Michiko Mirams

Intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a cross-sectional study.

BACKGROUNDnOsteoporosis research has focused on vertebral fractures and trabecular bone loss. However, non-vertebral fractures at predominantly cortical sites account for 80% of all fractures and most fracture-related morbidity and mortality in old age. We aimed to re-examine cortical bone as a source of bone loss in the appendicular skeleton.nnnMETHODSnIn this cross-sectional study, we used high-resolution peripheral CT to quantify and compare cortical and trabecular bone loss from the distal radius of adult women, and measured porosity using scanning electron microscopy. Exclusion criteria were diseases or prescribed drugs affecting bone metabolism. We also measured bone mineral density of post-mortem hip specimens from female cadavers using densitometry. Age-related differences in total, cortical, and trabecular bone mass, trabecular bone of cortical origin, and cortical and trabecular densities were calculated.nnnFINDINGSnWe investigated 122 white women with a mean age of 62.8 (range 27-98) years. Between ages 50 and 80 years (n=89), 72.1 mg (95% CI 67.7-76.4) hydroxyapatite (68%) of 106.5 mg hydroxyapatite of bone lost at the distal radius was cortical and 34.3 mg (30.5-37.8) hydroxyapatite (32%) was trabecular; 17.1 mg (11.7-22.5) hydroxyapatite (16%) of total bone loss occurred between ages 50 and 64 years (n=34) and 89.4 mg (83.7-101.1) hydroxyapatite (84%) after age 65 years (n=55). Remodelling within cortex adjacent to the marrow accounted for 49.9 mg (45.4-53.7) hydroxyapatite (47%) of bone loss. Between ages 50-64 years (n=34) and 80 years and older (n=33), cortical density decreased by 127.8 mg (93.1-162.1) hydroxyapatite per cm(3) (15%, p<0.0001) before porosity trabecularising the cortex was included, but 374.3 mg (318.2-429.5) hydroxyapatite per cm(3) (43%, p<0.0001) after; trabecular density decreased by 18.2 mg (-1.4 to 38.2) hydroxyapatite per cm(3) (14%, p=0.06) before cortical remnants were excluded, but 68.7 mg (37.7-90.4) hydroxyapatite per cm(3) (52%, p<0.0001) after.nnnINTERPRETATIONnAccurate assessment of bone structure, especially porosity producing cortical remnants, could improve identification of individuals at high and low risk of fracture and therefore assist targeting of treatment.nnnFUNDINGnAustralia National Health and Medical Research Council.

The skeleton: a multi-functional complex organ. The growth plate chondrocyte and endochondral ossification

Endochondral ossification is the process that results in both the replacement of the embryonic cartilaginous skeleton during organogenesis and the growth of long bones until adult height is achieved. Chondrocytes play a central role in this process, contributing to longitudinal growth through a combination of proliferation, extracellular matrix (ECM) secretion and hypertrophy. Terminally differentiated hypertrophic chondrocytes then die, allowing the invasion of a mixture of cells that collectively replace the cartilage tissue with bone tissue. The behaviour of growth plate chondrocytes is tightly regulated at all stages of endochondral ossification by a complex network of interactions between circulating hormones (including GH and thyroid hormone), locally produced growth factors (including Indian hedgehog, WNTs, bone morphogenetic proteins and fibroblast growth factors) and the components of the ECM secreted by the chondrocytes (including collagens, proteoglycans, thrombospondins and matrilins). In turn, chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand. This review discusses how the growth plate chondrocyte contributes to endochondral ossification, with some emphasis on recent advances.

Altered gene expression in early osteochondrosis lesions

Osteochondrosis is a condition involving defective endochondral ossification and retention of cartilage in subchondral bone. The pathophysiology of this condition is poorly characterized, but it has been proposed that the fundamental defect is failure of chondrocyte hypertrophy. The aim of the current study was to characterize phenotypic changes in chondrocytes associated with the initiation of osteochondrosis. Early lesions were induced in an equine model of osteochondrosis by feeding foals a high energy diet for 8 or 15 weeks. Lesions in articular‐epiphyseal growth cartilage were examined histologically and by quantitative PCR analysis of expression of a number of genes representative of pathways that regulate chondrocyte behavior during endochondral ossification. There were more cells present in clusters in the lesions compared to normal articular cartilage. Expression of matrix metalloproteinase‐13, type I collagen, type X collagen, and Runx2 mRNA was significantly greater in the lesions compared to normal cartilage from the same joint. Expression of vascular endothelial growth factor, type II collagen, connective tissue growth factor, aggrecan, Sox9, and fibroblast growth factor receptor 3 mRNA was not significantly different in lesions than in control cartilage. These observations suggest that osteochondrosis does not result from failure of chondrocytes to undergo hypertrophy.

Reversed T cell receptor docking on a major histocompatibility class I complex limits involvement in the immune response

The anti-viral Txa0cell response is drawn from the naive Txa0cell repertoire. During influenza infection, the CD8+ Txa0cell response to an H-2Db-restricted nucleoprotein epitope (NP366) is characterized by preferential expansion of Txa0cells bearing TRBV13+ Txa0cell receptors (TCRs) and avoidance of TRBV17+ Txa0cells, despite the latter dominating the naive precursor repertoire. We found two TRBV17+ TCRs that bound H-2Db-NP366 with a 180° reversed polarity compared to the canonical TCR-pMHC-I docking. The TRBV17 β-chain dominated the interaction and, whereas the complementarity determining region-3 (CDR3) loops exclusively mediated contacts with the MHC-I, peptide specificity was attributable to germline-encoded recognition. Nevertheless, the TRBV17+ TCR exhibited moderate affinity toward H-2Db-NP366 and was capable of signal transduction. Thus, the naive CD8+ Txa0cell pool can comprise TCRs adopting reversed pMHC-I docking modes that limit their involvement in the immune response.

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8+ T cells in aged mice.

Significance Compromised CD8+ T-cell immunity is associated with significant morbidity and mortality in the elderly. Whereas the number of naïve CD8+ T cells declines with age, the drivers of loss and consequences for clonal composition are unclear. We show that aging disproportionately impacts small naïve CD8+ T-cell populations. For one CD8+ T-cell population, loss of diversity was minimally attributable to expansion but rather was associated with diminished cell number and selective retention of cells exhibiting markers of heightened self, but not foreign, recognition. Thus, vaccine formulations for the elderly may benefit from targeting naïve antigen-specific populations with relatively high precursor frequency and self-reactivity, and retention of high-quality T cells may be achieved through repeated low-level T-cell receptor stimulation. In advanced age, decreased CD8+ cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8+ T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8+ T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8+ T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8+ T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8+ T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging.

Periostin expression distinguishes between light and dark hypertrophic chondrocytes

Hypertrophic chondrocytes exist in two forms detectable by electron microscopy, light and dark chondrocytes; the functional implications of the heterogeneous morphology are unknown. The aims of the study were to establish a method for separating light from dark hypertrophic chondrocytes and to identify genes differentially expressed between the two populations. Three-dimensional pellet cultures of chondrocytes from cartilage of neonatal rats were induced to undergo hypertrophy by treatment with triiodothyronine. Cultures were dissociated and subjected to density gradient centrifugation. The cell fraction with the lowest density comprised predominantly light hypertrophic chondrocytes, and the fraction with the highest density comprised predominantly dark hypertrophic chondrocytes. An Affymetrix GeneChip rat expression array was used to compare expression between dark cell-containing pellets and the light cell-enriched fraction. Genes identified on the array as putative dark cell-selective genes included genes encoding extracellular matrix proteins and enzymic modulators thereof. Expression of a subset of genes (Col1a1, periostin, osteoglycin, tPA/Plat, and Chst11) was confirmed as dark cell-selective using quantitative reverse transcriptase polymerase chain reaction. The most highly differentially expressed dark cell-selective gene was periostin. In immunocytochemical studies of light and dark cell-enriched fractions, periostin staining was detectable in dark, but not light hypertrophic chondrocytes. The results provide insight into molecular differences between light and dark hypertrophic chondrocytes.

Thoroughbred horses in race training have lower levels of subchondral bone remodelling in highly loaded regions of the distal metacarpus compared to horses resting from training

Bone is repaired by remodelling, a process influenced by its loading environment. The aim of this study was to investigate the effect of a change in loading environment on bone remodelling by quantifying bone resorption and formation activity in the metacarpal subchondral bone in Thoroughbred racehorses. Sections of the palmar metacarpal condyles of horses in race training (nu2009=u200924) or resting from training (nu2009=u200924) were examined with light microscopy and back scattered scanning electron microscopy (BSEM). Bone area fraction, osteoid perimeter and eroded bone surface were measured within two regions of interest: (1) the lateral parasagittal groove (PS); (2) the lateral condylar subchondral bone (LC). BSEM variables were analysed for the effect of group, region and interaction with time since change in work status. The meansu2009±u2009SE are reported. For both regions of interest in the training compared to the resting group, eroded bone surface was lower (PS: 0.39u2009±u20090.06 vs. 0.65u2009±u20090.07 per mm, Pu2009=u20090.010; LC: 0.24u2009±u20090.04 vs. 0.85u2009±u20090.10 per mm, Pu2009<u20090.001) and in the parasagittal groove osteoid perimeter was higher (0.23u2009±u20090.04% vs. 0.12u2009±u20090.02%). Lower porosity was observed in the subchondral bone, reflected by a higher bone area fraction in the LC of the training group (90.8u2009±u20090.6%) compared to the resting group (85.3u2009±u20091.4%, Pu2009=u20090.0010). Race training was associated with less bone resorption and more bone formation in the subchondral bone of highly loaded areas of the distal metacarpus limiting the replacement of fatigued bone. Periods of reduced intensity loading are important for facilitating subchondral bone repair in Thoroughbred racehorses.

Exercise-induced inhibition of remodelling is focally offset with fatigue fracture in racehorses

SummaryBone remodelling is inhibited by high repetitive loading. However, in subchondral bone of racehorses in training, eroded surface doubled in association with fatigue fracture and there was greater surrounding trabecular bone volume suggesting trabecular modelling unloads the bone focally, allowing damage repair by remodelling.IntroductionRemodelling replaces damaged bone with new bone but is suppressed during high magnitude repetitive loading when damage is most likely. However, in cortical bone of racehorses, at sites of fatigue fracture, focal porosity, consistent with remodelling, is observed in proportion to the extent of surrounding callus. Focal areas of porosity are also observed at sites of fatigue damage in subchondral bone. We hypothesised that fatigued subchondral bone, like damaged cortical bone, is remodelled focally in proportion to the modelling of surrounding trabecular bone.MethodsEroded and mineralizing surfaces and bone area were measured using backscattered scanning electron microscopy of post-mortem specimens of the distal third metacarpal bone in 11 racehorses with condylar fractures (cases) and eight racehorses in training without fractures (controls).ResultsCases had a two-fold greater eroded surface per unit area at the fracture site than controls (0.81u2009±u20090.10 vs. 0.40u2009±u20090.12xa0mm−1, Pu2009=u20090.021) but not at an adjacent site (0.22u2009±u20090.09 vs. 0.30u2009±u20090.11xa0mm−1, Pu2009=u20090.59). Area fraction of surrounding trabecular bone was higher in cases than controls (81u2009±u20092 vs. 72u2009±u20092xa0%, Pu2009=u20090.0020) and the eroded surface at the fracture site correlated with the surrounding trabecular area (adjusted R2u2009=u20090.63, Pu2009=u20090.0010).ConclusionIn conclusion, exercise-induced inhibition of remodelling is offset at sites of fatigue fracture. Modelling of trabecular bone may contribute to unloading these regions, allowing repair by remodelling.

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDYnEquine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes.nnnOBJECTIVEnTo optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo.nnnMETHODSnChondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification.nnnRESULTSnChondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture.nnnCONCLUSIONSnFreshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes.nnnPOTENTIAL RELEVANCEnThis culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.

Role of subchondral bone remodelling in collapse of the articular surface of Thoroughbred racehorses with palmar osteochondral disease

REASONS FOR PERFORMING STUDYnTo gain a better understanding of the aetiology of articular surface collapse in horses with palmar osteochondral disease.nnnOBJECTIVESnTo determine whether acceleration of focal bone resorption associated with reduced physical activity contributes to articular surface collapse in racehorses with palmar osteochondral disease.nnnSTUDY DESIGNnCross-sectional study comparing metacarpal bones from horses at varying stages of race training.nnnMETHODSnMetacarpal bones from 36 racing Thoroughbred horses were examined with high-resolution peripheral quantitative computed tomography to determine the proportion of the articular surface that had collapsed and with backscattered scanning electron microscopy to quantify porosity and eroded bone surface. Racing and training histories were obtained for comparison with imaging data.nnnRESULTSnIn 21 cases, inward collapse of the calcified cartilage layer was observed on backscattered scanning electron microscopy. An increased extent of articular surface collapse was associated with greater numbers of microfractures in the calcified cartilage and superficial subchondral bone (Spearmans correlation [rs ] = 0.62, P<0.001). In the deeper bone (6-10u2009mm), porosity was lower with a greater extent of articular surface collapse (rs = -0.38, P = 0.023), whereas in the superficial bone (0-4u2009mm) there was no association between articular surface collapse and porosity (rs = 0.19, P = 0.26). Both porosity (median 14, range 3.8-26 vs. 3.8, 1.6-17%, P = 0.008) and eroded surface (1.1, 0.74-4.5 vs. 0.64, 0.11-4.7u2009mm(-1) , P = 0.016) of the superficial subchondral bone were higher in resting than in training horses, and in some resting horses subchondral bone voids were highly concentrated, resulting in an apparent loss of support for the overlying calcified cartilage layer.nnnCONCLUSIONSnArticular surface collapse is common in cases of palmar osteochondral disease and is likely to be a sequel to fatigue injury of subchondral bone. Focal subchondral bone resorption appears to contribute to collapse of the calcified cartilage and is potentiated by a reduced-intensity exercise regimen.