Recombinant platelet-derived growth factor-BB and hyaluronic acid stimulates knee cartilage regeneration by forming higher chondrocytes count and lower YKL-40 level in rats model

Abstract

Objective

Osteoarthritis is caused by cartilage degeneration arising from cartilage degradation of type II collagen which synthesis also deteriorated. Nowadays, osteoarthritis is still difficult to handle because of the irreversibility and progressivity of the disease. Regenerative therapy offers a great challenge and better result for osteoarthritis treatment. This study aims to prove that the administration of recombinant platelet-derived growth factor-BB (rrPDGF-BB) with hyaluronic acid (HA) can stimulate the higher forms of chondrocyte and lower the YKL-40 levels as a specific marker of cartilage degradation in mouse knee osteoarthritis model.

Method

This was an experimental study, post-test only control group design using white Wistar rats as subjects that were induced by monosodium iodoacetate (MIA) to create osteoarthritis (OA). The treatment group was given treatment group was given rrPDGF-BB and HA, while no such treatment was given to the control group. The chondrocyte cell count was examined with routine histopathology, and YKL-40 levels were calculated by ELISA.

Result

Statistical analysis using independent t-test showed that the mean difference in the number of cell counts of chondrocytes was significantly higher than the control group. While the mean difference of YKL-40 level in the treatment group was significantly lower compared to the control group with.

Conclusion

We conclude that the administration of rrPDGF-BB and HA in mouse knee joint osteoarthritis model have a regenerative effect on knee joint cartilage characterized by a higher number of chondrocytes and lower YKL-40 levels.

1. Introduction

Osteoarthritis (OA) is a degenerative condition of the joint, involving articular cartilage, subchondral bone, ligament, and muscle. This condition affected over 30 million US people which occurs about 10% in men and 13% in women aged 60 years or older.¹ Considering the severity of the condition, targeted treatment holds a crucial role.

Pathogenesis of OA is initiated by a progressive destruction of articular cartilage through the elucidative molecular mechanism. There are several biomarkers available for diagnosis of OA in the early stages. However, none is suitable for monitoring its progressivity. In the last few years, chitinase and chitinase-like molecules named YKL-40 have been developed. Increased level of YKL-40 is associated with inflammation, tissue remodeling process and neoplastic process in several types of cancer, even though the actual biological activity is still unknown.^2,3

In arthritic condition, YKL-40 is expressed and secreted by inflammatory cells and chondrocyte, thus making it a suitable marker of synovium membrane replacement due to OA.^3,4 The relationship between YKL-40 and conventional parameters, such as proinflammatory cytokine and sonographic examination, showed that YKL-40 play a role in the development of the inflammatory process.³ Proinflammatory cytokines such as interleukin-6 (IL-6) and IL-17 in the synovial fluid will increase the production of YKL-40 in chondrocyte cells of primary OA, thus supporting its role as a diagnostic marker.

Considering the irreversibility of OA pathogenesis, a treatment modality that blocks the degenerative process is needed. Platelet-derived growth factor (PDGF) is known to have the ability to assist the regenerative process in cartilage undergoing degradation. There are a number of PDGF produced by platelet at the time of fracture, like PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD. PDGF-BB has the characteristics of chemotactic and mitogenic.^5,6 It also mediates the process of tissue repair through proliferation and cell differentiation (fibroblast, chondrocytes, smooth muscle cells, and capillary endothelial cells), induction of matrix synthesis (fibronectin, hyaluronic acid, proteoglycan, and collagen), and arrangement of production and secretion of fibroblast collagenase.^5,6

In order to increase the PDGF-BB working time during regeneration and metabolism of cartilage, hyaluronic acid (HA) may be utilized as growth scaffold and carrier for PDGF-BB.⁷ HA is known to assist in the reconstitution of synovial fluid, increasing elasticity, viscosity, and improve joint function.^8,9 It has a significant effect on the metabolism of cartilage.¹⁰ Despite its synergistic effect, there were no previous studies reporting the administration of a combination of PDGF-BB and HA for OA condition.

This study will investigate the effect of PDGF-BB and HA as a scaffold in influencing the progression of OA. The experiment was done on rat's osteoarthritic knee induced by monosodium iodoacetate (MIA).

2. Material and methods

This is an experimental study using randomized post-test only control group design. All procedures were approved by the Committee of Ethical Research of our University. A total of 36 rats of 12 weeks old were involved in this study.

All rats had their knees induced to create the condition of OA by administrating 0.3 mg/150 mg body weight of monosodium iodoacetate (MIA). The MIA was administrated intraarticularly under anesthesia using ketamine 44–100 mg/kgBW. The rat was fixed in supine position with one hind limb flexed in 90°. The MIA was injected into the knee joint through the patellar tendon. After the injection, the rats were placed above the warming pad until it recovers.

They were then divided into two groups. The rats in the treatment group (n = 16) were given rrPDGF-BB 10 ng/mL and HA 10 mg/mL intraarticularly once a week for two weeks, while those in the control group (n = 16) were not. After 4 weeks all rats were euthanized using intramuscular ketamine 132–300 mg/kgBW, and they were then tested for YKL-40 level and chondrocyte count.

The blood sample was taken from the femoral artery and the level of YKL-40 in the serum was assessed with ELISA method. The knee joint was exposed, and after the macroscopic sample was retrieved we went on with a microscopic count of chondrocytes. The specimens were stained with hematoxylin-eosin (HE)and reviewed under 400x magnification.

Statistical analysis was performed using an independent t-test to compare the mean post-test value of the control and treatment groups in order to perceive the effect of each variable. In all instances, p < 0.05 was considered statistically significant.

3. Result

All 32 rats were analyzed at the end of the study. There were no complications recorded in this study. The mean number of chondrocyte in the control and treatment of groups were 49.81 ± 6.99 and 58.25 ± 6.81 cells per field, respectively (p < 0.05). The level of serum YKL-40 in the control and treatment groups were 577.50 ± 130.99 and 378.24 ± 87.27 ng/mL, respectively, as seen in Table 1.

Table 1.

Chondrocyte count and level of serum YKL-40 in both groups (mean ± SD).

Variables	Groups(mean ± SD)		Mean difference	95% CI	p-value
	Treatment	Control
Number of chondrocyte cells (cells per field of view)	58.25 ± 6.82	49.81 ± 6.99	8.44	3.45–13.43	0,002
Level of serum YKL-40 (ng/mL)	378.24 ± 87.27	577.50 ± 130.99	199.26	280.13–118,39	<0,001

The data showed that the chondrocyte count was higher in the treatment group compared to the control group, with the 95% confident interval (CI) was 3.45–13.43 as seen in Fig. 1. And serum YKL-40 level was lower in the treatment group compared to the control group (95%CI = 280.13–118.40) as seen in Fig. 2 The histopathology of the knee cartilage showed degenerative changes that consistent with osteoarthritis in both groups. But the treatment group showed a slightly smooth cartilage surface with moderate surface chondrocyte cell population and lower clustered chondrocyte cells compared than control group as seen in Fig. 3, Fig. 4. The collagen type II matrix was fine arranged with a little vascular infiltration and the tidemark was not duplicate. This finding explained that the rrPDGF-BB with HA as scaffold may promote synthesis and decrease degradation of cartilage.

Fig. 1.

Individual value plot of the mean standard deviation of chondrocytes count with error bars represent 95% confident interval over the experiment group and control group.

Fig. 2.

Individual value plot of the mean standard deviation of the YKL-40 level with error bars represent 95% confident interval over the experiment group and control group.

Fig. 3.

TheHE-stained histopathology of knee cartilage in control group under a light microscope with 400x magnification.

Fig. 4.

The HE-stained histopathology of knee cartilage in experiment group with rrPDGF-BB and HA under a light microscope with 400x magnification.

4. Discussion

This study found that the combined administration of rrPDGF-BB and HA has a direct effect on chondrocytes isolated from cartilage. The experimental design of this study allowed to observe the direct changes as shown by increasing number of chondrocytes and decreased YKL-40 level which is related to reduced inflammation.

The previous study has described the significant increase of proliferation of chondrocyte culture to which rrPDGF-BB was administered.¹¹ Exposure of rrPDGF-BB in culture results in an increase of cell proliferation and production of proteoglycan in the cartilage. It also prevents accelerated maturation of cells along the endochondral line. In order to fill a defect with cartilage, it is very crucial for the cartilage to be able to proliferate and differentiate.³ The effect was mediated by tyrosine kinase, which was activated when a ligand binds to the receptor.

Another interesting addition to this study is hyaluronic acid, a substance which helps in the reconstitution of synovial fluid, increasing elasticity, viscosity and hence, the joint function. This substance is usually given in its salt form through intraarticular injection to the knee joint when OA does not respond to other therapy.

HA is a widely accepted biomaterial and it has been applied in various fields, including bioscaffold for tissue culture and cosmetic products. Therefore, there is a possibility of expanding its use in the management OA. Exogenous HA has the capacity to infiltrate articular cartilage in both normal individual and OA patients, binding HA receptor expressed on chondrocyte surface.¹²

However, HA, like other native materials, has its own limitation related to the sensitivity of these materials to processing condition. Furthermore, we must also consider its physical and chemical interaction with the growth factor applied to it.³In our study, rrPDGF-BB was combined with HA and resulted in a positive outcome. We would recommend investigating further the process of combining the 2 substances, which was applied in our study and how it benefited OA management.

In order to illustrate the inflammation process, we used YKL-40 as the marker. YKL-40 is expressed and secreted by inflammatory cells and chondrocytes in arthritis. It was reported to be found in a superficial and mid layer of cartilage undergoing OA, supporting the notion that matrix injury triggers YKL-40 production.⁴ The use of YKL-40 is expected to aid in the diagnosis of OA. We have shown in this study that the level of YKL-40 detected in serum decreased with the administration of intraarticular rrPDGF-BB, indicating its use in tracking the progress of inflammation in OA.

5. Conclusion

Intraarticular injection of rrPDGF-BB and HA helped to increase the number of chondrocytes in rat's knee OA model and lower YKL-40 levels. Further studies are needed to investigate the best dose and method of administration in order to reach the maximum benefit of this regimen.

Conflict of interests

The author declares that there is no conflict of interests regarding the publication of this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: