Association of inflammatory, genetic, and epigenetic markers with Kellgren-Lawrence grading in post-traumatic osteoarthritis of knee: A protocol

Table 1 Kellgren-Lawrence radiographic osteoarthritis grading[10]

Grades	Changes seen in X-ray
Grade 0	None. Absence of X-ray changes
Grade 1	Not severe. Doubtful narrowing of joint space and possible osteophytic lining
Grade 2	Minimal severity. Definite osteophytes and possible narrowing of joint space
Grade 3	Moderate severity. Moderate multiple osteophytes, definite narrowing of joint space and some sclerosis and possible deformity of bone ends
Grade 4	Severe. Large osteophytes, marked narrowing of joint space, severe sclerosis and definite deformity of bone ends

Table 2 List of biomarkers to be analyzed in the study

Biomarkers	Variable	Method of analysis
Cytokines	Serum IL-1β, IL-6, IL-10	ELISA
Collagen markers	Urine CTX-II	ELISA
Non collagenous proteins	Serum COMP	ELISA
Growth factors	Serum TGF-beta1	ELISA
Oxidative stress markers	Serum oxidized-LDL	ELISA
Enzymes	Serum MMP13	ELISA
miRNA	Plasma miR-146a-5p, miR-34a-5p	qRT-PCR
Genetic studies- SNP	Plasma GDF5, SMAD4	PCR

PCR: Polymerase chain reaction; IL-interleukin; CTX-II: C-terminal telopeptide of type II; COMP: Cartilage oligomeric matrix protein; TGF -beta1: Transforming growth factor beta 1; MMP13: Matrix metalloproteinase-13; GDF5: Growth differentiation factor 5; SMAD4: SMAD family member 4, Mothers against decapentaplegic homolog 4; ELISA: Enzyme linked immunosorbent assay; qRT-PCR: Quantitative real time polymerase chain reaction.

Table 3 Method specifications of analysis of biomarkers

Biomarker	Company code no	Measurement range	Sensitivity	CV% (Intra assay)	CV% (Inter assay)
IL-6	Elabscience E-EL-H6156	1.56-100 pg/mL	0.94 pg/mL	4.4-5.7	3.0-6.4
IL-1	Diaclone 850.006.096	15.6-500 pg/mL	6.6 pg/mL	4.5-5.3	8.7-9.9
IL-10	Diaclone 950.060	12.5-400 pg/mL	4.9 pg/mL	3.5-6.5	7.5-10.0
TGF-beta 1	Elabscience E-EL-0162	0.16-10 ng/mL	0.1 ng/mL	4.4-4.8	4.7-6.3
MMP13	Elabscience E-El-H6023	15.63-1000 pg/mL	9.38 pg/mL	4.5-5.3	4.7-5.7
Oxidized LDL	Elabscience E-EL-H-0654	62.5-4000 pg/mL	37.5 pg/mL	4.6-5.8	4.8-5.3
CTX-II	Elabscience E-EL-H0837	0.16-10 ng/mL	0.09 ng/mL	3.0-6.0	4.76-6
COMP	Elabsience E-EL-H0654	0.63-40 ng/mL	0.38 ng/mL	4.8-6.8	5.8-8.6

IL: Interleukin; TGF-beta1: Transforming growth factor beta 1; MMP13: Matrix metalloproteinase-13; LDL: Low density lipoprotein; CTX-II: C-terminal telopeptide of type II; COMP: Cartilage oligomeric matrix protein.