Lubricin expression in human osteoarthritic knee meniscus and synovial fluid: A morphological, immunohistochemical and biochemical study

Abstract

The purpose of this study was to investigate the expression of lubricin, the product of the human PRG4 (proteoglycan 4) gene, in menisci and synovial fluid from normal donors and patients with osteoarthritis (OA), using a combination of histology, immunohistochemistry, ELISA and Western blotting analysis, to provide further insight on the role of this protein in the progression of OA and pathological processes in the meniscus. Lubricin expression was studied in samples from 40 patients and in 9 normal donors after arthroscopic partial meniscectomy. Histological analysis confirmed normal microanatomy and the absence of structural changes in control samples. Menisci derived from OA patients showed evidence of structural alterations, fibrillations and clefts. Immunohistochemical analysis revealed very strong lubricin immunostaining in normal menisci in contrast to weak/moderate staining seen in osteoarthritic menisci. Quantitative ELISA and Western blot analysis confirmed the above results. The findings of this study support the notion that changes in lubricin expression and boundary-lubricating ability of cartilage is followed by the development of OA. This study could provide the biological foundation for the development of novel therapeutic treatments, to be applied before the surgery, for the prevention of post-traumatic cartilage damage.

Introduction

Knee osteoarthritis (OA) is characterized by progressive alterations in joint structures and low-grade inflammation (Berenbaum, 2013). OA is characterized as joint failure due to progressive changes in several musculoskeletal components that include, but are not limited to, articular cartilage (Berenbaum, 2013). Other joint structures, such as the menisci are altered in OA, but have not been as extensively studied as the articular cartilage (Loeser, 2011). In this study special attention was focused on the expression of lubricin in the meniscus, after partial meniscectomy, and in synovial fluid (SF) from OA patients in comparison to normal donors. Partial meniscectomy is a routine surgical procedure that is frequently carried out following damage to the meniscus. Before the introduction of arthroscopy, broken menisci were removed completely with grave consequences for joint articulation and biomechanics, facilitating subsequent progression of the arthritic knee (Musumeci et al., 2012). Today surgical procedures attempt to save as much meniscal tissue as possible, in order to preserve at least a component of its biomechanical function in the knee. Increasing evidence suggests that the meniscus may not represent a passive by-stander in the OA processes. Knee menisci are two semilunar biconcave disks (lateral and medial) that reside within the medial and lateral tibio-femoral articulations. They are divided into inner, middle and outer rim (Musumeci et al., 2013a). The inner rim is the most delicate part, because it is not vascularized. The lateral meniscus has the form of an almost complete circle and adheres to the two cruciate ligaments (Musumeci et al., 2013a). The medial meniscus has the form of a half moon and is more extensive than lateral, with its extremities adhering to anterior and posterior intercondylar areas. Between the two menisci, the medial meniscus is more subject to trauma, because it is less mobile than the lateral for the presence of the semimembranosus tendon, but also because usually we tend to have a slight valgus during gait (Musumeci et al., 2013a). Microscopically, the meniscus is composed of dense fibrocartilage, sparsely populated with cells called fibrochondrocytes because they exhibit characteristics of both fibroblasts and chondrocytes (Galanti et al., 2013). The meniscus plays an important role in the complex biomechanics of the knee joint. It is involved in load bearing, load transmission, shock absorption, joint stability, joint lubrication and joint congruity. Its loss results in higher peak stresses exerted on cartilage, and eventually in cartilage degeneration and OA (Musumeci et al., 2012). Lubricin, also known as superficial zone protein (SZP), is a chondroprotective, mucinous glycoprotein, the product of the proteoglycan 4 (PRG4) gene (Musumeci et al., 2013b). It has been found in several tissues including the synovial membranes and synovial fluid (SF) (Elsaid et al., 2005), the superficial zone of articular cartilage (Schumacher et al., 1994), tendon (Rees et al., 2002), ligament (Leonardi et al., 2012a), disk (Leonardi et al., 2012b, Leonardi et al., 2012c) and meniscus (Schumacher et al., 2005, Young et al., 2006, Zhang et al., 2011, Musumeci et al., 2013b). The essential role of lubricin is maintaining joint integrity. Lubricin was originally identified as a lubricating glycoprotein present in SF, specifically synthesized and expressed by articular chondrocytes of the superficial zone and it is recognized to play a major protective role in preventing cartilage wear and synovial cell adhesion and proliferation and reducing the coefficient of friction of the articular cartilage surface (Musumeci et al., 2011a, Musumeci et al., 2011b, Loeser, 2013). Lubricin is important for articular joint physiology, and the loss of lubricin accumulation may be involved in the pathology of OA. Previous studies demonstrated that important down-regulation of lubricin, as well as of other proteoglycans and synovial fluid biomarkers, develops in the human knee meniscus and anterior cruciate ligament (ACL) immediately after acute joint injury, underlining its involvement in the articular damage (Catterall et al., 2010, Musumeci et al., 2013b). A study in an animal model of OA demonstrated that recombinant lubricin improves the chondroprotection, which suggests that recombinant lubricin molecules may potentially be used in new approaches for the treatment of OA and associated cartilage disorders (Flannery et al., 2009). The purpose of our study was to investigate, in human patients, the expression of lubricin in menisci and synovial fluid from normal donors and patients with OA, using histology, immunohistochemistry, ELISA and Western blot analysis, to provide insights into the role of this protein in the progression of OA-related pathological processes in the meniscus.