Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair

We compared the changes in the ratio of type-I and type-II collagen in monolayer cultures of human articular chondrocytes (HAC). HAC were isolated from samples of cartilage from normal joints and cultivated in monolayer for up to 46 days. Expression of collagen type-I and type-II was determined by immunocytochemistry, Western blotting, and the nested reverse transcription polymerase chain reaction (RT-PCR), and quantified by real-time PCR. The transition from a spherical morphology to the flattened morphology of an anchorage-dependent culture was accompanied by a rapid change in the collagen phenotype with the replacement of collagen type II by collagen type I. This was confirmed by immunocytochemistry and Western blotting between days 21 and 28. Using techniques for the analysis of gene transcription (nested RT-PCR and real-time PCR), a complete switch of collagen gene expression was not observed. Expression of collagen type I increased 100-fold during the culture time. That of collagen type II was found during the entire period and decreased more than 100-fold. The main finding was that expression of the genes encoding collagen type I and II was highly time-dependent and the ratio of collagen type II to I (CII/CI), defined as an index of cell differentiation, was significantly higher (215- to 480-fold) at the beginning of the culture. At the end of the experimental culture time, ratios between 0.1 and 1 were reached

Caveolae, specialised regions of the cell membrane which have been detected in a wide range of mammalian cells, have not been described in bone cells. They are plasmalemmal invaginations, 50 to 100 nm in size, characterised by the presence of the structural protein, caveolin, which exists as three subtypes. Caveolin-1 and caveolin-2 are expressed in a wide range of cell types whereas caveolin-3 is thought to be a muscle-specific subtype. There is little information on the precise function of caveolae, but it has been proposed that they play an important role in signal transduction. As the principal bone-producing cell, the osteoblast has been widely studied in an effort to understand the signalling pathways by which it responds to extracellular stimuli. Our aim in this study was to identify caveolae and their structural protein caveolin in normal human osteoblasts, and to determine which subtypes of caveolin were present. Confocal microscopy showed staining which was associated with the plasma membrane. Transmission electron microscopy revealed the presence of membrane invaginations of 50 to 100 nm, consistent with the appearance of caveolae. Finally, we isolated protein from these osteoblasts, and performed Western blotting using anti-caveolin primary antibodies. This revealed the presence of caveolin-1 and -2, while caveolin-3 was absent. The identification of these structures and their associated protein may provide a significant contribution to our further understanding of signal transduction pathways in osteoblasts

A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA). The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3. Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3. We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.