226 Abstracts from the Bone and Tooth Society Meeting

We have previously reported that macrophage polykaryons, derived from a carcinoma of breast, were capable of bone resorption when cultured on slices of cortical bone. In order to determine if this is a general property of macrophage polykaryons, we extracted foreign body macrophage polykaryons from the joint capsule derived from a patient undergoing revision of a failed revision hip arthroplasty. This capsule contains a heavy inflammatory response (including macrophages and macrophage polykaryons) to polyethylene and acrylic cement fragments.

These macrophage polykaryons did not respond morphologically to calcitonin, and were stained immunohistochemically by EBM/II and other anti- macrophage markers. They produced numerous resorption pits both in the presence of and absence of hormonal mediators. These pits were similar in dimensions and morphology to those known to be produced by osteoclasts. These results suggest that foreign body macrophage polykaryons are capable of bone resorption.

P22. Monoclonal antibodies raised against ROS 1712.8 cells recognise a sub-population of marrow stromal cells in vitro M Gilligan, J Perry, E Green, H Docherty and D Heath Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B112TH

We have raised five monoclonal antibodies (Mabs), UBIM 1,2,3,12 and li: against the rat osteoblast-like cell line ROS 17/2.8. Immunohistochemical staining on cryostat sections of neonatal rat calvaria and long bone has shown that these Mabs recognise antigens restricted to normal rat osteoblasts (OBs) and chondrocytes’,‘.

The tissue distribution of the antigens recognised by these Mabs was further investigated by immunohistochemical staining of sections of 20 day old whole fetal rat. All five Mabs stained exclusively osteogenic and chondrogenic tissue with no staining of any other tissue seen. This further demonstrates the specificity of these Mabs.

Further studies were undertaken to determine if these Mabs were binding to the adherent stromal cell population derived from rat long bone marrow. Marrow was mechanically removed from long bones, nonadherent cells removed at seven days and the adherent cell population maintained through three passages. No binding was seen to the nonadherent cells. All five Mabs bound to the adherent cells, at a low level in primary culture and at increased levels after passaging.

To determine if these five Mabs recognised antigens present on a sub-population of cells in the adherent cell population on immunofluorescent technique was used. No binding was seen in primary cultured marrow but all five Mabs bound to a sub-population of cells in passaged marrow. Whether these positive cells represent osteogenic cells remains to be determined.

References 1. Presented in part at the 21st European Symposium on Calcified Tissues, Jerusalem, Israel, March 12-16,1989. 2. Perry, Gilligan, Green, Docherty, Heath (1989). American Iournal of Bone and Mineral Research, paper in press.

P23. pH and resorption revisited C A Walsh, W E Dawson and J A Gallagher Department of Human Anatomy and Cell Biology, University of Liverpool

Metabolic acidosis is accompanied by hypercalcuria and negative calcium balance. It has been suggested that this is the result of excessive breakdown of the skeleton by osteoclasts, activated by lowering of plasma pH (Arnett & Dempster 1986 Endrocrinol: 119).

We have used the in vitro assay system first developed by Boyde et al, and Chambers et al, to study the effect of pH on avian embryonic cells - taking into account efficacity of buffers, time of incubation and the effects of cells and bone slices on pH. The pH of Minimum Essential Medium with Hanks salts, buffered with 1OmM Pipes or Hepes buffer was adjusted using 1M sodium hydroxide or 1M hydrochloric acid.

Results obtained (using Pipes buffer) suggest that after a 24 hour incubation period there may be a slight increase in osteoclast activity at pH 6.50. However over a longer incubation period the optimal pH for resorption was Z30. Over the pathophysiological range of pH the resorptive activity of the osteoclasts was relatively constant, it was only at extremes of pH that activity was inhibited.

pH (to): MEAN AREA OF RESORPTION (%)

6.73 0.14 1.75 7.30 0.20 2.80 7.62 0.11 0.26

P24. Breast carcinoma cells secrete factors which stimulate and inhibit osteoblasts and osteoclasts C E Evans, C Ward and I P Braidman* *Departments of Urthopaedics and Medicine (Endocrinology), Hope Hospital, Eccles Old Road, Salford M6 8HD

Metastatic bone disease is a common complication of breast cancer. It is frequently manifested by osteolytic bone lesions, caused by increased osteoclast numbers, but often bone formation is stimulated. The mechanisms for these changes, however, are still unclear. In preliminary experiments, we have examined the effects of conditioned medium (CM) from a breast carcinoma line (MCF7) and primary cultures of cancer cells from two patients, GB and MH, on human osteoblast-like cells (BDC) and osteoclast (0~1) recruitment in cultured 19 day foetal rat calvariae’. BDC were derived from human bone and have many osteoblastic characteristics2. Ocl are responsive to calcitonin and resorb bone’. BDC proliferation, DNA synthesis and alkaline

Abstracts from the Bone and Tooth Society Meeting 227

phosphatase activity (ALP) were determined and expressed here as a ratio of test/control + SD. Ocl number (mean cell no/parietal bone f SD) and area ($/cell+SD) were measured by image analysis of parietal bones from whole calvariae reacted for acid phosphatase with tartrate. CM from MCF7 cells inhibited BDC replication (ratio 0.53f0.22) and DNA synthesis (ratio 0.59f0.03). Ocl recruitment, however, was markedly enhanced (cell no. 104f21, area 1537+492; controls 51-t@ 892+243, p<O.OOl). Similar results were observed with CM from GB, which also stimulated ALP by 46%. In contrast, CM from MH stimulated BDC replication (ratio 1.4550.33) and DNA synthesis (1.29?0.38), but inhibited Ocl recruitment (cell no. 13+4, area 596-tl73; controls as above, p<O.OOl). CM from transformed human fibroblast lines and small cell lung carcinomas had no effect in either system. Our results show that in vitro, breast carcinoma cells secrete factors which stimulate, or inhibit, BDC proliferation independently of Ocl. Ocl recruitment was, however, also influenced. We do not know if this occurs via bone forming cells, or was due to the same (or other) factors secreted by the carcinoma cells acting directly on the Ocl.

References 1. I P Braidman, C Rothwell, D M Webber, P Crowe and D C Anderson (1989) Location of osteoclast precursors in foetal rat calvaria cultured on collagen gels J Bone and Mineral Research (in press). 2. C E Evans, C Ward and C S B Galasko (1988) The effects of soluble factors produced by myeloma cells on bone derived cells in vitro. Brit J Cancer 58 235.

P25. Does mandibular condylar cartilage regenerate? I’ D Robinson Department of Oral and Maxillofacial Surgery, Guys Hospital, London SE1 9RT

Further to clinical observations that anatomical remodelling and a return to full function is a common outcome following trauma or degeneration affecting the articular surface of the mandibular condyle, this study set out to determine whether adult primate condylar cartilage has the capacity for regeneration.

Fifteen young adult marmosets underwent open arthrotomy of both temporomandibular joints (TMJ). The articular surface of one condyle was breached through all cartilage layers into subchondral bone with a dental drill. The opposite condyle was left uninjured as a control. For comparison with an alternative joint model, the femoral components of the knees were similarly wounded.

After sacrifice at post-operative intervals between 3 days and 12 months the relevant joints were taken en bloc, fixed in formalin, decalcified and embedded in paraffin wax prior to sectioning at 4 and staining with H and E, Alcian Blue and Herovici’s modified Van Gieson.

By 2 weeks the TMJ wounds had lost their outline form as repair tissue filled the defect and the margins began remodelling. Mature patterns of collagen fibres

appeared at 4 weeks and new cartilage at 6 weeks. All cell layers were reconstituted by 8 weeks.

The resurfaced condyle showed further maturation with no evidence of breakdown up to 1 year.

P26. A preliminary study of normal and osteoporotic trabecular bone using frequency domain analysis J A P Jayasinghe and A Boyde Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1 E 6BT

In osteoporosis, selective thinning and loss occurs in differently oriented groups of trabeculae, but there is no efficient method to analyse these changes quantitatively The present study examined the use of two dimensional Fast Fourier Transform analysis (2DFFT) to study changes in the spatial frequency of trabeculae as a function of orientation. Post-mortem samples were obtained from clinically normal premenopausal and osteoporotic postmenopausal females. The images used were contact radiographs of <>5mm vertical sections from femoral heads: precision 3mm sagittal sections of cleaned, defatted 4th lumbar vertebral bodies: and 100Irm extended- focus images of the latter from a beam scanning confocal microscope. X-ray images of 5mm sections are unsatisfactory because there is too much overlap in the projection. The 100u.m optical sections were limited by the area scanned - a maximum of 24mm?. The radiographs of the 3mm sections were the most useful images used so far. Templating reverse FFTs permitted both qualitative and quantitative analysis of discrete orientations of trabeculae in the original image. 2DFFT can be employed to analyse changes during osteoporosis: it remains to determine the optimum image input, but we suggest that it might derive from an object scanning confocal microscope.

P27. A new, simple and rapid technique for the quantitative study of collagen orientation in cortical bone C M Riggs’ and A Boyde Department of Anatomy and Developmental Biology, University College London, Cower Street, London WClE 6BT *Department of Surgery and Obstetrics, Royal Veterinary College, North Mymms, Nr Hatfield, Herts AL9 7TA

Technical limitations of previous optical techniques for determining the preferred orientation of collagen fibres in cortical bone slices have restricted research in this field. We have developed a new technique to permit the rapid investigation of fibre orientation patterns in large numbers of large bone cross-sections. lOOpm, plane parallel cross-sections of a bone, cleaned of soft tissue and mounted in DPX, are viewed between crossed left and right handed circular polarisers. The resultant image of the entire cross- section is captured with a CCD TV camera, transferred to an image analysing computer and converted to a 5122 pixels, 256 grey level image. Analysis of pixel intensities within and between sections allows a direct