J. Kumm

Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Biobank samples

Abstract Background: Blood biomarkers are subject to pre-analytical variability. In many cases, the stability of important new tissue biomarkers during freeze cycles and storage has not been studied sufficiently. Methods: To test the stability of matrix metalloproteinases-7 (MMP-7) and their tissue inhibitors (TIMP-1), vascular growth factors (VEGF) and VEGF-receptor, serum samples were frozen and then thawed up to six times. The impact of storage temperature was investigated using an accelerated stability testing protocol. Stability at –20°C and –75°C was calculated using the Arrhenius equation. Results: The average concentration of TIMP-1 was stable, even after six freeze/thaw cycles. One thawing did not change the concentration of MMP-7 and VEGF-receptor. However, repeated freeze/thaw cycles increased the measured values significantly. Decreases in VEGF concentrations were dramatic, even after the first freeze/thaw cycle. According to the Arrhenius calculation, MMP-7 showed excellent stability, at least 5 years at –20°C and several 100 years at –75°C. The VEGF-receptor maintains 90% of its initial concentration at –20°C over 3 months, and decades at –75°C. TIMP-1 and VEGF showed poor stability with cryopreservation, even at –75°C. Conclusions: The stability of MMP-7, TIMP-1, VEGF or VEGF-receptor in biobanking is highly variable, and this should be taken into account in the interpretation of results. A temperature –20°C is unsuitable for prolonged storage of the biomarkers investigated, and repeated thawing of sera is not recommended. VEGF is especially unstable and should be quantitated using serum that has never been frozen.

Natural History of Intrameniscal Signal Intensity on Knee MR Images: Six Years of Data from the Osteoarthritis Initiative.

PURPOSE To assess the natural history of intrameniscal signal intensity on magnetic resonance (MR) images of the medial compartment. MATERIALS AND METHODS Both knees of 269 participants (55% women, aged 45-55 years) in the Osteoarthritis Initiative without radiographic knee osteoarthritis (OA) and without medial meniscal tear at baseline were studied. One radiologist assessed 3-T MR images from baseline and 24-, 48-, and 72-month follow-up for intrameniscal signal intensity and tears. A complementary log-log model with random effect was used to evaluate the risk of medial meniscal tear, adjusting for age, sex, body mass index, and knee side. RESULTS At baseline, linear intrameniscal signal intensity in the medial compartment was present in 140 knees (26%). Once present, regression only in a single knee was observed. In 31 knees (19%) with linear intrameniscal signal intensity at any of the first three time points, the signal intensity progressed to a tear in the same segment, and in a single knee, the tear occurred in an adjacent segment. The corresponding number of tears without prior finding of intrameniscal signal intensities was 11 (3%). In the adjusted model, the hazard ratio for developing medial meniscal tear was 18.2 (95% confidence interval: 8.3, 39.8) if linear intrameniscal signal intensity was present, compared when there was no linear signal intensity. There was only one of 43 knees with injury reported in conjunction with the incident tear. CONCLUSION In middle-aged persons without OA, linear intrameniscal signal intensity on MR images is highly unlikely to resolve and should be considered a risk factor for medial degenerative meniscal tear.

Diagnostic and prognostic value of bone biomarkers in progressive knee osteoarthritis: a 6-year follow-up study in middle-aged subjects

OBJECTIVE To determine the value of bone markers in early-stage progressive knee osteoarthritis (OA), a population-based cohort of middle-aged subjects with chronic knee complaints was followed over 6 years (two consecutive two 3-year periods). METHODS Tibiofemoral (TF) and patellofemoral (PF) radiographs were graded in 128 subjects (mean age at baseline 45 ± 6.2 years) in 2002, 2005 and 2008. Bone formation was assessed by the serum concentration of procollagen type I amino-terminal propeptide (sPINP); bone resorption by the level of the C-terminal cross-linked telopeptides of type I collagen (sCTx-I); and bone mineralization by the values of osteocalcin (sOC) by electrochemiluminescence immunoassay. A novel marker of bone resorption, urinary osteocalcin midfragments (uMidOC), was assayed using enzyme linked immunosorbent assay (ELISA). RESULTS Several diagnostic associations were found between the bone markers (PINP, OC, MidOC) and progressive OA expressed by TF osteophytosis. The increasing output of MidOC demonstrated several-fold higher risk for progressive TF osteophytosis [odds ratio (OR) 5.32; 95% confidence interval (CI) 1.41-20.06, P = 0.014] than other bone markers. The values of PINP had prognostic value for subsequent more severely expressed knee OA progression [r(s) = 0.460, P = 0.005]. CONCLUSIONS Bone metabolism is activated in early-stage knee OA. OA progression was preceded by the enhanced bone formation (by PINP) and accompanied by the activation of bone formation (by PINP), non-collagenous bone resorption (by MidOC), as well as by changes in mineralization (by OC). All three bone markers had diagnostic value, and one of them, PINP, had also a predictive value for knee OA progression, especially for progressive osteophytosis.

Risk factors for meniscal body extrusion on MRI in subjects free of radiographic knee osteoarthritis: longitudinal data from the Osteoarthritis Initiative

OBJECTIVE To determine risk factors associated with increased meniscal body extrusion on knee magnetic resonance (MR) images in subjects free of radiographic osteoarthritis (OA). METHODS We selected 340 subjects (aged 45-55 years, mean [SD] body mass index 26.7 [4.4], 51% women) with Kellgren-Lawrence grade 0 in both knees and bilateral knee MR images available at the baseline, 24 months, 48 months, and 72 month exam from the Osteoarthritis Initiative (OAI). We assessed mid-coronal 3-T MR images from baseline through the 72-month exam. One observer measured widths of the tibia plateau and medial or lateral meniscal body extrusion for baseline and 72 months follow-up. Another observer assessed meniscal integrity at all four time points. We calculated an extrusion ratio ([meniscal body extrusion]/[tibia width] × 100) to account for knee size. We evaluated risk factors for increased meniscal body extrusion ratio from baseline to 72 months by a multivariable linear regression mixed model for medial and lateral compartment, respectively. RESULTS In the medial compartment female sex (β = 0.35; 95% confidence interval [CI] 0.16-0.53), incident meniscal tear (β = 0.29; 95% CI 0.22-0.55), and the baseline value of the extrusion ratio (β = 0.63; 95% CI 0.56-0.70) were associated with increased extrusion ratio by 72 months. Results were similar for the lateral compartment. CONCLUSIONS Only female sex, incident meniscal tear, and higher baseline value of extrusion are risk factors for increased meniscal body extrusion in subjects free of radiographic OA. The results suggest that meniscal extrusion may contribute to and mediate the well-known increase in knee OA incidence in middle-aged women.

The ADAM12 is upregulated in synovitis and postinflammatory fibrosis of the synovial membrane in patients with early radiographic osteoarthritis

OBJECTIVE To investigate the possible association between ADAM12 (disintegrin and metalloproteinase domain12) expression in the synovium and the histological synovitis of patients with radiographic knee osteoarthritis (rKOA). METHODS The synovial biopsy samples were harvested from 44 subjects with chronic knee complaints during arthroscopy. In all subjects, the radiographs of both knee joints were performed for rKOA assessment. Histological features of synovitis were graded 0-3 in synovial samples. Messenger RNA (mRNA) of two ADAM12 splice variants [ADAM12-S(hort) and ADAM12-L(ong)] and the identical region for both-ADAM12-B(oth) were measured by real-time reverse transcription-PCR in all synovial samples (TaqMan® gene expression assay). Immunohistochemical staining of the synovial membrane with ADAM12 antibody was performed in 42 subjects. RESULTS ADAM12 mRNA was expressed in all synovial samples, whereas the main part of overall expression consisted of its long isoform (ADAM12-L). ADAM12 protein expression was detected in 80% of the synovial samples and correlated with mRNA expression (ρ=0.30, P<0.05). The expression of ADAM12 mRNA and protein in synovium correlated with the severity of histological synovitis (ρ=0.28, P<0.05 for ADAM12-B mRNA, R2=0.20, P<0.05 for ADAM12 protein). Out of several features of synovitis the expression level of both splice variants correlated only with the grade of fibrosis in the synovium (ρ=0.30, P<0.05 for ADAM12-L and ρ=0.33, P<0.05 for ADAM12-S). CONCLUSIONS ADAM12 is upregulated in the synovial tissue during synovitis on mRNA and protein level. We suggest that ADAM12 could be implicated in the development of KOA-associated synovitis, especially in the occurrence of postinflammatory fibrosis.

Two Single-Nucleotide Polymorphisms in ADAM12 Gene Are Associated with Early and Late Radiographic Knee Osteoarthritis in Estonian Population

Objectives. To investigate associations of selected single-nucleotide polymorphisms (SNPs) in ADAM12 gene with radiographic knee osteoarthritis (rKOA) in Estonian population. Methods. The rs3740199, rs1871054, rs1278279, and rs1044122 SNPs in ADAM12 gene were genotyped in 438 subjects (303 women) from population-based cohort, aged 32 to 57 (mean 45.4). The rKOA features were evaluated in the tibiofemoral joint (TFJ) and patellofemoral joint. Results. The early rKOA was found in 51.4% of investigated subjects (72% women) and 12.3% of participants (63% women) had advanced stage of diseases. The A allele of synonymous SNP rs1044122 was associated with early rKOA in TFJ, predominantly with the presence of osteophytes in females (OR 1.57; 95% CI 1.08–2.29, P = 0.018). The C allele of intron polymorphism rs1871054 carried risk for advanced rKOA, mostly to osteophyte formation in TFJ in males (OR 3.03; 95% CI 1.11–7.53, P = 0.018). Also the CCAA haplotype of ADAM12 was associated with osteophytosis, again mostly in TFJ in males (P = 0.014). For rs3740199 and rs1278279, no statistically significant associations were observed. Conclusion.   ADAM12 gene variants are related to rKOA risk during the early and late stages of diseases. The genetic risk seems to be predominantly associated with the appearance of osteophytes—a marker of bone remodelling and neochondrogenesis.

Urinary osteocalcin and other markers of bone metabolism: the effect of risedronate therapy

Objective. Serum osteocalcin (S‐OC) is widely used as an index of bone formation. However, there is evidence that some urinary fragments of OC reflect resorption and might be useful in monitoring antiresorptive therapy. Here, we report 6‐month changes in urinary midfragments of osteocalcin (U‐MidOC) and other bone turnover markers in response to risedronate treatment. Material and methods. The study group comprised 19 patients with postmenopausal osteoporosis, aged 49–66 years, and receiving risedronate therapy. Fifty‐four premenopausal women served as controls. Osteoporosis was diagnosed by lumbal bone mineral density (BMD). Urinary osteocalcin was measured by the U‐MidOC assay for midfragments. Bone formation was assessed by S‐PINP and S‐OC, and resorption by S‐CTx‐I. Results. At baseline, U‐MidOC was significantly correlated only with S‐OC. After the 1st month of therapy, a similar decrease was observed in the values of U‐MidOC and S‐CTx‐I, but in formation markers S‐P1NP and S‐OC only after three months. The rapid decrease in U‐MidOC, analogous to S‐CTX‐I, and the different kinetics for urinary and serum OC suggest that urinary OC midfragments are more associated with resorption than S‐OC. An association was also observed between the 1‐month change in U‐MidOC and 12‐month gain in lumbar BMD. The response in U‐MidOC after only the 1st month of therapy makes it a potential marker for monitoring the effect of risedronate, presumably reflecting different aspects of bone resorption than S‐CTx‐I does.

Structural abnormalities detected by knee magnetic resonance imaging are common in middle-aged subjects with and without risk factors for osteoarthritis

Background and purpose — Few data are available regarding structural changes present in knees without radiographically evident osteoarthritis (OA). We evaluated the prevalence of findings suggestive of knee OA by magnetic resonance imaging (MRI) in middle-aged subjects without radiographic OA with or without OA risk factors. Patients and methods — 340 subjects from the Osteoarthritis Initiative, aged 45–55 years (51% women) with Kellgren–Lawrence grade 0 in both knees, who had 3T knee MR images were eligible. 294 subjects had risk factors and 46 were without risk factors. MR images were assessed using the MOAKS scoring system. Results — At least 1 MR-detected feature was found in 96% (283/294) of subjects with risk factors and in 87% (40/46) of those without. Cartilage damage (82%), bone marrow lesions (60%), osteophytes (45%), meniscal body extrusion (32%), and synovitis–effusion (29%) were the most common findings in subjects with risk factors, while cartilage damage (67%), osteophytes (46%), meniscal body extrusion (37%), and bone marrow lesions (35%) were most common in subjects without. The prevalence of any abnormality was higher in subjects with OA risk factors than in subjects without (prevalence ratio adjusted for age and sex 1.3 [95% CI 1.1–1.6]), so was prevalence of subchondral cysts and bone marrow lesions. MR-detected structural changes were more frequent in patellofemoral joints. Interpretation — Our findings highlight the great challenge in distinguishing pathological features of early knee OA from what could be considered part of “normal ageing.” Bone marrow lesions were more frequently found in subjects with multiple OA risk factors.

105 KNEE OSTEOARTHRITIS PROGRESSION DURING 6 YEARS FOLLOW-UP: THE PROGNOSTIC VALUE OF BONE AND CARTILAGE BIOMARKERS

Purpose: To investigate the prognostic value of bone-cartilage biomarkers in predicting knee OA progression during 6 years follow-up. Methods: A population cohort aged 35-55 years (n=141) was investigated at baseline and after 3 and 6 years follow-up. Tibiofemoral (TF) and patellofemoral (PF) radiographs were graded for OA. Progression was documented if presence of osteophytes and/or joint space narrowing (JSN) was described in subjects with no previous radiographic changes or in case of radiographic deterioration during 6 years. At baseline and at 3 years follow-up, biomarkers of bone resorption (CTx-I), formation (PINP), osteocalcin (OC) were assessed by ECLIA and cartilage oligomeric matrix protein (COMP) by ELISA. Specific markers of cartilage degradation (U-CTx-II) and synthesis (PIIANP) were assayed by ELISA only at 3-years follow-up. For statistics nonparametric methods were used. Results: Within 6 years follow-up, progression in TF joint was observed in 38% of cases, mostly from OA grade 0 to 1. At 3 years follow-up we documented radiographic OA progression in 16 men and 38 women out of 141, and at 6 years follow-up in 21 men and 49 women out of 131. In women, the progression in TF osteophytes during 6 year follow-up was related to higher levels of COMP compared to non-progressors (p=0.011). The higher COMP, OC and U-CTx-II levels in case of TF osteophyte progression was found already at 3 years follow-up. The women who developed TF JSN progression had lower PINP and higher PIIANP levels compared to non-progressors (p levels 0.030 and 0.031, respectively). Progression in PF JSN was related to higher COMP and U-CTx-II values (p levels 0.047 and 0.003, respectively). Increased U-CTx-II levels was found in women who had progression in TFOA and PFOA simultaneously (p=0.030). Baseline PINP levels were higher in those who developed TFOA and PFOA during 6 years follow-up (p=0.007). Baseline CTx-I levels were higher in case of PF osteophyte progression (p=0.037). In men, the progression in TF and PF osteophytes was related to higher levels of COMP (p levels 0.003 and 0.025, respectively). The progression in PF osteophytes during 6 years was related to the increase in PIIANP (p=0.019). The progression in JSN was associated with lower OC levels (p=0.045). Conclusions: • The 6 years follow-up demonstrated changes in radiographic stage as well as in the levels of the set of biomarkers. • The pattern of biomarkers differed by gender. • In both genders, S-COMP levels that reflect the status of several soft joint tissues was associated with the development of TF osteophytes. • In women, increased degradation of articular cartilage based on U-CTx-II associated with progression in PF JSN. The same process was found in cases when TFOA and PFOA progression was combined. • Increased synthesis of articular cartilage based on S-PIIANP was related to progression in TF JSN that might refer to enhanced reparative process. • The activation of bone metabolism was demonstrated in future knee OA progressors already at baseline by the increase in osteoblast-related biomarkers S-OC and S-PINP. This study is supported by ECTS/Czech Society Young Investigator Grant. 106