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P11. Bone morphometry and in situ hybridisation for the assessment of bone forming surfaces
690
Abstracts
P8. ILI~ increases nitric oxide and IL6 production in human intervertebral disc cells in vitro IK Ashton, HL Craster, I Holt', SM Eisenstein
Centre for Spinal Studies and *Charles Salt Research Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shf'opshire SYIO7AG Nitric oxide (NO) is a multifunctional messenger molecule generated by the nitric oxide synthases (NOS). The induction of NOS by cytokines such as ILIB has been demonstrated in bone derived cells and articular chondrocytes but not previously in cells of the human intervertebral disc. Cells were isolated by sequential enzymic digestion from the annulus fibrosus of intervertebral discs removed during surgery for back pain and grown in supplemented Ham's F-12 for 9-12 days. Low basal levels of NO (measured colorimetrically as nitrite) were observed after 48-72h unless cells were stimlllated with ILII3 (101000U/ml) when NO production rose to 5-10 nmoles/105 cells/24h. The response was dose related and substantially inhibited by 0.2mM LNMMA (a competitive inhibitor of NO synthesis from arginine) and dexamethasone (IFM). IL6 production, measured by the B9 cell bioassay, increased in the presence of IL113to >10Ong/ml and this was also inhibited by LNMMA and dexamethasone. NO is a powerful modulator of such diverse processes as neurotransmission, inflammation and bone resorption and appears to mediate in part the suppressive effect of ILII3on cartilage matrix synthesis. Factors regulating NO synthesis in intervertebral disc, a tissue which shares structural and regulatory features with cartilage, may be relevant to disc metabolism or degeneration.
Pg. Treated hyperthyroidism: are the bones still at risk? DJ Grant, MET McMurdo, PA Mole*, CR Paterson*
Departments of Medicine and *Biochemical Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY Our aim in this cross-sectional study was to measure forearm bone mineral density (BMD) in postmenopausal women with a previous history of hyperthyroidism, comparing those remaining euthyroid after treatment and those requiring thyroxine therapy with the local control population. Our 106 subjects were divided into four groups: treated with radioiodine, remaining euthyroid (RU, n=15); treated with radioiodine and receiving thyroxine for at least five years (RT, n=46); treated with surgery, remaining euthyroid (SU, n=21); treated with surgery and receiving thyroxine for at least five years (ST, n=24). There were 102 control subjects. Mean z-scores at distal and ultradistal sites were as follows: -0.6l and -0.81 in group RU; -0.58 and -0.56 in group RT; -0.27 and -0.30 in group SU; -0.81 and -0.57 in group ST. Subjects in groups RU, RT and ST, but not SU, had significantly lower BMD than controls (p~.05). Postmenopausal women with previous hyperthyroidism treated with radioiodine had reduced BMD, whether or not receiving thyroxine. Such patients should be targeted for densitometry and protective therapy with oestrogen should be considered. Those treated with surgery appeared to be at less risk; this may be because most were diagnosed and treated whilst premenopausal.
P10. Early r e s p o n s e s to mechanical load in selectively-bred embryonic chicks differ AA Pitsillides, SCF Rawlinson, LE Lanyon
Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU Poultry selectively bred either for maximum egg (ET) or meat (MT) production suffer from growth abnormalities and skeletal i~adequacies are common. To investigate whether this is related to mechanically adaptive bone (re)modelling we have studied early resident bone cell responses associated with applied mechanical strain (1200,E at IHz for 10rain), in embryonic tibiotarsi from" commercially-bred and wild-type (WT) chicks. We have previously shown strain magnitude-related increases in glucose 6-phosphate dehydrogenase (G6PD) activity, assessed microspectrophotometrically, and that this response is imitated by prostacyclin (PGI2).
Bone Vol. 19, No. 6 December 1996:685-701 In this study, WT and MT periosteal osteoblasts in tibiotarsi both responded in a biphasic manner to exogenous PG[2, with maximal stimulation of G6PD activity at 10-7 and 10-6M PGI2. However, ET chick osteoblasts showed smaller, progressive increases up to 10-SM PGI 2. Load increased G6PD activity in tibiotarsi of WT and ET chicks, whilst MT chicks failed to show any significant increases. Load-related nitric oxide (NO) release from these different strains, paralleled G6PD activity load-responsiveness. Skeletal abnormalities which develop in rapidly growing MT chicks may reflect a compromised ability to produce PGI2 (and/or NO) in response to load. Osteopaenia in ET chick bones may reflect insufficient load-bearing stimuli.
Pll. Bone morphometry and in situ hybridisation for the assessment of bone forming surfaces V Kusec, AS Virdi, JT Triffitt
MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD Measurement of the extent of bone surfaces involved in bone formation is an important histomorphometric index. This is usually achieved by planned administration of fluorochromes, such as tetracyclines. Previous studies have suggested cytochemical localisation of alkaline phosphatase as a useful mean of identifying bone forming surfaces (Bradbeer et al, J Bone Miner Res 7: 905, 1992), however this is not a reliable indicator (Ballanti et al, Bone 16: 493, 1995). In the present study we have investigated the use of the major osteoblast products Type I collagen and osteonectin to demonstrate active surfaces, by in situ hybridisation. The findings were compared with in viz~ labelling with fluorochromes on the same sections. Young rabbits were injected intraperitoneally with fluorochromes and killed up to six days later. Bone tissue was fixed in 4% paraformaldehyde, embedded in plastic and sectioned. In situ hybridisation was carried out using digoxigenin labelled riboprobes for Type 1 collagen and osteonectin. Fluorochromes were assessed by ultraviolet microscopy and bone surface measurements performed for comparison. Type l collagen and osteonectin were colocalised at a large proportion of the bone surfaces. Expression of mRNAs of these proteins correlated with the extent of fluorochrome labelling. This suggests that histomorphometric assessment of bone forming activity may be possible by in situ hybridisation procedures using a variety of osteoblast products. P12. Identification of novel response genes in mechanically loaded articular cartilage: studies using differential display PCR ICEMcGuigan. PS Grabowski, RM Aspden*, SH Ralston
Department of Medicine and Therapeutics, and *Orthopaedic Surgery, University of Aberdeen Medical School, Foresterhill, Aberdeen An9 2ZD Background. Abnormal mechanical loading has been implicated in the initiation and progression of diseases such as osteoarthritis. Our previous work has shown that articular chondrocytes in explant culture respond rapidly (+ 4hr) to mechanical load by modifying production of matrix components. The mechanisms underlying this response are unclear. In order to identify response genes which are activated by mechanical loading of articular cartilage, we used reverse-transcription polymerase chain reaction (RT/PCR) to study expression of cytokines and growth factors in loaded articular cartilage, coupled with differential display PCR (ddPCR) to identify novel genes whose expression is upregulated by loading. Methods: Total RNA was extracted, and cDNA prepared from explants of bovine articular cartilage which had been subjected to 1 MPa load, applied cyclically (@ 0.5 Hz) or statically for 1hr. A series of unloaded explants were used in each experiment as controls. Results: There was no consistent expression of [L-113, TNFa, TGFb, IGF-[ under any conditions, whereas bFGF was almost invariably expressed under both loaded and unloaded conditions. Analysis by ddPCR has identified 4 mRNA's whose expression is differentially regulated by loading as shown in the table below: Control Static Cyclic
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Abstracts
P8. ILI~ increases nitric oxide and IL6 production in human intervertebral disc cells in vitro IK Ashton, HL Craster, I Holt', SM Eisenstein
Centre for Spinal Studies and *Charles Salt Research Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shf'opshire SYIO7AG Nitric oxide (NO) is a multifunctional messenger molecule generated by the nitric oxide synthases (NOS). The induction of NOS by cytokines such as ILIB has been demonstrated in bone derived cells and articular chondrocytes but not previously in cells of the human intervertebral disc. Cells were isolated by sequential enzymic digestion from the annulus fibrosus of intervertebral discs removed during surgery for back pain and grown in supplemented Ham's F-12 for 9-12 days. Low basal levels of NO (measured colorimetrically as nitrite) were observed after 48-72h unless cells were stimlllated with ILII3 (101000U/ml) when NO production rose to 5-10 nmoles/105 cells/24h. The response was dose related and substantially inhibited by 0.2mM LNMMA (a competitive inhibitor of NO synthesis from arginine) and dexamethasone (IFM). IL6 production, measured by the B9 cell bioassay, increased in the presence of IL113to >10Ong/ml and this was also inhibited by LNMMA and dexamethasone. NO is a powerful modulator of such diverse processes as neurotransmission, inflammation and bone resorption and appears to mediate in part the suppressive effect of ILII3on cartilage matrix synthesis. Factors regulating NO synthesis in intervertebral disc, a tissue which shares structural and regulatory features with cartilage, may be relevant to disc metabolism or degeneration.
Pg. Treated hyperthyroidism: are the bones still at risk? DJ Grant, MET McMurdo, PA Mole*, CR Paterson*
Departments of Medicine and *Biochemical Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY Our aim in this cross-sectional study was to measure forearm bone mineral density (BMD) in postmenopausal women with a previous history of hyperthyroidism, comparing those remaining euthyroid after treatment and those requiring thyroxine therapy with the local control population. Our 106 subjects were divided into four groups: treated with radioiodine, remaining euthyroid (RU, n=15); treated with radioiodine and receiving thyroxine for at least five years (RT, n=46); treated with surgery, remaining euthyroid (SU, n=21); treated with surgery and receiving thyroxine for at least five years (ST, n=24). There were 102 control subjects. Mean z-scores at distal and ultradistal sites were as follows: -0.6l and -0.81 in group RU; -0.58 and -0.56 in group RT; -0.27 and -0.30 in group SU; -0.81 and -0.57 in group ST. Subjects in groups RU, RT and ST, but not SU, had significantly lower BMD than controls (p~.05). Postmenopausal women with previous hyperthyroidism treated with radioiodine had reduced BMD, whether or not receiving thyroxine. Such patients should be targeted for densitometry and protective therapy with oestrogen should be considered. Those treated with surgery appeared to be at less risk; this may be because most were diagnosed and treated whilst premenopausal.
P10. Early r e s p o n s e s to mechanical load in selectively-bred embryonic chicks differ AA Pitsillides, SCF Rawlinson, LE Lanyon
Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU Poultry selectively bred either for maximum egg (ET) or meat (MT) production suffer from growth abnormalities and skeletal i~adequacies are common. To investigate whether this is related to mechanically adaptive bone (re)modelling we have studied early resident bone cell responses associated with applied mechanical strain (1200,E at IHz for 10rain), in embryonic tibiotarsi from" commercially-bred and wild-type (WT) chicks. We have previously shown strain magnitude-related increases in glucose 6-phosphate dehydrogenase (G6PD) activity, assessed microspectrophotometrically, and that this response is imitated by prostacyclin (PGI2).
Bone Vol. 19, No. 6 December 1996:685-701 In this study, WT and MT periosteal osteoblasts in tibiotarsi both responded in a biphasic manner to exogenous PG[2, with maximal stimulation of G6PD activity at 10-7 and 10-6M PGI2. However, ET chick osteoblasts showed smaller, progressive increases up to 10-SM PGI 2. Load increased G6PD activity in tibiotarsi of WT and ET chicks, whilst MT chicks failed to show any significant increases. Load-related nitric oxide (NO) release from these different strains, paralleled G6PD activity load-responsiveness. Skeletal abnormalities which develop in rapidly growing MT chicks may reflect a compromised ability to produce PGI2 (and/or NO) in response to load. Osteopaenia in ET chick bones may reflect insufficient load-bearing stimuli.
Pll. Bone morphometry and in situ hybridisation for the assessment of bone forming surfaces V Kusec, AS Virdi, JT Triffitt
MRC Bone Research Laboratory, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD Measurement of the extent of bone surfaces involved in bone formation is an important histomorphometric index. This is usually achieved by planned administration of fluorochromes, such as tetracyclines. Previous studies have suggested cytochemical localisation of alkaline phosphatase as a useful mean of identifying bone forming surfaces (Bradbeer et al, J Bone Miner Res 7: 905, 1992), however this is not a reliable indicator (Ballanti et al, Bone 16: 493, 1995). In the present study we have investigated the use of the major osteoblast products Type I collagen and osteonectin to demonstrate active surfaces, by in situ hybridisation. The findings were compared with in viz~ labelling with fluorochromes on the same sections. Young rabbits were injected intraperitoneally with fluorochromes and killed up to six days later. Bone tissue was fixed in 4% paraformaldehyde, embedded in plastic and sectioned. In situ hybridisation was carried out using digoxigenin labelled riboprobes for Type 1 collagen and osteonectin. Fluorochromes were assessed by ultraviolet microscopy and bone surface measurements performed for comparison. Type l collagen and osteonectin were colocalised at a large proportion of the bone surfaces. Expression of mRNAs of these proteins correlated with the extent of fluorochrome labelling. This suggests that histomorphometric assessment of bone forming activity may be possible by in situ hybridisation procedures using a variety of osteoblast products. P12. Identification of novel response genes in mechanically loaded articular cartilage: studies using differential display PCR ICEMcGuigan. PS Grabowski, RM Aspden*, SH Ralston
Department of Medicine and Therapeutics, and *Orthopaedic Surgery, University of Aberdeen Medical School, Foresterhill, Aberdeen An9 2ZD Background. Abnormal mechanical loading has been implicated in the initiation and progression of diseases such as osteoarthritis. Our previous work has shown that articular chondrocytes in explant culture respond rapidly (+ 4hr) to mechanical load by modifying production of matrix components. The mechanisms underlying this response are unclear. In order to identify response genes which are activated by mechanical loading of articular cartilage, we used reverse-transcription polymerase chain reaction (RT/PCR) to study expression of cytokines and growth factors in loaded articular cartilage, coupled with differential display PCR (ddPCR) to identify novel genes whose expression is upregulated by loading. Methods: Total RNA was extracted, and cDNA prepared from explants of bovine articular cartilage which had been subjected to 1 MPa load, applied cyclically (@ 0.5 Hz) or statically for 1hr. A series of unloaded explants were used in each experiment as controls. Results: There was no consistent expression of [L-113, TNFa, TGFb, IGF-[ under any conditions, whereas bFGF was almost invariably expressed under both loaded and unloaded conditions. Analysis by ddPCR has identified 4 mRNA's whose expression is differentially regulated by loading as shown in the table below: Control Static Cyclic
Band #1 +++ ++
Band #2 +++ +++
Bancl #3
Band #4
+++ +++