Elsevier

Joint Bone Spine

Volume 78, Issue 6, December 2011, Pages 604-610
Joint Bone Spine

Original article
Comparing different physical factors on serum TNF-α levels, chondrocyte apoptosis, caspase-3 and caspase-8 expression in osteoarthritis of the knee in rabbits☆

https://doi.org/10.1016/j.jbspin.2011.01.009Get rights and content

Abstract

Objective

To study the therapeutic effects that different physical factors may have on rabbits with osteoarthritis of the knee.

Methods

A total of 64 rabbits were randomised and organised into eight groups, eight of which were each assigned a different physical factor, in which the rabbits received one type of physical therapy: millimetre waves for 20 min, pulsed electromagnetic fields, millimetre waves for 40 min, ultrasound, low-level laser therapy or ultrashort wave diathermy. The two remaining groups, the normal group and the model group, served as controls. The efficacy of the different treatments were determined by observing the configuration and structure of the cartilaginous tissue by haematoxylin and Eosin staining, measuring the serum tumour necrosis factor-α levels by enzyme immunoassay, evaluating the expression levels of caspases-3 and -8 by immunohistochemistry, and calculating the ratio of chondrocytes apoptosis by TdT-mediated dUTP nick end labelling. The values obtained for each assessment of the eight groups were analysed by a One-way ANOVA.

Results

By applying upmentioned physical treatments, the organisational configuration and structure of cartilage cells from the knees of rabbits with osteoarthritis increased. These treatments also decreased serum tumour necrosis factor-α levels, reduced the expression of caspase-3 and caspase-8 and reduced chondrocyte apoptosis, resulting in an overall delay in osteoarthritis development.

Conclusion

The application of pulsed electromagnetic fields, millimetre waves for 40 min, ultrasound, or low-level laser therapy had significant effects in improving osteoarthritis; in particular, treatment with pulsed electromagnetic fields or ultrasound yielded the greatest therapeutic effect.

Introduction

Osteoarthritis (OA) is the most common joint disease and is a leading cause of chronic disability [1]. This disease is compounded by age, genetic factors, and mechanical forces, and is characterised by distinct changes in both superficial and interior cells within the cartilage [2]. The pathogenesis of OA remains unclear, but recent studies have revealed that apoptosis is associated with the onset and development of OA [3]. Apoptosis, a form of programmed cell death, is critical not only during development and tissue homeostasis, but also in the pathogenesis of a variety of diseases [4]. Blanco et al. have found that the proportion of apoptotic cells in OA is greater when compared to that in normal or healthy cartilage [5]. The greater proportion of apoptotic cells in OA implies that apoptosis plays an important role in the development of OA. In molecular studies, the expression of several caspases, such as caspase-3 and caspase-8, is increased in human osteoarthritic cartilage and in animal models [6], [7]. Additionally, tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine that plays a central role in inflammation and apoptosis [8].

Millimetre waves (MW), pulsed electromagnetic fields (PEMFs), ultrasounds (US), low level laser therapy (LLLT) and short wave diathermy (SWD) are non-invasive techniques that are considered safe and effective forms of physiotherapy for the management of OA [9], [10], [11], [12], [13], [14].

We hypothesised that an effective treatment can significantly improve the progression of OA and can be monitored through the analysis of several key disease indicators, such as histological evaluation of the articular cartilage, serum TNF-α levels, expression of caspase-3 and caspase-8 and apoptosis of chondrocytes. In this study, we quantitively evaluated the effects of MW for 20 min, PEMFs, MW for 40 min, US, LLLT and SWD therapies on serum TNF-α levels, chondrocyte apoptosis, and caspase-3 and caspase-8 expression in rabbits with osteoarthritis of the knee. Our results may shed light on the efficacy of various physical therapies available to patients in the management OA of the knee.

Section snippets

Animals

A total of 64 New Zealand White (NZW) rabbits, 32 male and 32 female, 3 months of age and weighing 2–2.5 kg, were used for this study.

All animals were given a standard laboratory diet with drinking water and housed in individual cages with a 12-hour light-dark cycle at 20–26 °C. The research complied with national legislation and with Ministry of Health of the People's Republic of China Guide for the Care and Use of Laboratory Animals, and had local ethical committee approval. All the rabbits

The appearance of cartilage sections by H&E staining

In Fig. S1 (see the supplementary material associated with this article online), we found that normal rabbit cartilage (Group G) appeared as a thick layer of cartilage neatly arranged in a grid-like pattern. In the model group (Group H), the cartilage layer was thinner and contained fewer cartilage cells; these cells appeared in a less ordered structure. After treatment, the number of cartilage cells increased and the cells re-established an ordered pattern. The modified Mankin scores of the MW

Discussion

In this study, we evaluated the effects of six intervention factors on OA in the knee by using four indices: histological evaluation of the articular cartilage by H&E staining, analysis of serum TNF-α levels by ELISA, assessment of caspase-3 and caspase-8 expression by immunohistochemistry and apoptosis of chondrocytes by TUNEL. According to the graded scores based on the four indices (shown in Table 6), PEMFs and US were confirmed as the two most optimal physical intervention factors, whereas

Disclosure

We declare that we have no conflict of interest statement.

Authors’ contributions

Hua Guo conceived the study, performed the statistical and data analysis and drafted the manuscript. Qinglu Luo, Jinglong Zhang and Haidan Lin participated in experimental design of the study and data acquisition. Chengqi He conceived the study and is the primary author of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Yuqing Wang for the statistical and data analysis. We thank the National Natural Science Fund (No. 30672215) for the financial support.

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    Chengqi He, the corresponding author certifies that all authors agree to the content, presentation, and decision to submit the manuscript.

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