterstained with haematoxylin and dehydrated again before mounting.
Positive control with human tonsil tissue and negative control specimens were assessed as well.
Only structures that morphologically appeared as vascular and stained with either immunomarker (CD34/VEGF) were taken into account when determining the vessel count.
Fukushima (2017)19mRNA:
TNFα, IL-1β, IL-6, ADAMTS-4, MMP1, MMP3
Tissue: synovial membraneTotal RNA was extracted from harvested synovial samples using TRIzol (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions and was used as a template for first-strand cDNA synthesis using SuperScript III RT (Invitrogen).Transcript analysisThe PCR reaction mixture consisted of 2 µL cDNA, the specific primer set (0.2 µM final concentration) and 12.5 µL SYBR Premix Ex Taq (TaKaRa, Kyoto, Japan) in a final volume of 25 µL. Quantitative PCR was performed using a real-time PCR detection system (CFX-96; Bio-Rad Laboratories). The PCR cycle parameters consisted of an initial denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. mRNA expression was normalised to the level of GAPDH mRNA.
Hashimoto et al (2013)12mRNA: IL-1β, IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, CCL3L1, MMP-13, ADAMTS-4, COL2A1, ACANTissue: articular cartilageCartilage sample obtained from anterolateral femoral head–neck junction at site of mechanical impingement.
Sample immersed promptly in TRIzol reagent (Invitrogen) to avoid RNA degradation.
Transcript analysisRNA clean-up using RNeasyMini Kit (Qiagen).
Reverse-transcription performed with SuperScript II reverse transcriptase (Invitrogen) to synthesise first-strand cDNA.
qPCR performed using cDNA with 20 mL of reaction mixture containing SYBR Green PCR MasterMix (Applied Biosystems, Foster City, California, USA) and primers on a 7500 Fast Real-Time PCR system (Applied
Biosystems).
Results normalised to GAPDH levels.
Comparative Ct method used to evaluate the expression level of each target gene relative to control.
Shapiro et al (2016)17Proteins: FAC (OD), IFN-γ, IL-6, IL-1RA, IL-1β, MCP-1, Eotaxin, MIP-1β, IP-10, PDGF-BB, RANTES, TNFα, VEGFSynovial fluidCollection of synovial fluid done at time of hip arthroscopy by injecting 10 mL of sterile normal saline intra-articularly and aspirating back the fluid.
Lavasate placed in 2 mL tubes with 130 µL of protease inhibitor (Roche Diagnostics), dissolved in phosphate-buffered saline solution (0.045 tablet/mL sample) at pH 7.4 and frozen at −80°C.
ELISAHuman multiplex inflammatory cytokine panel and the BioPlex 200 System (Bio-Rad Laboratories) used to determine biomarker concentration.
The assay was performed through the use of antibody linked polystyrene beads with various fluorophore levels (validated against standard ELISA).
Heterogeneous sandwich ELISA used to determine FAC concentration, reported as OD.
  • ACAN, aggrecan; ADAMTS-4, a disintegrin and metalloproteinase with thrombospondin motifs-4; CCL3, chemokine (C-C) motif ligand 3; CCL3L1, chemokine (C-C) motif ligand 3-like 1; COL1A1, collagen type I alpha 1; cDNA, complementary DNA; COL2A1, collagen type II alpha 1; COMP, cartilage oligomeric matrix protein; CRP, C reactive protein; Ct, threshold cycle; CXCL1, chemokine (C-X-C) motif ligand 1; CXCL2, chemokine (C-X-C) motif ligand 2; CXCL3, chemokine (C-X-C) motif ligand 3; CXCL6, chemokine (C-X-C) motif ligand 6; FAD, fibronectin–aggrecan complex; FAI, femoroacetabular impingement; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; IL-1β, interleukin-1 beta; IL-8, interleukin-8; IP-10, interferon-inducible protein 10; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; MMP-3, matrix metalloproteinase-3; MMP-13, matrix metalloproteinase-13; OA, osteoarthritis; OD, optical density; PDGF-BB, platelet-derived growth factor-BB; qPCR, quantitative PCR; RANTES, regulated on activation normal T cell expressed and presumably secreted; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.