J. Denton

Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis

Aims: Preliminary studies have suggested that there is an increase in adipocytic tissue in osteoporotic (OP) bone, supporting in vitro evidence for a switch in differentiation of stromal cells from the osteoblastic to the adipocytic lineage. To investigate this the variation of the ratio of adipose tissue to haemopoietic/stromal tissue in OP bone was measured. Methods: The ratio of adipocytic to haemopoietic/stromal tissue (A/H) was measured by semi-automated image analysis in iliac crest biopsies from 127 patients with osteoporosis (84 female patients, 48 male patients; mean age, 55 years; range, 5–80). Fourteen patients with normal histomorphometric data (nine women; five men; mean age, 48 years; range 21–70) acted as controls. Results: The ratio of A/H was higher in OP bone than in the normal controls (OP mean 43.06% v normal mean 22.4%; p < 0.001). Multiple regression analysis showed that 98.5% of the variability in the A/H ratio was the result of age and several measures of bone formation, including cancellous wall thickness, osteoid volume, cancellous thickness, cortical wall thickness, cancellous apposition rate, and bone formation rate, together with cancellous separation (each significant at p < 0.001). Those with the greatest effect on the A/H ratio (in decreasing order) were cancellous apposition rate, osteoid volume, and age. Conclusions: Cancellous apposition rate, osteoid volume, and age were associated with the increase in the proportion of adipose tissue present in OP bone. Of these, cancellous apposition rate reflects osteoblast activity, indicating that the increase in the volume of adipose tissue in osteoporosis is associated with reduced bone formation, supporting the postulated switch in differentiation of stromal cells from the osteoblastic to the adipocytic pathway in osteoporosis.

Age related histomorphometric changes in bone in normal British men and women.

AIMS--To define the iliac crest histomorphometry of static variables in 234 individuals aged 16-100 years (91 men, 143 women) and of dynamic variables in 84 individuals aged 19-94 years (33 men, 51 women) from the North West of England. METHODS--Iliac crest biopsy specimens were sectioned, undecalcified, and examined using image analysis. RESULTS--The decrease in the quantity of cortical and trabecular bone and the connectivity of trabecular bone was more pronounced in women than men. This was associated with a reduction in bone formation and increased bone resorption which was greater in women at both the tissue and cellular level. Some of these histomorphometric differences first became evident at the natural menopause, and therefore provide clues as to the cause of the high prevalence of osteoporosis in postmenopausal women. CONCLUSIONS--These results show an age and sex dependent variation both in static and dynamic parameters, which differ, in some respects, from other studies and confirm the need for large regional studies to provide a database of normal morphometric results for a specific population.

Detection and distribution of aluminium in bone.

The distribution of aluminium has been investigated using a new histochemical technique in transiliac bone biopsy specimens from 20 patients with aluminium related bone disease and 17 with other metabolic bone diseases. The method, which employs solochrome azurine at acid pH (acid solochrome azurine), showed a wider distribution of aluminium within cortical and trabecular bone than other histochemical techniques. The results compared well with atomic absorption spectrophotometric analysis of these biopsy specimens. Wavelength dispersive electron probe analysis of serial sections validated the procedure. The method has certain advantages over both these analytical techniques.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low (∼50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)–PCR was confirmed, especially for abundant transcripts, and RT–PCR validated the regulation pattern for 19 of 24 candidate genes (overall R2=0.4662). RT–PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET – whose combined expression carried greater prognostic value than tumour grade – and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Synthesis of the active metabolite of vitamin D, 1,25(OH)2D3, by synovial fluid macrophages in arthritic diseases.

Synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown in cells from knee joint synovial fluid of 20 patients with inflammatory rheumatoid disease, reactive or psoriatic arthritis, or gout, all of which had high synovial fluid cell counts, and by cells from a patient with aseptic necrosis of a femoral condyle after short term (less than 24 hours) or long term (seven days) primary culture. Cells from 18 patients with inflammatory arthritis, five of which had low synovial fluid cell counts and cells from six patients with osteoarthritis were unable to synthesise this metabolite from 25-hydroxyvitamin D3 (25(OH)D3). Macrophages are believed to be the cells responsible for synthesising 1,25(OH)2D3 because these were significantly more numerous in samples that formed 1,25(OH)2D3; they were also the predominant cell type present in the aseptic necrosis sample and the only cell type present in preparations maintained for one week in monolayer culture.

Effects of hydrated lime and quicklime on the decay of buried human remains using pig cadavers as human body analogues

Recent casework in Belgium involving the search for human remains buried with lime, demonstrated the need for more detailed understanding of the effect of different types of lime on cadaver decomposition and its micro-environment. Six pigs (Sus scrofa) were used as body analogues in field experiments. They were buried without lime, with hydrated lime (Ca(OH)(2)) and with quicklime (CaO) in shallow graves in sandy loam soil in Belgium and recovered after 6 months of burial. Observations from these field recoveries informed additional laboratory experiments that were undertaken at the University of Bradford, UK. The combined results of these studies demonstrate that despite conflicting evidence in the literature, hydrated lime and quicklime both delay the decay of the carcass during the first 6 months. This study has implications for the investigation of clandestine burials and for a better understanding of archaeological plaster burials. Knowledge of the effects of lime on decomposition processes also has bearing on practices involving burial of animal carcasses and potentially the management of mass graves and mass disasters by humanitarian organisations and DVI teams.

Retained surgical swab debris in post-laminectomy arachnoiditis and peridural fibrosis

We examined soft tissue biopsies from 26 patients with symptomatic nerve root fibrosis and arachnoiditis after a previous laminectomy. Dense fibrous connective tissue was found about the nerve roots and in 14 cases (55%) fibrillar foreign material was seen within it. This material had the histochemical characteristics of cotton fibres from swabs and neurosurgical patties. In two other cases nerve root fibrosis was associated with residual radiopaque lipid thought to derive from earlier myelography. Our findings suggest that risks may be associated with the introduction of foreign material into the vertebral canal, and that microscopic fragments of surgical swabs and patties may have a role in the pathogenesis of postoperative periradicular fibrosis.

Aluminium deposition in bone after contamination of drinking water supply

Two healthy individuals who drank water accidentally contaminated at source with aluminium sulphate solution were investigated 6-7 months later. Bone biopsy specimens showed discrete lines of positive staining for aluminium, the distribution being compatible with acute exposure some months previously. These findings show that under certain conditions normal individuals can absorb aluminium via the gut, and that such aluminium can be deposited in bone.

QUANTIFICATION OF VITAMIN D RECEPTOR mRNA IN TISSUE SECTIONS DEMONSTRATES THE RELATIVE LIMITATIONS OF IN SITU-REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION

In situ‐reverse transcriptase‐polymerase chain reaction (IS‐RT‐PCR) is a recently described technique that is used to localize low levels of mRNA within cells and tissue sections. One of the major criticisms levelled at this technique is that positive results may be meaningless, as amplification is required to demonstrate the transcripts of interest. The use of IS‐RT‐PCR to demonstrate mRNA for receptors for 1,25‐dihydroxyvitamin D3 (VDR) in sections of human kidney and bone has previously been described. To ascertain whether the levels of VDR mRNA detected following IS‐RT‐PCR were transcriptionally significant, computerized image analysis was used to determine the mean silver grain density in human kidney and bone cells following conventional in situ hybridization and after various cycles of IS‐RT‐PCR. Only a few cycles of PCR were needed to produce an optimum signal, but amplification of signal following IS‐RT‐PCR was found to be relatively inefficient. Following the optimum number of cycles of IS‐RT‐PCR in kidney sections, there was a less than four‐fold increase in signal. Similarly, in bone, the optimum signal detected was only approximately five times greater than that found with conventional in situ hybridization. These results clearly demonstrate that the increase in signal following IS‐RT‐PCR follows a more linear pattern and is relatively inefficient, compared with the usual exponential increase with conventional solution phase RT‐PCR.

Possible clearance of effete polymorphonuclear leucocytes from synovial fluid by cytophagocytic mononuclear cells: implications for pathogenesis and chronicity in inflammatory arthritis.

A feature common to all forms of chronic inflammatory arthritis, irrespective of the possible underlying cause, is the persistent exudation of large numbers of polymorphonuclear leucocytes (PMNL) into synovial fluid. These cells possess potent degradative enzymes and proinflammatory mediators, and their removal is vital to normal inflammatory resolution. A major route of disposal of extravasated PMNL appears to be programmed cell death (apoptosis), followed by their rapid recognition, and intact phagocytosis, by mature tissue macrophages. Such macrophages, containing PMNL (cytophagocytic mononuclear cells (CPM)), long recognised in synovial fluid as Reiter cells, are commonly found in reactive arthritis, spondyloarthritis, and crystal arthritides, but only rarely in rheumatoid disease. In a retrospective analysis of 187 knee synovial fluid cytospins, the relation between the formation of CPM and the presence of apoptotic (pyknotic) PMNL was investigated. As long as the synovial fluid examined was fresh there was a high correlation between numbers of CPM (as a percentage of macrophages) and pyknotic numbers of PMNL in fluids containing CPM. This suggests that the formation of CPM occurs in vivo and is involved in the disposal of PMNL. Numbers of pyknotic PMNL increased rapidly in stored synovial fluid without a significant change in numbers of CPM, and were highest in synovial fluid which did not contain CPM. The presence or absence of CPM, or their disease associations, could not be explained simply by limiting numbers of macrophages, or apoptotic PMNL in synovial fluid. These findings are consistent with a regulatory role for CPM in synovial fluid, where they may be important in preventing autolysis of PMNL, and thus local tissue damage.