Effects of transforming growth factor β, basic fibroblast growth factor and dexamethasone on the...
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682 Abstracts Bone Vol. 16, No. 6 June 1995:679--695 P1. Factors modulating chondrogenesis in micromass cultures from developing chick limbs PSA White, TR Amett Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT The actions of retinoids in developing limb tissues are well known. We investigated whether potential roles could exist for thyroid hormone (T3), 1,25-(OH)2D3 or glucocorticoids as developmental modulators in chick embryos using micromass culture 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~aldroplet in 24 multiwell dishes, then cultured for 3-6d with additional medium containing hormone or vehicle and 10% serum. Chondrogenic nodules were visualised by alcian blue staining; the absorbance of bound dye eluted with guanidine HCI was measured at 65Onto. As expected, 9-cis retinoic acid (9-cis RA) strongly inhibited chondrogenesis and also reduced cell proliferation assessed by [3H]-thymidine incorporation. No effects of 1,25-(OH)2D3 or the analogues EB1089 and KH1060 alone, or in combination with 9-cis RA, were observed. However. T3 (10-250nM) exerted a range of actions including stimulation of chondrogenesis, reversal of the inhibition of chondrogenesis by 9-cis RA, as well as increasing cell proliferation. Inhibition of T3-induced chondrogenesis was seen in the presence of 10nM 1,25-(OH)2D3. Concentrations of dexamethasone (dex) as low as 0.2nM caused large increases in numbers of chondrogenic nodules; this effect was reversed by 9- cis RA. Micromass cultures provide a useful screening method for detecting functional responses to potential modulators of skeletal development, although the results with 9-cis RA show that this system may not directly reflect in vir~ findings. P2. Effects of transforming growth factor !3, basic fibroblast growth factor and dexamethasone on the proliferation and differentiation of rat marrow stromal fibroblasts RM Locklin, MC Williamson, JN Beresford, JT Triffitt, ME Owen MRC Bone Research Laboratory, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD The differentiation of stromal fibroblastic stem cells is highly regulated by systemic and local factors. We have examined the control of rat marrow stromal cell differentiation in vitro by TGF% bFGF and dexamethasone. Marrow was flushed from the femora of young rats and primary cultures established in c~MEM with 15% FCS and 50~ag/ml ascorbic acid supplemented with bFGF (Sng/ml), TGFB (lng/ml), or dexamethasone (10nM), either alone or in various combinations. Dexamethasone and TGF!3 reduced cell proliferation and sth'nulated alkaline phosphatase activity, bFGF was mitogenic, resulting in typically fibroblastic cells with markedly reduced alkaline phosphatase levels. Dexamethasone stimulated the generation of adipocytes, which were absent under all other conditions, and adipocyte formation was further increased by the addition of bFGF. The presence of TGF[~ completely inhibited adipocyte formation and reversed dexamethasone-induced adipogenesis. TGFI3 alone promoted osteogenesis with reduced cell proliferation and increased alkaline phosphatase and collagen production. These results confirm a central role for bFGF and TGFf~ in the regulation of osteogenesis and adipogenesis in rat bone marrow stromal cells and emphasise the importance of local factors in the regulation of bone cell differentiation. P3. Bone formation by stimulated periosteal cells is a consequence of gene repression JA Hoyland, K Morris, K Morgan, L Andrew, S Hopkins, G Andrew, D Marsh, AJ Freemont Bone Disease Research Centre, University of Manchester Manchester M13 9PT We have studied the time course of mRNA expression for collagens type l, 11, IX and X, osteocalcin, BMP2 by in situ hybridisation by cells in the early stages of periosteal bone formation in 30 human fractures, 24 rabbit laminectomies and 25 rats with type II collagen induced tenosynovitis. 5urn formalin fixed, EDTA decalcified, paraffin embedded serial sections were stained H and E and toluidine blue and probed using 3sS labelled cDNA probes, disclosed by autoradiography. First cellular proliferation with little evidence of matrix deposition but high levels of expression of collagen [ mRNA was seen, followed by formation of a variable connective tissue matrix, resembling osteoid, fibro-cartilage or one of uncertain type. In every case cells co-expressed collagens 1, 11 and IX, osteocalcin and BMP2. With time the matrix became more clearly cartilage or bone and the cells displayed an appropriate osteoblastic or chondroblastiq genotype. In some lesions new bone started to. remodel and in others type X collagen gene expression evidenced endochondral ossification. These data demonstrate that when stimulated by trauma or inflammation periosteal stern cells express a ubiquitous phenotype before full differentiation; formation of a definitive matrix results from repression of gene expression rather than stimulation. P4. Nitric oxide production is stimulated by mechanical loading AA Pitsillides, SCF Rawlinson, RFL Suswillo, LE Lanyon Department of Veterinary Basic Science, The Royal Veterinary College, London Mechanical loads result in functionally adaptative alterations in skeletal architecture and mass. The mechanism coordinating and controlling such responses is ill-defined. Nitric oxide (NO) is a mediator with diverse messenger functions. The role, function and mechanism of action, make it a potential mediator and integrator of mechanical stimuli. This study examines the effect of physiological mechanical loads on NO release from bone- derived primary osteoblast-like cells and bones in organ culture. Cells were derived by outgrowth from cortical explants of rat limb (LOBS) and calvariae (COBS), passaged onto plastic strips, cultured for 24 hours and the strips mechanically strained (4000,E; 1Hz for 600 cycles). Cells subjected to cyclic perturbation of medium served as controls. Rat ulnae were cultured for 5 hours and mechanically loaded (1200-4000~E; 1Hz for 8mins). Control bones were not loaded. The concentration of nitrite (NO2-) in medium, sampled at specified times, was assessed by chemiluminesence Strained cells showed immediate stimulation of NO2- release (0- 20min), which decreased to control levels (between 20-40rain) and remained low (40-80rain) thereafter. In control cells no stimulation in NOT release was evident. Increased NO 2- release appeared to be more marked from COBS (229%) than from LOBS (95%). Cultured rat ulnae also produced significant loading- related increases of NOT accumulatio'n; 0.90_+0.24,M in controls, 1.42+_0.42,M in loaded bones, Load-related increases of NO2- concentration were inhibited in the presence of L-NAME (an NO-synthase inhibitor; 1raM). Increased NO2 release was also evident in medium collected over discrete 5rain periods from canine cancellous bone cores (S pairs), maintained m vitro and superfused with medium whilst subjectcd to 15min of mehanical loading (5000~JE, 1Hz). These results indicate that mechanical strain results in rapid and transient NO release from isolated osteoblast-like cells derived from both long bones and calvariae, and from rat ulnae and canine cancellous bone cores in organ culture. This suggests that the loading-related adaptive changes in bone cell behaviour may be mediated at least in part by NO. P5. Variations in bone density in Europe and the risk of spinal deformity M Lunt, J Adams, L Benevolenskaya, J Cannata, 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 important risk factor for osteoporotic fracture. Wide variation across Europe has been shown in the rates of hip fracture (MEDOS) and prevalent spine deformities. (EVOS). We have investigated the effect of bone mineral density (BMD) on the risk of spine deformity. Thirteen centres randomly selected samples of 17% to 86% of
Transcript of Effects of transforming growth factor β, basic fibroblast growth factor and dexamethasone on the...
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
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