682 Abstracts Bone Vol. 16, No. 6 June 1995:679--695

P1. Factors modula t ing chondrogenes i s in micromass cultures from deve lop ing chick l imbs PSA White , TR A m e t t Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT

The ac t ions of re t ino ids in deve lop ing limb tissues are well known. We invest igated whe the r potential roles could exist for t h y r o i d h o r m o n e (T3), 1 ,25-(OH)2D3 or g lucocor t i co ids as deve lopmen ta l m o d u l a t o r s in chick embryos us ing micromass cul ture assays. Mesenchymal cells from stage 21-27 chick embryo limb buds were plated at 1.5-2.0 x 105 per well in a 10~al droplet in 24 mul t iwe l l d ishes , then cu l tu red for 3-6d wi th add i t iona l m e d i u m c o n t a i n i n g h o r m o n e or vehic le a n d 10% se rum. C h o n d r o g e n i c nodu le s were visual ised by alcian blue staining; the abso rbance of b o u n d d y e eluted wi th guan id ine HCI was m e a s u r e d a t 65Onto. As expected, 9-cis retinoic acid (9-cis RA) s t r o n g l y i n h i b i t e d c h o n d r o g e n e s i s a n d a l so r e d u c e d cell p ro l i f e ra t ion assessed b y [ 3 H ] - t h y m i d i n e i nco rpo ra t i on . No effects of 1,25-(OH)2D3 or the ana logues EB1089 and KH1060 a lone , or in c o m b i n a t i o n w i th 9-cis RA, were observed . H o w e v e r . T3 (10-250nM) exerted a range of act ions inc luding s t imu la t i on of c h o n d r o g e n e s i s , reversa l of the inhibi t ion of c h o n d r o g e n e s i s b y 9-cis RA, as well as inc reas ing cell prol iferat ion. Inhibit ion of T3-induced chondrogenes i s was seen in the p r e s e n c e of 10nM 1,25-(OH)2D3. C o n c e n t r a t i o n s of dexame thasone (dex) as low as 0.2nM caused large increases in number s of chond rogen i c nodules; this effect was reversed by 9- cis RA. Micromass cul tures p rov ide a useful screening method for de t ec t ing func t iona l responses to potent ia l modu la to r s of skeletal deve lopmen t , a l t hough the results wi th 9-cis RA show that this sys tem m a y not direct ly reflect in vir~ findings.

P2. Effects of t ransforming growth factor !3, basic f ibroblast growth factor and d e x a m e t h a s o n e on the prol i ferat ion and differentiation of rat marrow stromal fibroblasts RM Locklin, MC Wil l iamson, JN Beresford, JT Triffitt, ME Owen MRC Bone Research Laboratory, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD

The d i f ferent ia t ion of s t romal f ibroblast ic s tem cells is h ighly regula ted by systemic and local factors. We have examined the control of rat m a r r o w s t romal cell d i f ferent ia t ion in vitro by TGF% bFGF and dexamethasone . Mar row was f lushed from the femora of y o u n g rats and p r imary cul tures established in c~MEM with 15% FCS a n d 50~ag/ml ascorbic acid s u p p l e m e n t e d with bFGF (Sng/ml) , TGFB ( l n g / m l ) , or dexamethasone (10nM), either a lone or in va r ious combina t ions . Dexame thasone and TGF!3 reduced cell prol i fera t ion and sth 'nulated alkal ine phospha t a se activi ty, bFGF was mitogenic , resul t ing in typical ly fibroblastic cells w i th m a r k e d l y r e d u c e d a lka l ine p h o s p h a t a s e levels. Dexamethasone s t imula ted the genera t ion of adipocytes , which were absent u n d e r all o ther condit ions, and adipocyte formation was fur ther increased by the addi t ion of bFGF. The presence of TGF[~ comple te ly inhibi ted ad ipocy te format ion and reversed d e x a m e t h a s o n e - i n d u c e d ad ipogenes i s . TGFI3 alone p r o m o t e d os t eogenes i s wi th r e d u c e d cell p ro l i f e ra t ion and increased a lka l ine p h o s p h a t a s e and col lagen p roduc t ion . These results con f i rm a centra l role for bFGF and TGFf~ in the regulat ion of osteogenesis and ad ipogenes i s in rat bone mar row stromal cells and emphas i se the impor t ance of local factors in the regulat ion of bone cell differentiat ion.

P3. Bone f o r m a t i o n by s t imula ted per iosteal ce l l s is a consequence of gene repression JA H o y l a n d , K Morr is , K M o r g a n , L A n d r e w , S H o p k i n s , G A n d r e w , D Marsh, AJ Freemont Bone Disease Research Centre, University of Manchester Manchester M13 9PT

We have s t ud i ed the t ime course of m R N A express ion for col lagens type l, 11, IX and X, osteocalcin , BMP2 by in situ hybr id i s a t i on b y cells in the ear ly s tages of per iosteal bone format ion in 30 h u m a n fractures, 24 rabbit laminectomies and 25 rats with type II collagen induced tenosynovitis. 5urn formal in fixed, EDTA decalcified, paraffin embedded serial

sections were s ta ined H and E and to luidine blue and probed us ing 3sS labelled cDNA probes, disclosed by autoradiography. First ce l lu l a r p r o l i f e r a t i o n wi th little ev idence of matr ix deposi t ion but h igh levels of expression of collagen [ mRNA was seen, fo l lowed by fo rmat ion of a var iab le connect ive t issue matr ix , r esembl ing os teoid , f ibro-car t i lage or one of uncer ta in type. In every case cells co-expressed col lagens 1, 11 and IX, os teoca lc in a n d BMP2. With t ime the matr ix became more clearly car t i lage or bone and the cells d isplayed an appropr ia te osteoblast ic or chondrob las t iq genotype . In some lesions new bone s ta r ted to. r emode l and in others type X col lagen gene express ion ev idenced endochond ra l ossification. These da ta d e m o n s t r a t e tha t w h e n s t imula ted by t r a u m a or i n f l a m m a t i o n p e r i o s t e a l s tern cells express a u b i q u i t o u s p h e n o t y p e before full differentiat ion; format ion of a definit ive matr ix results f rom repress ion of gene expression ra ther than s t imu la t i on .

P4. Nitric oxide production is stimulated by mechanical load ing AA Pitsillides, SCF Rawlinson, RFL Suswillo, LE Lanyon Department of Veterinary Basic Science, The Royal Veterinary College, London

Mechanical loads result in funct ional ly adapta t ive alterations in skeletal a r ch i t ec tu re and mass . The m e c h a n i s m coord ina t ing and control l ing such responses is ill-defined. Nitric oxide (NO) is a med ia to r with diverse messenger functions. The role, function and m e c h a n i s m of act ion, make it a potential media to r and in tegra tor of mechanica l stimuli. This s tudy examines the effect of phys io log ica l mechan ica l loads on NO release from bone- der ived p r ima ry osteoblast-like cells and bones in organ culture. Cells we re der ived by o u t g r o w t h f rom cortical explants of rat l imb (LOBS) and calvar iae (COBS), passaged onto plastic strips, cu l tu red for 24 hour s a n d the s t r ips mechan ica l ly s t ra ined (4000,E; 1Hz for 600 cycles). Cells subjected to cyclic perturbat ion of m e d i u m served as controls . Rat u lnae were cul tured for 5 hours and mechan ica l ly loaded (1200-4000~E; 1Hz for 8mins). Cont ro l bones we re not loaded. The concen t ra t ion of nitr i te (NO2-) in med ium, sampled at specified times, was assessed by c h e m i l u m i n e s e n c e Strained cells showed immedia te s t imulat ion of NO2- release (0- 20min), w h i c h decreased to control levels (between 20-40rain) and r e m a i n e d low (40-80rain) thereaf ter . In control cells no s t imula t ion in N O T release was evident. Increased NO 2- release appea red to be more marked from COBS (229%) than from LOBS (95%). C u l t u r e d rat u lnae also p r o d u c e d s ignif icant loading- related increases of N O T accumulatio 'n; 0.90_+0.24,M in controls, 1.42+_0.42,M in loaded bones, Load-re la ted increases of NO2- concen t ra t ion were inhibi ted in the presence of L-NAME (an N O - s y n t h a s e inhibitor; 1raM). Increased NO2 release was also evident in m e d i u m collected over discrete 5rain per iods from canine cancel lous bone cores (S pairs) , main ta ined m vitro and s u p e r f u s e d w i t h m e d i u m wh i l s t sub jec tcd to 15min of mehanical loading (5000~JE, 1Hz). These results indicate that mechanical strain results in rapid and t ransient N O release from isolated osteoblast-l ike cells derived f rom both long bones a n d calvar iae , and from rat ulnae and canine cancel lous bone cores in o rgan culture. This suggests that the loading-re la ted adap t ive changes in bone cell behaviour may be media ted at least in part by NO.

P5. Var ia t ions in bone density in Europe and the risk of sp ina l deformi ty M Lunt , J A d a m s , L Benevolenskaya , J Canna ta , J Dequeker, C Dodenhof , J Falch, D Felsenberg, J Kanis, P Masaryk, T O'Neill, H Pols, G Poor, J Reeve, D Reid, C Scheidt -Nave, A Silman, K Weber European Prospective Osteoporosis Study (EPOS), Institute of Public ttealth, Cambridge CB2 2SR

Low bone mass is an impor t an t risk factor for os teoporot ic fracture. Wide var ia t ion across Europe has been shown in the rates of hip fracture (MEDOS) and prevalent spine deformities. (EVOS). We have invest igated the effect of bone mineral densi ty (BMD) on the risk of spine deformity. Thir teen centres r a n d o m l y selected samples of 17% to 86% of