3. Studies with cells derived from growing deer antler

Bone, 10, 143- 154 (1989) Printed in the USA. All rights reserved.

8756-3282189 $3.00 + .OO Copyright 0 1989 Maxwell Pergamon Macmillan plc

BONE AND TOOTH SOCIETY AUTUMN SEPTEMBER 23,1988 Venue:

University

MEETING,

of Wales College of Medicine, Heath Park, Cardiff

LocalOrgunizer:Dr. Juliet Compston, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff. 1. Location and differentiation of osteoclast precursors in foetal rat calvariae cultured on collagen gels I P Braidman, C Rothwell, P Crowe, J Denton,* and D C Anderson Departments of Medicine, Hope Hospital, Eccles Old Road, Salford M6 8HD, and Rheumatology’, University of Manchester School of Medicine

resistance acid phosphatase are unreliable markers for osteoclastic differentiation G Hattersley and T J Chambers Department of Histopathology, St GeorgeS Hospital Medical School, London, UK The osteoclast is known to derive from bone marrow, and recent experiments have enumerated tartrateresistant acid phosphatase (TRAP) positive multinucleate cells that develop in cultures of haemopoietic tissue to analyse the regulation of osteoclast generation. These cells have never been characterised as osteoclasts, however, and we elected to assess the relationship between bone resorption, TRAe and multinuclearity in mouse bone marrow cultures. Mouse bone marrow cells, and/or peritoneal macrophages, were incubated on plastic coverslips or bone slices in the presence or absence of la,25dihydroxyvitaminD3 (la,25(OH)>D3). Osteoclast generation (bone resorption) occurred in marrow cultures only with la,25(OH),D?. However, TRAPpositive multinuclear cells developed both with and without the hormone. The multinuclear cells bound F4/80, a marker for macrophages that does not bind to osteoclasts. Peritoneal macrophages became multinucleate and developed TRAP-positivity in culture to levels similar to those observed in freshly-isolated osteoclasts, especially with la,25(OH)>D3, but remained non-resorptive. In cultures of marrow cells incubated with la,25(OH)>D3 bone resorption was more extensive than could be accounted for by the number of multinucleate cells present, suggesting a major contribution by mo&onuclear cells with osteoclastic function. Thus, while TRAP and multinuclearity are reliable markers for osteoclastic phenotype in bone, they are unreliable markers in culture.

In foetal rat calvariae, mature osteoclasts only appear in the last 48 hours of gestation. We wished to investigate the origin of these cells and their mechanism of differentiation. Calvariae were cultured on type 1 collagen geis (l.Sml/mg) in MEM, containing p glycerophosphate (5mM), sodium ascorbate (O.lmg/ml), proline (IOmM) and 5% FCS, conditions which prevented the deterioration in morphology and enzyme loss observed with liquid media. 19 day calvariae contain few osteoclasts but after 48 hours culture, numerous large neutral red positive cells were seen on the endocranial bone surface, similar to those in calvariae at 21 days gestation. These cells contracted within 15 minutes of incubating tissue with calcitonin and contained high tartrate resistant acid phosphatase activity. Osteoclasts were therefore detected by reacting whole tissue (mean cell number *SD and mean area $f SD/parietal bone, measured by image analysis) or cryostat sections (mean absorbance ? SD, measured by quantitative cytochemistry) for acid phosphatase activity and by inspection of 2~ sections of fixed tissue. Thus, after 48 hours culture, osteoclasts increased in number and area from 9 +3 and ll$ +5 respectively to 79 fll and 63~’ +24 respectively and acid phosphatase activity rose from 0.12 kO.003 to 0.42 f0.13. This was not seen when endocranial membranes (containing meningeal blood vessels) were stripped away from the 19 day calvariae. Co-culture of these “stripped” bones with the membranes or with 16 day calvariae (before mineralisation) for 48 hours produced increased cell numbers (86 ?21,78 +19 respectively) cell area (57$ +21,52$ f22 respectively) and enzyme activity (0.37 +O.ll). These were not observed if a nucleopore filter (0.22mp) was inserted between the membranes and the “stripped” bone. We conclude that precursor osteoclasts are present from at least 16 days gestation and that endocranial membranes are a major source of these cells.

3. Studies with cells derived from growing deer antler T R Arnett, C M Gray, M L Taylor, M A Horton and A S I Loudon Department ofAnatomy and Developmental Biology, University College London, ICRF Haemopoiesis Research Group, Department of Haematology, St Bartholomew’s Hospital and Institute of Zoology, Londotz Little is known about the cell biology of deer antler and the factors regulating the remarkable rates of growth seen in this mineralised tissue. In this study we have

2. Generation of osteoclastic function in mouse bone marrow cultures: multinuclearity and tartrate143

144

examined the hormonal regulation of cells cultured from the growing antler tissue of deer culled at Whipsnade zoo. Routine histology revealed highly active osteoblast-like cells on many surfaces of the forming trabeculae in the middle portion of the antler and an abundance of multinucleate osteoclasts. Positive immunocytochemical staining of multinucleate cells in tissue imprints or isolated cell preparations from a 20 cm fallow deer antler was seen with the osteoclast-specific monoclonal antibodies 13C2 and 23C6. Cells were mechanically disaggregated from a small piece of midportion mineralising antler tissue and incubated on slices of devitalised sperm whale dentine in medium of reduced bicarbonate content in a 5% CO2 atmosphere for 24 h, in the absence or presence of synthetic salmon calcitonin (sCT). The slices were fixed and stained for tartrate-resistant acid phosphatase (TRAP) and then with toluidine blue. Numerous, TRAP-positive multinucleate cells were clearly seen to be associated with extended resorption trails in the surface of control dentine slices. Resorption was reduced on slices cultured with sCT, but was not abolished, even by hormone concentrations as high as 100 ng/ml. Only a proportion of TRAP-positive multinucleate cells exhibited the characteristic contracted morphology associated with calcitonin inhibition of rat or rabbit osteoclasts. Cell outgrowths from fragments of tissue explanted from the mineralising mid-region of a 25 cm Pere David’s deer antler were cultured at passages 2-3 in phenol red-free medium with 5% stripped fetal calf serum containing ethanol vehicle, testosterone, 17B oestradiol or 1,25(OH)2D4 After 5 days in culture, cell proliferation, determined by ‘H-thymidine uptake, was significantly inhibited by 10-RM testosterone and 10.‘M 17B-oestradiol (~~0.02) but was unaffected by 10e9M 1,25. Dose- and time-dependent inhibitions of cell number, measured as methylene blue binding to cell monolayers, were observed in the presence of testosterone at concentrations around the physiological range: cell numbers were reduced to 57% of the control value (~~0.02) after 5 days in culture with 10-RM testosterone. The alkaline phosphatase activity of these antler-derived cells was very low and was not significantly altered by the sex hormones or 1,25(OH)2D3. These data are consistent with the observation that cessation of antler growth in vivo is associated with a rise in plasma testosterone levels and contrasts with the recently described stimulatory actions of the sex steroids on skeletal bone cell function in z&o.

4. Sex differences in the induction of vitamin D deficiency by calcium deprivation Claire D Hickey, M R Clements and E Barbara Mawer Department ofMedicine, UniversityofManchester Medical School, Manchester Ml3 9PT The hepatic inactivation and biliary excretion of 25(OH)D are enhanced by calcium deprivation. To investigate the time course of these events Wistar rats were fed on either standard rodent pellets or wholemeal

Abstracts from the Bone and Tooth Society Meeting diet until they weighed approx 170g. They were then transferred to vitamin D-deficient diets containing either 0.02% or 0.5% Ca and blood samples were drawn at intervals from day 0 to 28. Mean serum [25(OH)D] in rats on pellet diet was 42.7 + 2.7 ng/ml (+SEM) and subsequently declined with a mean plasma elimination half-time (t’/z) of 26.0 days on 0.5% Ca diet compared with 13.4 days on a 0.02% Ca diet. Between days 0 and 28 there was a greater fall in mean serum [Ca2+] in males on 0.02% Ca diet (2.64 to 2.35 mmol/l) than in the corresponding females (2.55 to 2.41 mmoV1). This was associated with a higher mean serum [1,25(OH),D] on day 28 in males (195 f 17pg/ml) than in females (139 k 8 pg/ml). Rats on wholemeal diet were given a S.C. injection of 5pg D? 7 days before exclusion of vitamin D from the diet. Mean serum [25(OH)D] was 20.6 ? 2.5 ng/ ml at the time of transfer and the animals became deficient within a further 28 days. As before mean serum [1,25(OH),D] was higher in the males (288 f 23 pg/ml) than in the females (204 * 22 pg/ml) on 0.02% Ca diet whilst values for t’/z-25(OH)D were 21% shorter in the females compared with the males. These findings again confirm that the increased metabolic clearance of 25(OH)D on calcium-deficient diets is not due to its consumption by the increased production of 1,25(OH)2D. They are consistent with the evidence that the enhanced hepatic inactivation of 25(OH)D in calcium deprivation is mediated by a direct effect of 1,25(OH)*D on the liver cell and that the expression of the 1,25(OH)2D receptor in hepatocytes may be stimulated by oestrogen.

5. Reduced bone formation in non-steroid treated patients with rheumatoid arthritis S Vedi, P I Croucher, M M O’Sullivan and J E Compston Departments of Pathology and Rheumatology, University Hospital of Wales and College of Medicine, Cardifi UK Generalised bone loss occurs in patients with nonsteroid treated rheumatoid arthritis (RA) but its cellular basis has not been established. We have measured mean wall thickness (MWT), which reflects the amount of bone formed per remodelling unit, in iliac crest trabecular bone from 45 non-steroid treated patients with RA, aged 34-71 years (mean 53). Eight-micron sections stained with haematoxylin and eosin were examined at x 100. The mean interstitial bone thickness (MIRT), which is related to resorption depth, was calculated from mean trabecular width and MWT. Mean wall thickness was significantly reduced in both male and female patients when compared to controls (42.9 f 4.3 vs 50.4 k 6.3 pm, p