Abstract
Background: Age and oxidative stress have been implicated as the main causes of the cartilage damage in osteoarthritis (OA). The osteogenic protein–1(OP–1), a member of the bone morphogenetic family (BMP), plays a major role in cartilage repair.
Aim: To correlate the OP–1 in the synovial fluid with the age and antioxidants like superoxide dismutase (SOD), glutathione peroxidase (GPx) and uric acid (UA) in the blood and with the cartilage metabolic markers like hyaluronic acid (HA) and keratan sulphate (KS) in the synovial fluid of osteoarthritic patients.
Method: Seventy five osteoarthritic patients were taken up for the study. Heparinized blood samples were collected for the estimation of SOD, GPx and UA. Synovial fluid was aspirated for the estimation of HA, KS and OP–1. OP–1 was estimated by using a polyclonal antibody (anti–OP–1) which was produced against OP–1 in the mouse ascitic fluid by sandwich ELISA.
Result: Age and UA showed a significant correlation with OP–1. On the other hand, the correlations between OP–1 and the antioxidants (SOD and GPx) and the cartilage metabolic markers (HA and KS) were insignificant in osteoarthritic patients.
Conclusion: The study suggests about the important role of OP-1 in cartilage aging and degeneration, which may help us in understanding the potential for the therapeutic interventions in the treatment of cartilage degenerative processes.
Keywords: Osteogenic protein-1, Antioxidants, Cartilage metabolic markers
Introduction
Osteoarthritis (OA) is characterized by the degradation of articular cartilage, which causes pain and disability [1], among which knee OA is the most common form [2]. The degradation of the articular cartilage is mainly caused by excess loading, aging, and a metabolic imbalance in the tissues [3–5]. The molecules derived from the cartilage, which are present in the synovial fluid, may be a markers of the biosynthetic changes or of degradative changes [6]. Keratan sulphate (KS) and Hyaluronic acid which are present in the synovial fluid have been studied as cartilage metabolic markers [7]. Reactive oxygen species have been seen to play a role in the pathogenesis of OA by causing oxidative damage which is generated by the cells within the joints [8]. To prevent the ROS toxicity, our body possess well co-ordinated antioxidant systems like SOD and GPx and non enzymic antioxidants like UA. The regeneration and repair of the cartilage have become one of the major obstacles in the current orthopaedics. OP-1, which is known as the Bone morphogenetic protein -7 (BMP-7), belongs to the transforming growth factor (TGF- β) superfamily, plays a crucial role in the cartilage repair and the maintenance of the articular cartilage integrity [9]. Many biochemical markers have been studied for monitoring the cartilage destruction [10,11]. The relationship between OP-1 in the synovial fluid and the antioxidants in the blood and the cartilage metabolic markers in the synovial fluid among the osteoarthritic patients, has not been studied much.
The present study aimed at finding the correlation between OP-1 and age, the cartilage metabolic markers and the antioxidants in osteoarthritic patients.
Material and Methods
This study was conducted in the Department of Biochemistry, Sikkim Manipal Institute of Medical Sciences (SMIMS),India.
Patient Selection
Seventy five clinically diagnosed patients with primary knee osteoarthritis, who were of the age group of 40–80 years, were selected from among the patients who attended the Sir Thutop Namgyal Memorial Hospital (STNM) and the Central Referral Hospital (CRH), Gangtok, India. Informed consents were obtained from the participants prior to the study.
Inclusion Criteria
The patients were selected according to the criteria which was described by the American College of Rheumatology [12].
Exclusion Criteria
Smokers, alcoholics, diabetics, hypertensives and the patients who were suffering from any other systemic diseases, which included the patients with post traumatic osteoarthritis, were excluded from the study.
The Sample Collection and Processing
Heparinized venous blood samples were collected and a part of it was centrifuged and the plasma was used for the estimation of UA (Uricase Method, RFCL, India) [13]. The other part of the venous blood was haemolyzed and it was used to estimate the activity of SOD by using the Ransod kit and the activity of GPx by using the Ransel kit (Both were obtained from the Randox Laboratory Ltd, UK) [14,15]. The levels of the antioxidants were analyzed in a semi-auto analyzer (mod: CHEM-5 plus V2, Transasia, ERBA, India). The synovial fluid was aspirated and centrifuged to remove the debris and the supernatant was used for the estimation of HA and KS by doing a sandwich enzyme linked Immunosorbent (ELISA) (Blue Gene Biotech Co Ltd, Shanghai, China) [16]. Their levels were read in an ELISA reader (RFCL, India).
OP–1 Estimation
OP–1 was estimated by using anti–OP–1(IgG) which was produced against OP–1 in the mouse ascitic fluid in our laboratory, as was described by Cheirmaraj et al., [17], and anti–OP–1(sc-9305, Santa Cruz, USA) which was conjugated with horse radish peroxidase (HRP, Bangalore Genie,Bangalore,India), by Sandwich ELISA, in the synovial fluid of osteoarthritic patients .To calculate the concentrations of OP–1 in the synovial fluid, a standard curve was prepared by using known concentrations of OP–1(78.4 ug/ml) which were diluted in TBS/Tween at concentrations which ranged from 0 ng/ml to 250 ng/ml.
Statistical Analysis
SPSS, version 16 was used for the statistical analysis. Pearson’s correlation was used to study the correlation between OP–1 and age, the cartilage metabolic markers and the antioxidants in osteoarthritic patients. A p value which was <0.05 was taken as significant in our study.
Results
The correlation between OP–1 in the synovial fluid and the antioxidants (SOD, GPx, UA) in the blood and the cartilage metabolic markers (HA, KS) in the synovial fluid and the ages of the osteoarthritic patients have been shown in the [Table/Fig-1, 2 and 3].
[Table/Fig-1]:
Correlation between OP-1 in the synovial fluid and antioxidants (SOD, UA, GPx) in the blood of osteoarthritic patient. p< 0.05 considered significant*, not significant°
| n=75 | OP–1(ng/ml) | |
|---|---|---|
| r–value p value | ||
| SOD(u/ml) | - 0.067 | 0.567° |
| GPx(u/l) | 0.139 | 0.235° |
| UA(mg/dl) | 0.237 | 0.041* |
[Table/Fig-2]:
Correlation between OP-1 and the cartilage metabolic markers in the synovial fluid of osteoarthritic patient p< 0.05 considered significant*, not significant°
| n=75 | OP–1 | |
|---|---|---|
| r value p value | ||
| HA (ng/ml) | 0.78 | 0.507° |
| KS(ng/ml) | 0.65 | 0.581° |
[Table/Fig-3]:
Correlation between OP-1 in the synovial fluid and age of osteoarthritic patients P<0.05 significant, Significant*
| n=75 | OP–1 | |
|---|---|---|
| r value p value | ||
| Age | -0.283 | 0.041* |
In the study, OP–1 showed a negative significant correlation with the age (r= -0.283). While the correlation between OP–1 and Uric acid (r=0.237) was positively significant, GPx, HA and KS (r=0.139, 0.78 and 0.65) showed an insignificant positive correlation. Whereas the correlation between OP–1 and SOD (r= - 0.067) was negatively insignificant.
Discussion
The presence of OP–1 was documented for the first time in the human synovial fluid [7]. OP–1 has been implicated to have a wide range of anabolic and anti-catabolic activities [9]. In this study, the correlation between OP–1 and the age of the osteoarthritic patients was found to be negatively significant. This could be the result of the increase in the cartilage aging and degeneration [18–20], which had led to the low level of OP–1, thus contributing to the development of osteoarthritis. On the other hand, OP–1 in the synovial fluid showed a significant positive correlation with the UA in the blood of osteoarthritic patients, which may indicate the genetic and hormonal influence [21, 22] of uric acid.
We could not find a significant correlation between OP–1 in the synovial fluid with the antioxidants (SOD, GPx) in the blood and with the cartilage metabolic markers (HA and KS) in osteoarthritic patients. This may be due to the limitations of our study, where the antioxidant assay and OP–1 were studied in different body fluids. In the future, further studies would be required to correlate the antioxidants, the cartilage metabolic markers and OP–1 in the blood and the synovial fluid, to get a true picture about the relationship between these parameters in OA.
Conclusion
The results of our study suggest the important role of OP-1 between aging and OA. The relationship of OP–1 with the age and UA may help us in understanding the mechanisms of the development and the progression of OA, which may pave way for developing different preventive and therapeutic strategies.
Financial or Other Competing Interests
None.
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