The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively. © 2014 Springer Science+Business Media, LLC.

Objective

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism.

Methods

Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm² power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca²⁺ influx was measured by using a Fluo-4 probe.

Results

The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca²⁺ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BK_ca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes.

Conclusion

These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca²⁺ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

Introduction

Joint haemorrhage is the principal clinical manifestation of haemophilia frequently leading to advanced arthropathy and arthrofibrosis, resulting in severe disability. The degree and prevalence of arthrofibrosis in hemophilic arthropathy is more severe than in other forms of arthropathy. Expression of connective tissue growth factor (CTGF) has been linked to many fibrotic diseases, but has not been studied in the context of haemophilic arthropathy.

Aim

We aim to compare synovial tissues histologically from haemophilia and osteoarthritis patients with advanced arthropathy in order to compare expression of proteins that are possibly aetiologic in the development of arthrofibrosis.

Methods

Human synovial tissues were obtained from 10 haemophilia and 10 osteoarthritis patients undergoing joint surgery and processed for histology and immunohistochemistry.

Results

All samples from haemophilia patients had synovitis with hypertrophy and hyperplasia of synovial villi. Histologically, synovial tissues contained hyperplastic villi with increased cellularity and abundant haemosiderin- and ferritin-pigmented macrophage-like cells (HMCs), with a perivascular localization in the sub-surface layer. CTGF staining was observed in the surface layer and sub-surface layer in all haemophilia patients, exclusively co-localizing with HMCs. Quantification showed that the extent of CTGF-positive areas was correlated with the degree of detection of HMCs. CTGF was not observed in any of the samples from osteoarthritis patients.

Conclusion

Using histological analysis, we showed that CTGF expression is elevated in haemophilia patients with arthrofibrosis and absent in patients with osteoarthritis. Additionally, we found that CTGF is always associated with haemosiderin-pigmented macrophage-like cells, which suggests that CTGF is produced by synovial A cells following the uptake of blood breakdown products.