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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 106 - 106
1 Aug 2012
Li S Caterson B Hayes A Hughes C
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Introduction

Novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) have been shown to be putative biomarkers of progenitor/stem cell sub-populations (Hayes et al., 2007; Dowthwaite et al., 2005). Also, recent studies show that unique CS sulphation motifs are localized in putative stem/progenitor cell niches at sites of incipient articular cartilage & other musculoskeletal tissues (Hayes et al., 2011), which indicates their potential importance in cell differentiation during development. In this study, we investigated the importance of CS in the differentiation of bone marrow stem cells to the chondrogenic phenotype in vitro using p-nitrophenyl xyloside (PNPX) as a competitive inhibitor of CS substitution on matrix PGs.

Methods

Bovine bone marrow stem cells (BMSCs) were isolated from 7-day-old cow hock joints and cultured as monolayer for 4 weeks with chondrogenic medium ± 0.25mM PNPX. DMMB assay, real-time PCR, Western Blotting & immunohistochemistry (IHC) were used to analysis the chondrogenic markers. The expression and distribution of structural CS proteoglycans (CS-PGs) were analysed by immunofluorescent staining combined with confocal microscopy scanning.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 434 - 434
1 Nov 2011
Angibaud L Stulberg B Mabrey J Covall D Steffens J Hayes A Weisenburger J Haider H
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A tibial insert with choices in posterior slope, size, and thickness is proposed to improve ligament balancing in total knee arthroplasty. However, increasing slope, or the angle between the distal and proximal insert surfaces, will redistribute ultra-high molecular weight poly-ethylene (UHMWPE) thickness in the sagittal plane, potentially affecting wear. This study used in-vitro testing to compare UHMWPE wear for a standard cruciate-retaining (CR) tibial insert (STD) and a corresponding 6° sloped insert (SLP). Our hypothe sis was that slope variation would have little effect on wear.

Two of each style inserts were tested on an Instron-Stanmore knee simulator with a force-control regime. The gait cycle and other settings followed ISO 14243-1 & 2, except for the reference position, which was posteriorly shifted 6 mm to simulate the worst-case scenario. The STD insert was tilted 6° more than the SLP to level the articular surfaces. Wear was gravimetrically measured at intervals according to strict protocol.

No statistical difference (p=0.36) was found between wear for the STD (9.5 ±1.8 mg/Mc) and SLP (11.4 ±0.5 mg/Mc) inserts.

The overall wear rate measured was higher than previously published rates using implants similar to the STD inserts. This may relate to the shift in the reference position and the 6° slope, leading to increased shear loads. This is the first time the effect of tibial insert slope on wear has been evaluated in-vitro. When limited to 6°, wear testing suggests that al tering the tibial insert slope will have a minor effect on UHMWPE wear.


Orthopaedic Proceedings
Vol. 92-B, Issue SUPP_I | Pages 59 - 59
1 Mar 2010
Caterson* B Hayes A Tudor D Nowell M Hughes C
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In the mid-1980s we produced and characterised several monoclonal antibodies ‘mAbs 3-B-3(−); 4-C-3, 6-C-3 & 7-D-4) that recognised unique native sulphation motifs in chondroitin sulphate (CS) glycosaminoglycan (GAG) chains on connective tissue proteoglycans (PGs).

These antibodies were shown to specifically locate CS-PGs in the pericellular regions surrounding putative sites where haemopoietic stem cells were undergoing lymphopoiesis in the Bursa of Fabricius of embryonic chicks. In later studies, we also observed immunostaining for some of these mAbs ‘3-B-3(-) & 7-D-4’ in chondrocyte clusters present in tissue sections from late-stage osteoarthritic cartilage from canine and human patients. In a recent study ‘Hayes et al (2008), J. Histochem Cytochem. 56: 125–128’ we have used these anti-CS sulphation motif mAbs to specifically identify stem/chondroprogenitor cells in the surface/superficial zone of hyaline articular cartilage. Furthermore, we used these mAbs in FACS analyses to sort and isolate chondroprogenitor cells for potential pluripotent cell enrichment in tissue engineering/tissue regeneration technologies. We have also used several of these mAbs to identify stem/progenitor cells in different anatomical and functional regions of the tendon; i.e. where the tendon wraps around bone in compressed regions where the cells exhibit a more chondrogenic phenotype and also in the outer zones of the tendon surrounding pericytes where vascularisation occurs. In the developing intervertebral some of these mAbs specifically recognise stem/progenitor cells at the interzone between the outer and inner anulus an also the boundary of the nucleus with the inner annulus, these results indicating their use for stem/progenitor cell identification and isolation in other musculoskeletal tissues. Interestingly, these mAbs also immunostained the pericellular environment (stem cell niche) in the crypts of the gut and the limbus of the eye where stem cells reside. Collectively, this data strongly suggests that these mAbs recognising CS sulphation motifs can be used as biomarkers to identify stem cell niches in numerous tissues of the body and that they can be used for stem/progenitor cell isolation for use in tissue engineering/regeneration procedures.

This work was supported by BBSRC and ARC funding.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 145 - 145
1 Mar 2008
Duffy P McQueen M Hayes A
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Purpose: External fixation is a popular treatment method of unstable distal radius fractures. There has been much debate and confusion however regarding the use of bridging versus non-bridging fixation. The aim of this study is to define the indications for bridging and non-bridging external fixation in the treatment of unstable distal radius fractures. The study also endeavours to evaluate the complications and pitfalls associated with this treatment and to determine if non-expert surgeons can reproduce successful outcomes.

Methods: Between January 1995 and December 2000, 641 patients with fractures of the distal radius were treated at our institution with external fixation. The fractures were treated either by bridging or non-bridging external fixation. Demographic data was collected prospectively for these patients including their hospital number, date of birth, gender, age at injury, mode of injury, type of external fixator and whether the fracture was an open or closed injury. Further information was collected retrospectively from review of case notes and x-rays and included AO classification, status of the operating surgeon, duration of fixation, and complications.

Results: Patients treated with bridging external fixation had significantly more mal unions in terms of dorsal angulation and shortening. The non-bridging fixators were better able to maintain and in some cases improve on the immediate post external fixation measurements. Minor pin tract infections were more common in the non-bridging group.

Conclusions: Non-bridging external fixation is the treatment of choice for unstable fractures of the distal radius with sufficient space for the placement of pins in the distal fragment. A predictable outcome with low complication rate can be expected.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_III | Pages 380 - 381
1 Oct 2006
Day M Cao J Li S Hayes A Hughes C Evans R Dent C Caterson B
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Introduction: Kashin-Beck disease (KBD) is a special endemic osteoarthropathy whose main pathologic changes occur in growth plate cartilage and articular cartilage of human limbs and joints where it is manifested as cartilage degeneration and necrosis. Past and current research suggests that KBD, and its endemic geographic distribution in China, is due to the combined presence of fungal mycotoxins (on stored food ingested by affected populations) and a regional selenium deficiency in the environment providing local food sources. Thus, we hypothesise that the presence of fungal mycotoxins and the absence of selenium in the diet specifically affects chondrocyte metabolism in the growth plate during limb and joint development and in articular cartilage of adults, which leads to localised tissue necrosis, and the onset of degenerative joint disease. The aim of this study was to examine the effects of mycotoxins; e.g. Nivalenol (NIV), selenium and NIV in the presence of selen! ium in in vitro chondrocyte culture systems to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD.

Methods: Chondrocyte tissue cultures were established using cartilage explant cultures either in the presence or absence of selenium (0.5–1.5 microg/ml) and the mycotoxin nivalenol (0.5–1.5 microg/ml) and culture for 1 to 4 days. Medium was harvested daily at day 1 through 4 and analysed for glycosaminoglycan (GAG) release and the presence of aggrecanase or MMP activity using RT-PCR for gene expression and monoclonal antibodies that detect their respective enzyme-generated neo-epitopes on cartilage aggrecan metabolites.

Results: Our studies to date have shown that NIV exposure induces catabolic changes in chondrocyte metabolism with an increased expression of aggrecanase activity. Addition of selenium did not affect mRNA expression of the aggrecanases ADAMTS-4 & 5. Parallel studies involving immunohistochemical analyses of articular cartilage from KBD showed an increase in aggrecanase activity.

Conclusions: These studies demonstrate that induction of aggrecanase activity as one of the molecular mechanisms involved is the pathogenesis of KBD. However, the addition of selenium does not alter aggrecanase gene expression indicating that its beneficial effects are occurring in other areas of cartilage metabolism.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_III | Pages 413 - 413
1 Oct 2006
Hodgson P Hughes C Day M Hayes A Cao J Li S Evans R Dent C Caterson B
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Introduction: Kashin-Beck disease (KBD) is an endemic osteoarthropathy with pathological changes occurring in growth plate and articular cartilage in humans. It manifests as cartilage degeneration and necrosis. It is postulated that KBD is due to fungal mycotoxins infiltrating the diet and a regional selenium deficiency in the environment providing food sources in a broad belt across China. Previous work has established an in vitro system in which chondrocytes are cultured and an ex vivo cartilage graft is produced. Subjecting these chondrocytes to either selenium (SEL), Nivalenol (NIV) or in combination during the growth of the graft was found to alter the morphology of the cartilage graft. In addition, the quantity of the large aggregating proteoglycan, was significantly reduced in a dose dependent manner in the presence of Nivalenol. This study aimed to examine the composition of aggrecan from grafts grown in the presence of NIV or SEL alone, or in combination to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD.

Methods: Chondrocytes (from 7 day old bovine cartilage) were seeded at high density in MilliCell filter inserts (12mm diameter; Millipore, MA). Cultures were maintained for 4 weeks in DMEM supplemented with 20% heat–inactivated FBS, ascorbate (100μg/ml) and TGFß2 (5ng/ml) or additionally supplemented with either SEL , NIV or both at concentrations of 0.01, 0.05 and 0.1μg/ml. Media was refreshed thrice weekly and later analysed. At 4 weeks the cartilage grafts were harvested, weighed and extracted in 4M guanidium chloride (with an inhibitor cocktail) for biochemical analysis of matrix molecules. Residues were papain digested. Glycosaminoglycan concentration was determined using the DMMB assay in all media samples, guanidine extracts and papain digests. Aggrecan and GAG composition was determined using Western blotting with a panel of antibodies recognising chondroitin sulphate (CS), keratan sulphate (KS) and protein core epitopes present in aggrecan.

Results: The total GAG synthesised in a 4week period was substantially reduced in chondrocytes cultured in the presence of NIV at 0.05 and 0.1μg/ml and to a lesser extent in those cultures exposed to the highest dose of SEL. However, the amount of GAG released into the media remained fairly constant within the treatment groups, but a marked reduction was apparent in the guanidine extracts of the cartilage grafts. Western blot analysis with a series of antibodies on guanidine extracted aggrecan showed no substantial changes in the core protein molecular weights however analysis demonstrated that KS was reduced in NIV treated cultures. Results also indicated that NIV treated cultures appeared to contain less CS substitutions on the aggrecan core protein.

Discussion: The GAG concentration data indicates that there is an inability of the GAG to remain within the cartilage grafts extracellular matrix. when treated with NIV. Western blot analysis indicates minor changes in the composition of the aggrecan in relation to protein core length and CS/KS side chain substitutions or length. Further work will investigate the proportion of aggrecan able to form high molecular weight aggregates, the metabolism of link protein and hyaluronan.