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P52. Ovariectomy causes profound bone loss from the tibiae but not the calvariae of rats
Bone Vol. 19, No. 6 December 1996:685-701
P49. Effects of menopause and anti-osteoporosis therapy on circulating collagen pmpeptides MWJ Davie, M Worsfold, L Risteli*, J Risteli**, CA Sharp
Abstracts
P51. Transforming growth factor-beta expression in regenerating deer antler AM Parry, JS Price*, H Bentley, RGG Russell
Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust, Oswestry, Shropshire and Collagen Research Group, Departments of "Medical Biochemistry and "°Clinical Chemistry, University of Oulu, Finland
Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield and *Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London
Oestrogen status is considered to influence bone metabolism and may have direct effects on both bone tissue and clearance of metabolites such as the collagen propeptides which are cleared by the liver. We have investigated the effects of menol~ause and therapy for bone loss on the circulating collagen type I propeptide levels PINP, PICP and PIIINP (by RIA (pg/L), Orion, Finland) in 19 screened healthy premenopausal (41+4years, range 33-50years) and 46 postmenopausal women (63+1 |years, range 43-91years) and in two groups of postmenopausal women receiving therapy for osteoporosis: 44 had HRT (Estraderm 25&50) for I year, another 7 had bisphosphonate (Didronel) for up to I year. At menopause both PINP and PICP increase (36.1+10 vs 50.5+23, (42%) and 83.4+21 vs 109±28 (31%), both P<0.05). HRT (1 year) lowers PINP (56.5±26 vs 30-2:16 (47%), P<0.0001) and PICP (123+47 vs 100±27 (19%) P<0.05) levels. Similarly, after up to 1 year of bisphosphonate therapy both PINP and P|CP are reduced (58+10 vs 26-29 (55%) P=0.02 and 124_+_28vs 81±8 (35%) P=0.02). PIIINP is not significantly changed by either menopause or HRT. PostM oestrogen deficiency is associated with increased circulating PINP and PICP. Both HRT and bisphosphonate both cause a marked reduction of both propeptides to premenopausal levels within 1 year of treatment. PINP exhibits a greater response to therapy than does PICP, whose response is blunted possibly due to hormonal modulation of its clearance mechanism.
The annual regrowth of antlers in male deer provides a rare and interesting model of complete bone regeneration. However, the regulation of antler growth is poorly understood. The aim of this study was to determine the iocalisation of transforming growth factor-beta (TGF-I~) in this model of accelerated endochondral ossification, since the TGF-I~ family members are known to be multifunctional mediators of osteochondrogenesis. Antler tissue was obtained from male red deer (Cervus elaphus) throughout the annual growth cycle. Two rabbit anti-TGF-{~ antibodies were used, anti-LC(1-30), which recognises intraceilular TGF-I~ [1] and antiCC(1-30), which recognises extracellular TGF-I~i and TGF-1~3 [2]. Using the anti-LC(1-30) antibody, positive staining was detected in the epidermis, around the hair follicles and was associated with the dermal and perichondrial blood vessels. Faint staining was also detected in chondrocytes within both the non-mineralised and calcified cartilaginous zones. A similar staining pattern was obtained using the anti-CC(1-30) antibody, but with more diffuse staining in the dermal and perichondrial regions and intense staining in the cartilaginous matrix. These results suggest that TGFI~ isoforms synthesised by antler chondrocytes are sequestered in the surrounding extracellular matrix, where they may be involved in the regulation of antler growth. [1] Flanderset a11989, J. Cell Biol., 108:653-660. [2] McCuneet al, 1992,Hum. Pathol., 23: 13-20.
P52. Ovariectomy causes profound bone loss from the tibiae but not the calvariae of rats T Davies, RA Hillam, B Pemor, GK Wakley, TM Skerry
Department of Anatomy, University of Bristol, Bristol BS2 8EJ PS0. Regulation of differentiation of human bone marrow stromal cells into osteoblast-like cells by TGF-I~and 1,25(OH)2D3 P Liu, B Oyajobi, RGG Russell
DeptartnL,'nt of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield $10 2RX Osteoblasts are believed to evolve from committed progenitors in bone marrow, and the process is likely to be regulated by systemic hormones, locally derived growth factors and cytokines that are bone-active. However, the role of these agents in osteoblast development is poorly understood. We examined the effects of TGP-I~, IGP-I and IL-6 on the differentiation of human bone marrow-derived stromal cells into osteoblast-like cells in v/tr0 in the presence of dexamethasone (Dex) and 1,25(OH)2D3 (1,25D) using alkaline p h o s p h a t a s e (ALP) as a marker of osteogenic differentiation. Bone marrow was flushed out of ribs obtained from thoracic operations, mononuclear cells were isolated by density gradient centrifugation over Picoll-hypaque, and primary cultures of these were established in MEM supplemented with 10% FCS. At sub-confluence, the stromal cells were passaged into multi-well plates (5 x 104 cells/well) in media containing 2% FCS, vitamin K, ascorbate and treated with rhTGP-~ (0.01-10ng/ml), rhlGF-I (10-1010"7M) or rhlL-6 (0.05-10ng/ml) alone and in the presence of Dex or 1,25D. None of the three factors examined had any effect on ALP activity at the concentrations tested, and there was no further increase in ALP activity in the presence of Dex. ALP activity was enhanced 2-3 fold in the presence of 1,25D (50nM) alone. However, when TGF-I~was combined with 1,25D, there was a marked increase (up to 7 fold) in ALP activity which was dose-dependent (max. effect at 10ng/ml TGFrI~ and 50riM 1,25D) and independent of proliferation. However," neither IGF-I nor IL-6 induced such an increase in ALP activity in the presence of 1,25D. These data indicate that the interactions between TGF-I~ and 1,25D may be specific. We are currently investigating the nature of these interactions as they may be important in the differentiation of osteoprogenitors into osteoblast-like cells.
Different bones in the skeleton have different functions, and are subjected to varying levels of mechanical loading. The low levels of strain experienced by the skull would be associated with profound loss of bone due to disuse if applied to the tibia, suggesting the possibility that some bones are more susceptible to the effects of resorptive influences than others. We investigated the responses of tibiae and calvariae to ovariectomy (OVX). 12 female Wistar rats were divided into OVX: and sham operated groups. 20 months after surgery, the animals were killed and the tibiae and calvariae were embedded and sectioned for histomorphometric analysis. To summarise the results briefly, bone was lost from both bones in the ovariectomised rats, compared with sham operated controls. However, in the tibiae, 75% of the metaphyseal trabecular bone was lost, while in the calvariae only 10% of the bone was lost. Differences between groups and sites were highly significant. These results suggest that individual bones in the skeleton have different sensitivities to systemic mediators of resorption, which may be associated with some survival value associated with the bone in question. The mechanism underlying such a difference could provide a mechanism to manipulate bone mass artificially.
P53. Immunofluorescent detection of oestrogen receptors (ER) suitable for use with avian bone C Baris, BH Thorp +, J Denton, DH Carter**, AJ Freemont*, IP Braidman
University of Manchester Bone Disease Research Centre Departments of Medicine (Rheumatology), *Pathological Sciences and **Oral Pathology, Stopford Building, Oxford Road, Manchester M13 9PT and +Roslin Research Institu re, Edinburgh EH25 9PS Oestrogen-lnduced structural bone loss in poultry skeletons during egg lay causes considerable osteoporosis, but oestrogen target cells in hen bone are still unclear. Mammalian osteocytes contain ER, detectable by immunofluorescencet. As mammalian immune-
699
P49. Effects of menopause and anti-osteoporosis therapy on circulating collagen pmpeptides MWJ Davie, M Worsfold, L Risteli*, J Risteli**, CA Sharp
Abstracts
P51. Transforming growth factor-beta expression in regenerating deer antler AM Parry, JS Price*, H Bentley, RGG Russell
Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust, Oswestry, Shropshire and Collagen Research Group, Departments of "Medical Biochemistry and "°Clinical Chemistry, University of Oulu, Finland
Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield and *Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London
Oestrogen status is considered to influence bone metabolism and may have direct effects on both bone tissue and clearance of metabolites such as the collagen propeptides which are cleared by the liver. We have investigated the effects of menol~ause and therapy for bone loss on the circulating collagen type I propeptide levels PINP, PICP and PIIINP (by RIA (pg/L), Orion, Finland) in 19 screened healthy premenopausal (41+4years, range 33-50years) and 46 postmenopausal women (63+1 |years, range 43-91years) and in two groups of postmenopausal women receiving therapy for osteoporosis: 44 had HRT (Estraderm 25&50) for I year, another 7 had bisphosphonate (Didronel) for up to I year. At menopause both PINP and PICP increase (36.1+10 vs 50.5+23, (42%) and 83.4+21 vs 109±28 (31%), both P<0.05). HRT (1 year) lowers PINP (56.5±26 vs 30-2:16 (47%), P<0.0001) and PICP (123+47 vs 100±27 (19%) P<0.05) levels. Similarly, after up to 1 year of bisphosphonate therapy both PINP and P|CP are reduced (58+10 vs 26-29 (55%) P=0.02 and 124_+_28vs 81±8 (35%) P=0.02). PIIINP is not significantly changed by either menopause or HRT. PostM oestrogen deficiency is associated with increased circulating PINP and PICP. Both HRT and bisphosphonate both cause a marked reduction of both propeptides to premenopausal levels within 1 year of treatment. PINP exhibits a greater response to therapy than does PICP, whose response is blunted possibly due to hormonal modulation of its clearance mechanism.
The annual regrowth of antlers in male deer provides a rare and interesting model of complete bone regeneration. However, the regulation of antler growth is poorly understood. The aim of this study was to determine the iocalisation of transforming growth factor-beta (TGF-I~) in this model of accelerated endochondral ossification, since the TGF-I~ family members are known to be multifunctional mediators of osteochondrogenesis. Antler tissue was obtained from male red deer (Cervus elaphus) throughout the annual growth cycle. Two rabbit anti-TGF-{~ antibodies were used, anti-LC(1-30), which recognises intraceilular TGF-I~ [1] and antiCC(1-30), which recognises extracellular TGF-I~i and TGF-1~3 [2]. Using the anti-LC(1-30) antibody, positive staining was detected in the epidermis, around the hair follicles and was associated with the dermal and perichondrial blood vessels. Faint staining was also detected in chondrocytes within both the non-mineralised and calcified cartilaginous zones. A similar staining pattern was obtained using the anti-CC(1-30) antibody, but with more diffuse staining in the dermal and perichondrial regions and intense staining in the cartilaginous matrix. These results suggest that TGFI~ isoforms synthesised by antler chondrocytes are sequestered in the surrounding extracellular matrix, where they may be involved in the regulation of antler growth. [1] Flanderset a11989, J. Cell Biol., 108:653-660. [2] McCuneet al, 1992,Hum. Pathol., 23: 13-20.
P52. Ovariectomy causes profound bone loss from the tibiae but not the calvariae of rats T Davies, RA Hillam, B Pemor, GK Wakley, TM Skerry
Department of Anatomy, University of Bristol, Bristol BS2 8EJ PS0. Regulation of differentiation of human bone marrow stromal cells into osteoblast-like cells by TGF-I~and 1,25(OH)2D3 P Liu, B Oyajobi, RGG Russell
DeptartnL,'nt of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield $10 2RX Osteoblasts are believed to evolve from committed progenitors in bone marrow, and the process is likely to be regulated by systemic hormones, locally derived growth factors and cytokines that are bone-active. However, the role of these agents in osteoblast development is poorly understood. We examined the effects of TGP-I~, IGP-I and IL-6 on the differentiation of human bone marrow-derived stromal cells into osteoblast-like cells in v/tr0 in the presence of dexamethasone (Dex) and 1,25(OH)2D3 (1,25D) using alkaline p h o s p h a t a s e (ALP) as a marker of osteogenic differentiation. Bone marrow was flushed out of ribs obtained from thoracic operations, mononuclear cells were isolated by density gradient centrifugation over Picoll-hypaque, and primary cultures of these were established in MEM supplemented with 10% FCS. At sub-confluence, the stromal cells were passaged into multi-well plates (5 x 104 cells/well) in media containing 2% FCS, vitamin K, ascorbate and treated with rhTGP-~ (0.01-10ng/ml), rhlGF-I (10-1010"7M) or rhlL-6 (0.05-10ng/ml) alone and in the presence of Dex or 1,25D. None of the three factors examined had any effect on ALP activity at the concentrations tested, and there was no further increase in ALP activity in the presence of Dex. ALP activity was enhanced 2-3 fold in the presence of 1,25D (50nM) alone. However, when TGF-I~was combined with 1,25D, there was a marked increase (up to 7 fold) in ALP activity which was dose-dependent (max. effect at 10ng/ml TGFrI~ and 50riM 1,25D) and independent of proliferation. However," neither IGF-I nor IL-6 induced such an increase in ALP activity in the presence of 1,25D. These data indicate that the interactions between TGF-I~ and 1,25D may be specific. We are currently investigating the nature of these interactions as they may be important in the differentiation of osteoprogenitors into osteoblast-like cells.
Different bones in the skeleton have different functions, and are subjected to varying levels of mechanical loading. The low levels of strain experienced by the skull would be associated with profound loss of bone due to disuse if applied to the tibia, suggesting the possibility that some bones are more susceptible to the effects of resorptive influences than others. We investigated the responses of tibiae and calvariae to ovariectomy (OVX). 12 female Wistar rats were divided into OVX: and sham operated groups. 20 months after surgery, the animals were killed and the tibiae and calvariae were embedded and sectioned for histomorphometric analysis. To summarise the results briefly, bone was lost from both bones in the ovariectomised rats, compared with sham operated controls. However, in the tibiae, 75% of the metaphyseal trabecular bone was lost, while in the calvariae only 10% of the bone was lost. Differences between groups and sites were highly significant. These results suggest that individual bones in the skeleton have different sensitivities to systemic mediators of resorption, which may be associated with some survival value associated with the bone in question. The mechanism underlying such a difference could provide a mechanism to manipulate bone mass artificially.
P53. Immunofluorescent detection of oestrogen receptors (ER) suitable for use with avian bone C Baris, BH Thorp +, J Denton, DH Carter**, AJ Freemont*, IP Braidman
University of Manchester Bone Disease Research Centre Departments of Medicine (Rheumatology), *Pathological Sciences and **Oral Pathology, Stopford Building, Oxford Road, Manchester M13 9PT and +Roslin Research Institu re, Edinburgh EH25 9PS Oestrogen-lnduced structural bone loss in poultry skeletons during egg lay causes considerable osteoporosis, but oestrogen target cells in hen bone are still unclear. Mammalian osteocytes contain ER, detectable by immunofluorescencet. As mammalian immune-
699