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ABSTRACTS P-357 to P-536 POSTER PRESENTATIONS
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows: Odd numbers on Friday 11:00 - 12:00, Sunday 11:00 - 12:00
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Osteoporosis: Pathophysiology, Genetics, Epidemiology Continued...
EFFECT OF FLUOROURACIL ON BONE REMODELING IN RATS U. Cegiela*, W. Janiec, J. Folwarczna, I. Kaczmarczyk-Sedlak, M. Pytlik, B. Nowiñska, L. Sliwiñski Department of Pharmacology, Medical University of Silesia, Sosnowiec, Poland Intensification of bone resorption is often observed in neoplastic diseases. Several factors released by tumor cells, or by immune cells responding to tumor cells, are known to enhance osteoclastic activity. Chemotherapy, associated with the risk of damaging effect on normal, especially rapidly-dividing cells, may also contribute to disorders of bone remodeling. The aim of the present study was to examine the effect of fluorouracil (5-FU) on the macrometric and histomorphometric parameters of bones, as well as the mechanical properties of the femur. The experiments were carried out on 24 Wistar rats (11-week-old), divided into 4 groups: I Control, II FU-30 (5-FU: 30 mg/kg p.o. daily for 5 days every 2 weeks), III FU-15 (5-FU: 15 mg/kg i.m. daily for 5 days every 2 weeks), IV FU-65 (5-FU: 65 mg/kg i.m. once weekly). The animals were sacrificed after 4 weeks of the experiment. Fluorouracil disturbed bone remodeling in rats, inducing decreases in the width of trabeculae in the epiphysis and metaphysis of the femur, width of periosteal and endosteal osteoid, periosteal and endosteal transverse growth, and area of the transverse cross-section in the tibia. The calcium and mineral content in bones also decreased. The observed disorders of bone formation and mineralization, caused by 5-FU, led to worsening of mechanical properties of the femur (decreases in the load necessary to cause the fracture of the diaphysis and the neck of the femur). ASSOCIATION BETWEEN POLYMORPHISMS OF THE TCIRG1 GENE AND BONE MASS IN PERIMENOPAUSAL WOMEN C. Sobacchi1*, P. Vezzoni1, D. M. Reid2, F. E. A. McGuigan2, M. Mirolo1, A. Frattini1, O. M. E. Albagha2, A. Musio1, A. Villa1, S. H. Ralston2 1Istituto Tecnologie Biomediche, CNR, Milan, Italy 2Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK The TCIRG1 gene on chromosome 11q12-13 encodes a component of the osteoclast vacuolar proton pump. Previous work has shown that autosomal recessive osteopetrosis is due to inactivating mutations of the TCIRG1 gene in about 50% of cases. In order to investigate whether allelic variation in TCIRG1 contributes to the regulation of bone mass in normal individuals, we studied the relationship between bone mineral density (BMD) and polymorphisms of the TCIRG1 gene in a population based cohort of 739 perimenopausal women. Mutation screening of the promoter, exons and intron-exon boundaries identified five polymorphisms of TCIRG1; two in the promoter (G-1082A and G-900A); two within intron 4 (C3658T and A3900G) and one within intron 11 (G8645A). All polymorphisms were in strong linkage disequilibrium (LD) with the exception of C3856T where significant LD was observed only with A3900G. A nalysis of the genotype data using the Haplotyper program predicted 31 different haplotypes in the study population, although five common haplotypes accounted for 77.3% of alleles found in our population. There was a significant association between the G-1082A polymorphism and BMD at the lumbar spine (p=0.01) and femoral neck (p=0.042), with lower BMD values in carriers of the -1082A allele. This association remained after correcting for age, weight, menopausal status, smoking and HRT use (p=0.008 for spine BMD and p=0.03 for hip BMD). None of the other polymorphisms or common haplotypes were significantly associated with BMD. We conclude that, in this relatively large population, allelic variation at the G- 1082A polymorphic site at the TCIRG1 locus accounts for part of the heritable component of BMD, raising the possibility that TCIRG1 alleles act as a genetic determinant of bone mass in Scottish women. ROLE OF DIETARY CALCIUM INTAKE IN INFLUENCING BODY WEIGHT, BONE MASS AND PREVALENCE OF OSTEOPOROSIS IN EARLY POSTMENOPAUSAL WOMEN M. Varenna, L. Binelli, F. Zucchi, S. Casari*, L. Sinigaglia Department and Chair of Rheumatology, University of Milan, 'G. Pini' Institute, Milan, Italy Several studies have shown that a high level of calcium intake reduces bone loss in the appendicular skeleton of late postmenopausal women, but it seems to be ineffective in stopping vertebral bone loss in early postmenopausal women. However, this variable could be underestimated on the light of the results of epidemiological and biochemical studies about the inverse relationship between calcium intake and body weight, and overweight could play a protective role on bone mass in women with low calcium intake. To verify this hypothesis cases were recruited among women who were referred to our hospital for their first lumbar bone mineral density (BMD) measurement (Hologic QDR 4500) and had undergone spontaneous menopause five or fewer years earlier. Quantitative estimates of calcium intake were obtained from a 7-day food frequency questionnaire. By using calcium intake quartiles, we selected 465 women (age: 53.9 ±SD 2.9; age at menopause: 51.3 ±SD 2.2) in the upper quartile (calcium intake more than 750 mg/day), and 426 women (age: 53.7 ±SD 2.7; age at menopause: 50.9 ±SD 2.1) in the lower quartile (calcium intake less than 400 mg/day). No difference in age and age at menopause was found between the groups. As found in other epidemiological studies, women with lower calcium intake showed a greater Body Mass Index (BMI) and a higher prevalence of overweight (respectively 24.1 ±SD 3.4 vs 23.2 ±SD 3.3; p<0.001 and 37.3% vs 13.3%; p<0.001). Lumbar BMD was slightly lower in women with low calcium intake (0.86 ±SD 0.12 vs 0.89 ±SD 0.12), but no difference was found in Osteoporosis (OP) prevalence (20.9% vs 17.6%; p=0.2). Nevertheless, when BMI was entered in the analyses, both a generalized linear model for BMD value and a stepwise multiple logistic regression analysis for OP risk showed that calcium intake significantly influenced BMD values (p<0.001) and acted as significant predictive factor for OP risk (OR 1.38, 95% CI 1.03-1.85 for women in the lower quartile). In conclusion, the effective value of calcium intake in preventing bone loss in early postmenopausal women could be overshadowed by the protective role exerts by a greater body weight in women with low calcium intake. EFFECT OF CONCURRENT ADMINISTRATION OF ALENDRONATE SODIUM AND RETINOL ON THE MECHANICAL PROPERTIES OF THE FEMUR IN OVARIECTOMIZED RATS L. Sliwiñski*, M. Pytlik, I. Kaczmarczyk-Sedlak, W. Janiec, J. Folwarczna, B. Nowiñska, U. Cegiela, W. Pytlik Department of Pharmacology, Medical University of Silesia, Sosnowiec, Poland Alendronate sodium, an aminobisphosphonate with potent antiresorptive activity, is used in the treatment of postmenopausal osteoporosis. Retinol, as a component of multivitamin preparations, is frequently used especially by elderly people. There are no reports on the interaction of alendronate sodium and retinol. The aim of the present study was to investigate the effect of administration of alendronate sodium and retinol on mechanical properties of the femoral bone in bilaterally ovariectomized rats. The experiments were carried out on 3-month-old Wistar rats, divided into 7 groups: I - (C) sham-operated control rats, II - (OVX) ovariectomized control rats, III - (OVX + ALN) ovariectomy + alendronate sodium 3 mg/kg p.o., IV - (OVX + K700) ovariectomy + retinol 700 IU/kg p.o., V - (OVX + R3500) ovariectomy + retinol 3500 IU/kg p.o., VI - (OVX + ALN + R700) ovariectomy + alendronate sodium 3 mg/kg p.o. + retinol 700 IU/kg p.o., VII (OVX + ALN + R3500) ovariectomy + alendronate sodium 3 mg/kg p.o. + retinol 3500 IU/kg p.o. The drugs were administered to the rats by daily oral gavage (alendronate sodium in the morning, retinol in the afternoon) for 28 days. The mechanical properties of the diaphysis and neck of the femur, body mass gain, bone mass, bone mineral content and calcium content (in the tibia, femur and L-4 vertebra) were examined. Bilateral ovariectomy induced osteopenic changes in the rat skeletal system. Alendronate sodium (3 mg/kg p.o.) counteracted the development of osteopenia induced by ovariectomy. Retinol in both used doses unfavourably affected the examined bone parameters of ovariectomized rats. Retinol administered with alendronate sodium lessened the preventive action of alendronate sodium on the development of osteopenic changes in the skeletal system of ovariectomized rats. OESTROGEN AND THE CLUSTERING OF CORTICAL REMODELLING N. Loveridge1*, S. Vedi2, K. L. Bell1, J. E. Compston2, J. Reeve1 1Bone Research Group (MRC), Dept of Medicine, Addenbrookes Hospital (Box 157), Cambridge, UK 2Bone Research Group, Dept of Medicine, Addenbrookes Hospital (Box 157), Cambridge, UK Femoral neck fracture is strongly associated with the rapid appearance of large cortical pores. These pores are related to the spatial clustering of osteonal remodelling activity as identified by the presence of osteoid on the canal surface. However, the cause of this clustering remains an enigma. To determine whether oestrogen regulates clustering we have analysed iliac biopsies from post-menopausal women before and after 2 years treatment with HRT (n=10) and from pre-menopausal women before and after 6 months treatment with GnRH (n=10) for the presence of clustering of both resorbing and forming canals. For each biopsy the number and location of resorbing (crenellated) and forming (osteoid) canals were noted. Cluster analysis (JMP software) used 0.32mm (2x the mean inter-osteonal distance for all biopsies) as the cluster radius. For the HRT group, the percentage of resorbing and forming canals that were clustered was significantly higher than would be expected by chance (resorbing:- +22.2±7%. p=0.007; forming: +25±5%, p<0.0001). However, HRT treatment did not affect clustering of either resorbing or forming canals (Resorbing:- Pre 40.5±9%, Post 38.9±13% p=0.93; Forming:- Pre 49.9±7.5%, Post 57.7±7%, p=0.46). For the GnRH group there was significant clustering of both resorbing and forming canals (resorbing:- +14±6%. p=0.03; forming: +15±3%, p<0.0001;) but the inhibition of oestrogen secretion had no effect (Resorbing:- Pre 48.3±9%, Post 52±9% p=0.73; Forming:- Pre 53.1±7.4%, Post 55.1±7.5%, p=0.85.). Clustering was still significant at a radius of 0.16mm but was unaffected by treatment in either group. This study has shown for the first time, that in the ilium, a significant percentage of those canals undergoing resorption as well as those undergoing formation are spatially clustered. Neither the presence (HRT treatment) nor absence (GnRH treatment) of oestrogen affected the proportion of clustered canals. This suggests that oestrogen influences remodelling activity by regulating activation of complete clusters as single functional units rather than the individual canals of which clusters are composed. Therefore, estrogens prevent fractures by mechanisms that do not involve the prevention of clustering. Understanding how clustering is regulated may open the way to treatments for osteoporosis which augment the effectiveness of oestrogens and activators of oestrogen receptors. THE ANALYSIS OF ASSOCIATION BETWEEN ALLELES OF ERALPHA AND BGLAP GENES AND OSTEOPOROSIS IN POSTMENOPAUSAL WOMEN FROM RUSSIA M. V. Moskalenko*, M. V. Aseev, I. E. Zazerskaya Ott's Institute of Obstetrics and Gynecology RAMS, St-Petersburg, Russia Osteoporosis is a common multifactor disease with a strong genetic component. It is characterised by decreasing bone mineral density (BMD) and microarchitectural deterioration, which leads to increased susceptibility to fracture. Nowadays a number of genes have been identified, which play important role in regulation of Ca 2+ concentration and contribute in bone metabolism. For several genes exact mutations were discovered, which resulted in activity changes of corresponding proteins or in forming of functionally abnormal products. The allele of ERalpha and BGLAP (osteocalcin) genes in 69 non-related individuals without osteoporosis rates Northwest Russian population (control group) and in 40 postmenopausal women with osteoporosis (1 group) and 57 postmenopausal of women with severe form of osteoporosis (2 group) were investigated. The polymorphisms ERalpha and BGLAP genes were studied by PCR-RFLP method. The RFLPs were represented as X or x (XbaI) and P or p (PvuII) for ERalpha gene; as H or h (HindIII) for BGLAP gene, with the lower case letters signifying the presence of restriction site. We have not elucidated any authentic differences in frequencies of alleles of XbaI and PvuII polymorphism in 1 intron of ERalpha gene in group 1 of patients, compared to the group 2 and it's average frequency in population (p>0.05). The frequencies of alleles of this polymorphism were 25,6 % (X) and 38,9% (P) in control group; 38,7% (X) and 47,5% (P) in group 1; 35,1% (X) and 46,5% (P) in group 2. The frequency of functionally abnormal allele H (HindIII polymorphism of BGLAP gene) in group 2 was 23,7%, which is significantly higher (p<0,01) than only in group 1 (10,0%). The frequency of this allele in control group was 18,1% and did not significantly differ from the average in group 1 and group 2 (p>0,05). According to obtained data the ERalpha polymorphism did not demonstrate any major effect of on pathogenesis of osteoporosis of postmenopausal Russian women. The present study also suggests that the presence of the H allele of BGLAP gene is predictive factor for severe form of osteoporosis only. EFFECT OF ADMINISTRATION OF RETINOL AND ETIDRONATE ON BONE HISTOMORPHOMETRIC PARAMETERS IN OVARIECTOMIZED RATS I. Kaczmarczyk-Sedlak*, W. Janiec, M. Pytlik, L. Sliwiñski, J. Folwarczna, U. Cegiela, B. Nowiñska Department of Pharmacology, Medical University of Silesia, Sosnowiec, Poland Retinol belongs to factors affecting bone remodeling. The effect of retinol on the osseous tissue depends on the dose and duration of treatment. Retinol can cause bone damage and deformation. Retinol is frequently administered chronically in too high doses, sometimes by osteoporotic patients. The aim of the present study was to examine the interaction between retinol and an antiresorptive drug - etidronate disodium in bilaterally ovariectomized rats. The experiments were carried out on Wistar rats (200±30 g), divided into 7 groups: I - sham operated control rats, II - ovariectomized control rats (OVX), III - OVX + etidronate (10 mg/kg p.o.), IV - OVX + retinol (700 IU/kg p.o.), V - OVX + retinol (3500 IU/kg p.o.), VI - OVX + etidronate (10 mg/kg p.o.) + retinol (700 IU/kg p.o.), VII - OVX + etidronate (10 mg/kg p.o.) + retinol (3500 IU/kg p.o.). The drugs were administered for 4 weeks. Bone macrometric and histomorphometric parameters of the tibia (transverse growth, width of periosteal and endosteal osteoid, area of the transverse cross-section of the diaphysis and area of the transverse cross-section of the marrow cavity) and the femur (width of epiphyseal and metaphyseal trabeculae, width of epiphyseal cartilage) were examined. Etidronate partially counteracted the development of changes induced by the ovariectomy. Retinol (700 IU/kg p.o.) caused decreases in the area of the transverse cross-section of the marrow cavity and the width of osteoid, and an increase in the width of trabeculae. Retinol (3500 IU/kg p.o.) caused decreases in bone mass and the area of the transverse cross-section of the marrow cavity, and an increase in the width of trabeculae. Concurrent administration of etidronate and retinol in ovariectomized rats seemed not to affect bone histomorphometric parameters in a way suggesting any interaction between them. THE RELATIONSHIP BETWEEN BONE QUALITY AND OVARIAN SENESCENCE IN AGING MICE MODEL M. Grynpas1*, A. Jurisicova1, A. Taniuchi1, L. Wise1, X. Wang1, J. Canning2, G. Perez2, J. Tilly2 1Samuel Lunefeld Research Institute of Mount Sinai Hospital and University of Toronto, Canada 2Vincent Centre for Reproductive Biology, Massachusetts General Hospital and Harvard Medical School, USA The laboratory mouse is the model of choice for genetic studies in mammals due to the availability of many genetically defined inbred strains and ability to study the effects of over-expression or disruption of a given gene on cells or tissues. It has also been shown that 70% of bone mass is genetically controlled. Similarly the size of the primordial oocyte reserve is also genetically controlled. The aim of this study was to investigate the relationship between ovarian reserve, bone mass and bone quality with aging in inbred strains of mice. We examined bone mineral density (BMD), tissue mass (lean and fat mass) and bone mechanical properties in several inbred (129/Sv, C57BL/6, C3H, AKR/J) strains of 7 months old virgin mice. We also investigated primordial follicle endowment in the same strains (129/Sv, C57BL/6, AKR/J) at day 4 (neonatal) and at day 42 (young adult) postpartum. We found that AKR/J female mice had the lowest total number of follicles, highest body weight, bone mass, tissue mass, and the lowest toughness; whereas 129/Sv females possessed the highest total number of follicles (P<0.05 for AKR/J versus 129/Sv), but the lowest body weight, lean mass, elastic modulus, highest toughness and plastic energy. C3H mice had the thickest and shortest bone, highest elastic modulus, and elastic energy, but lowest plastic energy. C57BL/6 mice had the lowest BMD, fat mass, elastic modulus, and lowest elastic energy (see table). These results indicate that genetic modifiers play a major role in determining bone quality and ovarian reserve in aging mice. Our research will focus on the effect of aging on bone quality and ovarian function in these genetically distinct strains of mice.
OSTEOPROTEGERIN (OPG) AND RANK-L SERUM LEVELS AND OPG POLYMORPHISM IN JUVENILE IDIOPATHIC ARTHRITIS (JIA): A POTENTIAL MODULATING ROLE IN BONE DAMAGE OF CHILDREN WITH CHRONIC ARTHRITIS? L. M. Masi1*, E. Piscitelli1, F. Del Monte1, G. Simonini2, F. Marini1, A. Falchetti1, V. Ghinoi1, A. Amedei1, M. L. Brandi1, F. Falcini2 1Departement of Internal Medicine, University of Florence, Florence, Florence, Italy 2Department fo Pediatrics, University of Florence, Florence, Italy OPG has been identified as a novel cytokine that inhibits differentiation and activation of osteoclasts, while receptor activator of nuclear factor kB ligand (RANK- L) as a key mediator of joint destruction and bone loss. RANK-L and OPG are thought central regulators of osteoclast recruitment and activation. In the present study we evaluated the serum levels of OPG and RANK-L in different subset of JIA and correlated these values with disease activity parameters, radiological bone damage and bone mass. A polymorphism of OPG gene was evaluated and correlated with bone mass. 84 pts (66 girls and 18 boys) with JIA (38 oligoarticular and 46 polyarticular RF negative disease) and 40 sex and age-matched controls were enrolled. Serum OPG and RANK-L was measured using an ELISA method. OPG polymorphism was evaluated performing PCR reaction of genomic DNA and digestion of the product by Pst I enzyme. Patients with polyarticular disease had statistically significant higher OPG and lower RANK-L serum levels in comparison with subjects with oligoarthicular disease and controls. (p=0.001). No significant correlation was found between disease duration, ESR and CRP values either as OPG either RANK-L serum levels. On the contrary, we observed statistically significant high serum OPG levels between patients with and without erosions (72±22 vs. 50±18 pg/ml ; p=0,007). No correlation was found between OPG and RANK-L levels with DXA Z-score values. ANCOVA analysis and LSD test showed that patient with CC genotype had a LS-BMD higher in comparison with the TC (p=0.04) and TT genotype (p=0.02). Finally, no significant differences in levels of serum OPG were found between the three genotypes. In conclusion, we evaluated the amount of OPG and RANK-L in children with chronic arthritis. The higher OPG in JIA subjects might be the result of a compensatory production of OPG, to contrast the bone and cartilage damage, especially in patients with severe joint involvement and could represent a good marker of the disease but not a good predictor o of bone mass change. However, the OPG polymorphism may represent a marker in the identification of patient with a higher risk to loss bone mass. ASSOCIATION OF THE OSTEOPROTEGERIN GENE POLYMORPHISMS WITH BONE MINERAL DENSITY IN WOMEN WITH POSTMENOPAUSAL OSTEOPOROSIS B. Arko1*, J. Prezelj2, A. Kocijancic2, R. Komel3, J. Marc1 1Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 2Department of Endocrinology and Metabolic Diseases, Clinical Centre, Ljubljana, Slovenia 3Medical Centre for Molecular Biology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia Osteoporosis is a disease with a strong genetic component. The genes involved are however, still largely unknown. Osteoprotegerin (OPG) is a recently discovered member of the tumour necrosis factor receptor superfamily, which protects bone from excessive resorption by inhibiting the terminal stages of osteoclastogenesis, suppressing mature osteoclast activation and inducing their apoptosis. Due to its crucial role in the control of bone resorption the OPG gene might be a good candidate gene for osteoporosis. The aim of our work was to find possible sequence variations in the OPG gene and to evaluate their role in bone remodelling and the development of postmenopausal osteoporosis. Screening of the promoter region and all five exons of the OPG gene in a group of Slovenian women with postmenopausal osteoporosis by single-strand conformation polymorphism analysis indicated and direct DNA sequencing identified the presence of 13 sequence variations. Substitutions 209G/A, 245T/G, 1217C/T and 4501C/T were in complete linkage and so were the deletion 4752_4753 CT and substitutions 6893A/G and 6950A/C (Genebank Accession AB008821 and AB008822). Association with lumbar spine and femoral bone mineral density (BMD) and biochemical markers of bone turnover: osteocalcin, bone specific alkaline phosphatase and deoxypyridinoline was tested for polymorphisms 209G/A (promoter) and 950T/C (promoter) in 103 patients, and for polymorphisms 1181G/C (exon 1) and 6893A/G (exon 4) in 60 patients. Only polymorphism 209G/A was associated with BMD. Patients with genotype GA had lower lumbar spine BMD than those with genotype GG, (GG 0.726 ±0.082 g/cm2, GA 0.649 ±0.103 g/cm2, p = 0.005). However, there was no statistically significant association of this or any other polymorphism with biochemical markers of bone turnover. There are several sequence variations in the OPG gene. According to our study, the promoter polymorphism 209G/A seems to be the most promising candidate for the future research. Genotype 209 GA could be considered as a risk factor for genetic susceptibility to postmenopausal osteoporosis. EFFECT OF CIPROFLOXACIN ON THE SKELETAL SYSTEM IN RATS B. Nowiñska*, W. Janiec, M. Pytlik, J. Folwarczna, U. Cegiela, I. Kaczmarczyk- Sedlak, L. Sliwiñski, M. Górecka Department of Pharmacology, Medical University of Silesia, Sosnowiec, Poland Ciprofloxacin is a fluoroquinolone antibacterial agent. Fluoroquinolones have been reported to cause potent damage to articular cartilage in immature animals. As fluoroquinolones inhibited the activity of osteoblasts in vitro, their administration may be connected with unfavourable effects on the osseous tissue. The aim of the present study was to investigate the effect of ciprofloxacin on the osseous system in rats. The experiments were carried out on young (5-6-week-old) and adult (3-month-old) male Sprague-Dawley rats, divided into 2 respective control groups (receiving 0,9% NaCl) and 2 groups receiving ciprofloxacin in a dose of 210 mg/kg p.o. daily for 4 weeks. Bone length, diameter, mass, content of mineral substances and calcium, histomorphometric parameters in the tibia (area of the transverse cross-section of the marrow cavity and of the diaphysis, width of periosteal and endosteal osteoid, periosteal and endosteal transverse growth) and in the femur (width of trabeculae in the epiphysis and metaphysis, width of epiphyseal cartilage) and mechanical properties of the femur (load necessary to cause the fracture of the whole femur and the femoral neck, maximal deformation of the femur) were examined. The investigated parameters in the group of adult rats receiving ciprofloxacin did not markedly differ from those of the respective control group, indicating the lack of unfavourable effects on bone remodeling. Also in the group of young animals, no damaging effect of ciprofloxacin on the skeletal system was observed. DIFFERENT RISK FACTORS FOR TROCHANTERIC AND CERVICAL HIP FRACTURES IN MIDDLE AGED WOMEN A. H. Holmberg1*, O. Johnell1, JÅ Nilsson2, P. Nilson2, G. Berglund2 1Dpt of Orthopaedics, Malmö University Hospital, Sweden 2Dpt of Internal Medicine, Malmö University Hospital, Sweden Hip fracture is the most serious complication of osteoporosis, its incidence rising worldwide. Cervical and trochanteric fractures are in most studies summed up and counted as the same fracture type, but some papers have suggested that these fractures have different etiologies and different risk patterns. The aim of our study was to see if we could detect a different risk factor pattern to be able to use that in analysing the pathogenesis of the different fracture types. Malmö Preventive Study is a cardiovascular study with 10902 female probands. At screeening they went through a large questionnaire concerning health, life style, heredity and social status. Height and weight measuremetns were performed as well as blood sampling and pulmonary function tests. The probands were followed 11 years concerning death, malignancy and fracture. In this group 133 of the women suffered a hip fracture, 93 cervikal and 40 trochanteric fractures. The data were analysed with multiple age adjusted logistical regression. Significant risk factors for both cervical and trochanteric hip fractures were height and earlier fracture. Risk factors for trochanteric hip fractures only were smoking and diabetes, and for cervical fractures elevated pulse and earlier stomach ulcers. Signinficant protective factors for trochanteric hip fractures were BMI and weight, while for cervical hip fractures only high FVC (Forced Vital Capacity). (See table) These data suggest that trochanteric and cervical hip fractures have different risk factor patterns and that the pathogenesis also may be different. Since most studies have been made on subjects already suffering from a hip fracture, and risk factor analysis have been made in retrospect more studies are needed to certify the difference between the fractures and to plan separate strategies for preventing them.
IDENTIFICATION OF POLYMORPHISMS IN THE RUNX2/CBFA1 GENE AND ASSOCIATION WITH BONE MINERAL DENSITY AND FRACTURES V. Geoffroy1*, S. Branger1, J. L. Laplanche2, M. C. de Vernejoul1 1INSERM U349, Hopital Laribisiere, 2, rue Ambroise Paré, Paris Cedex 10, France 2Department of Biochemistry The RUNX2/CBFA1 gene encodes a transcription factor that is essential for osteoblast differentiation and function. RUNX2/CBFA1 deficiency results in complete lack bone formation and overexpression of RUNX2/CBFA1 in cells of the osteoblastic lineage to severe osteoporosis. Together these data suggest that bone formation and maintenance is highly related to Runx2/Cbfa1 transcriptional activity. Thus, RUNX2/CBFA1 is an appropriate candidate gene to investigate relationship to BMD and fractures. Some polymorphisms in the RUNX2/CBFA1 gene were already described within the exon 1, encoding a glutamine-alanine stretch, partly responsible for the transcriptional activity of the factor. We first try to identify polymorphisms within the Runx2/Cbfa1 exon 1 that could confirm this former report and second to determine if these polymorphisms are related to BMD and fractures in controls and post-menopausic osteoporotic patients. We observed 2 polymorphisms within the alanine stretch as compared to the normal GCG form (Allele G): a previous synonymous alanine codon polymorphism GCA (allele A) and a 18 bp deletion with 11 alanine residus (Allele 11Ala). We selected a population of 96 postmenopausal women for vertebral crushed fractures aged 67±9. Their mean BMD was 0.82±0.13 g/cm 2 at the lumbar spine and 0.68±0.10 g/cm2 at the femoral neck. The control women were 98 postmenopausal women aged 65±6. None of them had vertebral or wrist fracture. All of them had a T- score > -2.5. Their mean BMD was 1.06±0.13 g/cm2 at the spine and 0.82±0.10 g/cm2 at the femoral neck. The allele frequency were as followed (see table below). In conclusion, we observed the same polymorphisms as previously described and we could not identify in this sample a significant difference between vertebral fracture cases and controls.
INCREASED PHYSICAL ACTIVITY IN PATIENTS WITH INCIDENT FOREARM FRACTURE. A CASE CONTROL STUDY E. Waern1*, O. Johnell2, J. Jutberger1, J. Karlsson3, D. Mellström1 1Dep of Geriatric Medicin, University of Göteborg, Sweden 2Dep of Orthopaedics, University of Malmö, Sweden 3Dep of Orthophaedics, University of Göteborg, Sweden Background Distal forearm fracture is the most prevalent fracture in peri- and postmenopausal women. The increase in distal forearm fracture in this period of life has been attributed both to a reduction in bone strength caused by the accelerated phase of bone loss at the menopause and an incresaed liklehood of falling at the same period in life.Earlier studies have indicated that multiple riskfactors are important for hip fracture. One of these riskfactors is low physical activity. The question is if low physical activity is a riskfactor for forearm fracture. Population and methods All patients presenting with a fracture of the distal forearm at the orthopaedic clinic at Sahlgrenska University Hospital/Östra,Göteborg, over a two-years period, were offered to participate in this project. 200 patients were included. All patients had a questionnaire concerning known riskfactors for osteoporosis and fractures. BMD was measured by DXA, using Hologic 4500 A, in lumbar spine, proximal femur and total body in 200 patients (183 women,17 men) mean age 65,2 years (range 22-90). All patients were clinical assesed. 480 70-year-old women in a nordic osteoporosis study (Nordos) served as a controlgroup. Results Women (65-75 years of age) with a distal forearm fracture had an about half SD lower BMD in hip, spine and total body compared to the controlgroup. Weight, menopausal status, hormone replacement therapy, coffee consumption, smoking or dietary calcium intake were not associated with the risk for fracture of the distal forearm compared to control population. Previous fracture history, maternal hip fracture and use of corticosteroids were shown to be riskfactors for forearm fracture. Physical activity was negatively correlated to the risk for forearm fracture. Both measured as walking, OR 1.297 (1.060-1.587) and other forms of physical exercise, OR 1.361 (1.041-1.779). Conclusion In this case control study patients with forearm fracture had increased physical activity compared to a controlgroup. PILOT STUDY TO ASSESS BONE TURNOVER IN POSTMENOPAUSAL WOMEN ON HORMONE REPLACEMENT THERAPY FOR AT LEAST TWO YEARS C. Menendez1, C. De la Piedra2, F. I. Romero3, I. Aristegui3*, V. De Miguel4, I. Khan4, S. Palacios1 1Instituto Profesor Palacios, Madrid, Spain 2Fundacion Jimenez Diaz, Madrid, Spain 3Aventis Pharma, Spain 4Pivotal, Spain Objective: To evaluate in a group of postmenopausal (PM) women the percentage of patients with high bone turnover despite being on Hormone Replacement Therapy (HRT) for at least 2 years. This is a pilot feasibility study assessing patient availability for a subsequent clinical trial. Patients and Methods: Cross-sectional study assessing 50 PM women on HRT for at least 2 years with risk factors (RF) for osteoporosis (OP): Bone Mineral Density (BMD) < -1.0 T score and/or 2 or more non-skeletal RF at a gynaecological menopause unit. Previous BMD values were retrospectively collected on an available basis. BMD at lumbar spine (LS) and femoral neck (FN) was assessed by dual energy X-ray absorptiometry (DXA T-score). Bone remodelling status was evaluated by bone resorption (urinary N-telopeptide, NTx) and bone formation (serum bone specific alkaline phosphatase, BSAP) markers. Results: One of the 50 screened patients was not evaluable because of tibolone administration during 1 year. 78% of the women (mean age, 55 years) had been on HRT for < 10 years (median 6 ±3.76 years; 2-5 years, 46%; 6-10 years, 32%; over 11 years, 22%). Only 20% and 12% of the patients presented increased NTx (>50 nmol BCE/mMCreatinine) and BSAP (> 18 microg/L) values, respectively, with an acceptable correlation of these two parameters (p = 0.6). 54% and 46% of the patients had 2-3 and over 3 osteoporosis RF, respectively (mean ±SD, 3.5 ±3.46). Only 32 pre- baseline BMD assessments were available, all of them with T scores less than -1. FN and LS BMD T-scores less than -1.5 in 84% and 86% of the patients, respectively. Conclusion: Up to 20% of PM women on HRT with OP risk factors may have active bone turnover. Further studies with larger populations will be required to define 'HRT non responders' and identify possible related risk factors. EFFECT OF ACENOCUMAROL ON BONE METABOLISM OF MALE POPULATION M. Ciria1*, L. Perez-Edo1, J. Blanch1, I. Padró1, I. Aymar2, X. Garcia1, P. Benito1, J. Carbonell1 1Rheumatology Service. IMAS, Spain 2Internal Medicine Service. IMAS, Spain Background: Vitamin K is an important and necessary element for mineralisation of bone tissue. Use of antivitamin K (like acenocumarol) can affect bone metabolism. We report a cross-sectional, observational study pretending to assess the effect of acenocumarol on bone metabolism and bone mineral density (BMD) of male population. Patients and Methods: Males in treatment with SINTROM (acenocumarol) since more of 3 years, with functional class I-II. Patients with osteopenic diseases or usage of treatment that influences bone metabolism were excluded from the study. A double-photon densitometry with an Hologic QDR 4500 equipment and complete laboratory test that included levels of vitamin D, intact parathormone, alkaline phosphatase, tartrate-resistant acid phosphatase, osteocalcine, blood calcium and 24- hour urine calcium was performed to all patients. The control group was the normality curve of BMD in the spanish population (SEIOMM-FOHEMO). Cross-calibration between the Hologic 4500 equipment used for this study and the Hologic 1000 equipment used for the creation of the normal curve was performed. Obtained data was adjusted according to this calibration. Results: Forty males were included in this study. Age interval was from 51 to 81 years. Three age groups were formed to make the comparison with the normality curve possible. BMD values in lumbar spine and femoral neck in all patients did not differ from their reference population (Table 1). An elevated number of hiperparathyroidism (70%) was observed with no apparent relationship with levels of blood calcium, vitamin D, age or season. Conclusions: the sustained usage of acenocumarol can be associated with hiperparathyroidism by unknown mechanisms. In spite of this, in our sample it does not correlate with a low bone mass in hip or lumbar spine. Further large prospective studies are necessary to investigate this relation between acenocumarol and hiperparathyroidism.
BONE HISTOLOGY IN POSTMENOPAUSAL OSTEOPOROSIS - VARIATION IN BONE CELL ACTION P. Lozo1, D. Krpan2, A. Krvavica3, V. Kusec4* 1Health Centre Lozo, Zadar, Croatia 2General Hospital Sveti Duh, Zagreb, Croatia 3General Hospital Zadar, Zadar, Croatia 4Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre Zagreb, Zagreb, Croatia Bone was analysed in a group of 43 women with postmenopausal osteoporosis (44 -71 years) undergoing bone biopsy as a part of diagnostic procedure. Data were compared and grouped according to the published histomorphometric classification of postmenopausal osteoporosis. Densitometry of the lumbar spine and hip confirmed osteoporosis. The following histomorphometric parameters were assessed: bone volume (BV/TV, %), osteoid surface (OS/BS, %), osteoblast surface (Ob.S/BS, %), osteoid volume (OV/BV, %), osteoid thickness (O.Th, um), osteoclast surface (Oc.S/BS, %), mineral apposition rate (MAR, um/day). Histomorphometric analysis of bone biopsy demonstrated that on average bone resorption i.e. osteoclast surface was considerably increased and osteoid volume moderately increased. However, osteomalacia was not found in any of the patients. Other histomorphometric parameters studied were on average normal for age and sex as compared to published reference data. Percentage of patients in each group of histomorphometric classification was different from literature data, and most probably a consequence of sample size and choice. None of the patients had histomorphometric features of reduced osteoblastic and osteoclastic activity, but in 37% of postmenopausal women osteoclastic activity was increased while osteoblastic was normal, a feature not described in the original histomorphometric classification of postmenopausal osteoporosis. In conclusion bone resorption was a prevailing finding in women with postmenopausal osteoporosis. Histomorphometric analysis of bone biopsy in postmenopausal osteoporosis confirmed variation in bone cell actions. GAIN IN BONE MINERAL MASS IN PREPUBERTAL BOYS ONE YEAR AFTER DISCONTINUATION OF CALCIUM SUPPLEMENTATION : A FOLLOW-UP STUDY T. Chevalley1*, S. Ferrari1, D. Hans2, D. Slosman2, M. Fueg1, J. P. Bonjour1, R. Rizzoli1 1Division of Bone Diseases, WHO Collaborating Center for Osteoporosis and Bone Diseases, Dept. of Internal Medicine, University Hospitals Geneva, Geneva, Switzerland 2Division of Nuclear Medicine, Dept. of Radiology, University Hospitals Geneva, Switzerland Calcium supplementation (Calsup) enhances bone mineral mass accrual in both prepubertal girls and boys. The extent to which this beneficial effect persists beyond the period of supplementation in boys in not known. We conducted a one-year prospective double-blind placebo-controlled trial in 235 prepubertal boys aged 7.4±0.1 yrs (mean±SEM, range 6.5-8.5 yrs), with one year follow-up after stopping Calsup. The Calsup group (n=116) received 850 mg/day of calcium (Ca) provided by 2 food products and the placebo group (n=119) consumed similar products in terms of energy and protein but without added Ca. Spontaneous Ca intake was assessed by frequency questionnaires at baseline, 12 and 24 months. Areal BMD (aBMD) was determined by DXA using Hologic QDR 4500 at 6 skeletal sites: radius (distal metaphysis=RMet; diaphysis=RDia); hip (femoral neck=FN; trochanter=FT); femoral diaphysis (FDia) and L2-L4 vertebrae (LS) at baseline, at the end of supplementation (12 months) and one year after Calsup discontinuation (24 months). Mean baseline spontaneous Ca intake was 743±20 mg/day and remained quite stable during both the intervention and follow-up periods. The gain in aBMD (mg/cm2±SEM) at the end of the intervention was significantly (intention-to-treat analysis) higher in the Calsup (n=114) as compared with the Placebo (n=118) group at FDia (76±3 vs 64±3, p=0.005) and at the mean of 6 sites (32±1 vs 28±1, p<0.05). The Calsup effect was greater in the boys with a lower spontaneous protein intake. One year after the end of Calsup, there was a difference in aBMD gain between Calsup and placebo groups mainly in an active-treatment analysis (FDia: 135±5 vs 124±4, p=0,06, RMet: 25±2 vs 16±2, p<0.005, mean 6 sites: 55±2 vs 50±2, p<0.05). However, in an intention-to-treat analysis, the Calsup effect on aBMD gain was only detectable at RMet (Fdia: 127±6 vs 124±4, NS, Rmet: 25±2 vs 19±2, p<0.05, mean 6 sites: 53±2 vs 51±2, NS). Thus, depending on the statistical analysis applied, Calsup effect on aBMD gain in prepubertal boys might be maintained one year after Calsup discontinuation. ASSOCIATION BETWEEN TNFRSF1B ALLELES AND BMD IS DRIVEN BY 3' UTR HAPLOTYPES RATHER THAN CODING OR PROMOTER POLYMORPHISMS P. N. Tasker*, O. M. E. Albagha, F. E. A. McGuigan, D. M. Reid, S. H. Ralston Bone Research Group, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD, UK Previous linkage studies have identified a candidate locus for regulation of hip BMD on chromosome 1p36 and a strong positional candidate gene within this region is TNFRFS1B, which encodes the type 2 TNF receptor (p75). Several polymorphisms have been identified in TNFRSF1B including a VNTR in the promoter, a Met-Arg change at codon 196 (M196R) and polymorphisms at positions 593, 598 and 620 in the 3' untranslated region (UTR). The A593-T598-C620 3'UTR haplotype has previously been associated with BMD, but no studies have looked at the promoter VNTR and BMD, even though similar VNTR's have been found to act as a regulators of transcription in other genes. Here, we looked for evidence of allelic associations between TNFRSF1B polymorphisms and BMD in a population based study of 916 women aged 45-55. Raw BMD results were adjusted for age, height and weight and related to genotype group by GLM ANOVA. The promoter VNTR was in strong linkage disequilibrium (LD) with the M196R polymorphism (P<0.00001, D'=0.58), G593A (P<0.00001, D'=0.14) and T598G (P<0.00001, D'=0.83), but not with T620C (P=0.09, D'=0.06). Associations between the polymorphisms and BMD are shown in the table. None of the polymorphisms were associated with spine BMD (data not shown). The promoter polymorphism was weakly associated with femoral neck BMD, as were the 3' UTR polymorphisms when analysed individually(G593A; p=0.02; T598G; p=0.08; T620C; p=0.05). The 3'UTR ATC polymorphism was strongly associated with hip BMD however. The studies confirm that the ATC haplotype is primarily responsible for the association between TNFRSF1B and BMD, suggesting that this variation in the 3' UTR may be functional, possibly by affecting RNA stability.
SEXUAL DYSMORPHISM IN AGE-ASSOCIATED BONE PLASTICITY C. R. Russo*, F. Lauretani, B. Bartali, A. M. Corsi, C. Cavazzini, S. Bandinelli, L. Ferrucci Laboratory of Clinical Epidemiology, INRCA, Florence, Italy Background Age-associated bone loss is a well recognized phenomenon. Cadaver studies suggest that aging bones undergo adaptive geometrical changes in response to BMD reduction, aiming to prevent the development of fragility. We present in vivo cross-sectional data on age-associated differences in tibial bone geometry in a representative population of central Italy (the InCHIANTI study). Methods The InCHIANTI sample considered here comprises 960 subjects, 440 males, 520 females, age range: 20-102 years. Total and cortical bone area, medullary area, and density-weighted polar moment of inertia were obtained using a peripheral QCT device (XCT 2000) at the tibial shaft, 38% of the tibial length, proximal from the distal tibia end. Bone tissue was separated from the soft tissues using BonAlyse software with a density threshold of 180 mg/cm 3. Results In men, medullary area was 86.5 mm2 in the oldest age group and 60.7 mm2 in the youngest one, a 43% difference. Total and cortical bone area were greater in the oldest than in the youngest age group by 15% and 10%, respectively. Moment of inertia was slightly smaller in the old (by 2.4% ), compared to the young men. In women, medullary area was considerably greater in the oldest, compared to the youngest age group (103 vs 56 mm 2, a 107% difference). Total bone area was greater in the oldest (by 7.7%) than in the youngest age group, as in men. However, cortical bone area was smaller in the oldest, compared to the youngest age group, by 11.9%. Moreover, moment of inertia was smaller,by 5.4%, in the oldest, compared to the youngest age group. Conclusion Medullary area increased in the oldest men and women, not surprisingly more in women, the anatomical result of endo-cortical resorption. Bone size also increased as a result of periosteal formation, more in men than in women. The net effect is a preservation of bone strength in older men but not in older women. Thus, a sexual dysmorphism in bone plasticity, non-invasively measurable in vivo , may help explain the inter-sex difference in age-adjusted incidence of fragility fractures. NECK OF FEMUR FRACTURE: PREVIOUS HIP FRACTURE AND INSTITUTIONALISATION ARE IGNORED RISK FACTORS M. W. J. Davie1, M. Fletcher2, G. Clements3, F. Plant3*, W. J. C. Roberts4 1Robert Jones and Agnes Hunt Hospital, Oswestry, Shropshire, UK 2Dept Statitistics, School of Computing ,Staffordshire University, Stafford, UK 3R & D dept, Royal Shrewsbury Hospital, Shrewsbury, UK 4Meddygfa Padarn, Aberystwyth, Dyfed/Powys, UK Identification of patients for treatment to prevent hip fracture is important for optimal application of therapy. We are developing an instrument to estimate risk of neck of femur fracture from a subject's characteristics. In a preliminary analysis we investigated 90 consecutive female cases of fracture of the femoral neck over 3 months for age together with where they were living when they fractured and compared the details with patients with hip fracture in general practice. 88 patients 50yr and over with hip fracture were investigated. 5 had traumatic or pathological fracture and were excluded. Of 83 remaining 27 (32.5%) had a previous fracture, 10 of which were hip fractures. Non hip fractures (n=17, of which wrist=8) had occurred (mean, SD, median) 13,13.8,9 yr and hip fractures 8.7,11.2,5 yr previously. 58 (69.9%) of hip fractures occurred in women >80yr. 22 of these women had had a previous fracture (37.9%), 6 of which were hip fractures. 8 patients had had previous therapy with any bone active agent with no significant difference between patients with previous hip fracture, non-hip fracture or no previous fracture. 22 (37.9%) of women came from nursing/residential (residential) homes. Census data for Shropshire showed that, in this age group, 7784 live in the community and 1948 in residential homes with more hip fractures coming from residential homes (Residential/Community Odds ratio = 2.46(1.43-4.22). To investigate the importance of previous fracture, records of all female patients over 80yr (n=104) from a general practice in an adjoining district were reviewed. 6 patients with hip fracture were identified, 2 of whom had experienced a previous fracture (33.3%). 35 other patients without hip fracture had suffered a previous non hip fracture (34%). These data suggest that age over 80yr and the habitation of the patient at the time of fracture are important risk factors for hip fracture. Previous non- hip fracture is frequent in the over 80 age group and may be less important as risk factor but previous hip fracture, common amongst current hip fracture admissions, had not led to initiation of anti osteoporotic treatment. NO ASSOCIATION BETWEEN VITAMIN A INTAKE AND RATE OF BONE LOSS IN 1760 RECENTLY POSTMENOPAUSAL WOMEN A. P. Hermann1*, P. Vestergaard2, C. Brot3, N. U. Kolthoff4, B. Abrahamsen1, K. Brixen1, P. Charles2 1Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark 2Department of Endocrinology and Metabolism, Aarhus University Hospital, Aarhus, Denmark 3Department of Endocrinology , Hvidovre Hospital, Copenhagen, Denmark 4Department of Clinical Physiology, Hilleroed Hospital, Hilleroed, Denmark The aim of this study was to evaluate the effect of dietary and supplementary vitamin A on bone loss in recent postmenopausal women. We analysed 5-year follow- up data on BMD in a national comprehensive cohort study including 2016 early postmenopausal women. Initially the women were allocated to a randomised or a non- randomised arm by their own choice. In both arms treatment was given as open labelled, cyclic combined estrogen and gestagen. At study entry the participants filled in a 7-days food record. From these, current daily intake of vitamin A (retinol and beta-carotene) was estimated, using the Dankost software. Retinol values only were used for further calculations. Supplementary vitamin intake was also recorded. BMD in the whole body, spine, and hip were measured at time 0 and after 5 years, using Hologic 1000W and 2000 equipment. Bone loss during the five-year follow up period is given as the absolute difference between BMD at study entry and at the five-year visit. 1802 women attended the five-year visit. 1760 of these had a complete food record. 493 women had completed 5 years hormone treatment, 961 were untreated during all 5 years, and 306 had changed treatment status during the period. In the hormone treated group an increase in BMD of 0.02 g/cm2 was found in whole body and spine, while BMD of the femoral neck was unchanged. In the control group we found a decline in BMD of 0.03, 0.07, and 0.05 g/cm2 in the whole body, spine, and femoral neck respectively. The mixed group had a decreased BMD of 0.03 g/cm2 in the whole body and spine but unchanged status in the femoral neck. Median dietary retinol intake was 529 ìg/day and median total retinol intake was 1198 ìg/day. There was no association between dietary or total retinol intake and bone loss in any region, neither in linear regression analysis or when fitting quadratic, cubic or other curves. The data plots showed no evidence of threshold value. In conclusion we found no association between vitamin A intake and rate of bone loss in treated or untreated recently postmenopausal women. PATIENTS KNOWLEDGE OF OSTEOPOROSIS. RESULTS FROM THE SPANISH COMPLIANCE OSTEOPOROSIS STUDY IN POSTMENOPAUSAL OSTEOPOROSIS (PROCUOS) C. Turbi1*, G. Herrero Beaumont2, J. C. Acebes2, G. Graña3, A. Torrijos4 1Lilly Spain, Eli Lilly and Company. Alcobendas, Madrid, Spain 2Fundación Jiménez Díaz, Madrid, Spain 3Hospital Juan Canalejo, A Coruña, Spain 4Hospital La Paz, Madrid, Spain INTRODUCTION: Over the last decade there has been increasing awareness of osteoporosis, both in the medical profession and among the general public. For its effective management, patients must understand the disease and its complications. OBJECTIVES: To assess the knowledge of osteoporosis in postmenopausal women, aged 55 years or over with increased risk of osteoporotic fractures. MATERIAL AND METHODS: Study subjects were postmenopausal women participating in a 12-month observational, prospective, multicenter, and open-label study. Participants had not received treatment with bone active agents for at least 3 months prior to study participation and, after enrolment, were initiated on either raloxifene (Evista) or alendronate, (Fosamax) as determined by the treating physician. A modified questionnaire was used to evaluate the patients disease knowledge, and included the following questions: 1. Is osteoporosis a disease which you will have for the rest of your life. 2. Can you prevent osteoporosis by changing your lifestyle or using medicines. 3. State a complication of this disease. It was considered 'acceptable knowledge' when patients answers 'yes' in question 1, 2 and knew at least one complication of osteoporosis. It was considered 'unacceptable knowledge' if the patient did not know at least one complication of the disease. Other answers were considered as 'moderately acceptable knowledge'. RESULTS: Overall, 87.8% of patients answered that osteoporosis is a disease that they will have for the rest of their lives. Likewise, 88.0% thought that they could prevent osteoporosis by changing their lifestyle or using medicines. Finally, 68.7% of patients stated one complication of osteoporosis. A total of 428 patients (49.3%) had an acceptable knowledge of osteoporosis, 124 (14.3%) had moderately acceptable knowledge and 317 (36.5%) had an unacceptable knowledge. CONCLUSIONS: Educational intervention is required in more than a half of postmenopausal women, to increase their understanding of osteoporosis and its complications, and to assist with its effective management. PEAK BONE MASS IN THE CALCANEUS IN MEN IS FOLLOWED BY A SMALL BUT SIGNIFICANT BONE LOSS U. Pettersson1*, R. Lorentzon1, K. Landin-Wilhelmson2, L. Hulthén2, O. Johnell3, R. Kullenberg4, E. Norjavaara5, L. Samuelsson5, D. Mellstrom2 1Sports Medicine Unit, Umeå University, Umeå, Sweden 2Department of Geriatrics & Internal Medicine, Göteborg University, Göteborg, Sweden 3Department of Orthopaedics, Malmö University, Malmö, Sweden 4Department of Radiophysics, Göteborg University, Göteborg, Sweden 5The National Service Administration, Göteborg, Sweden We have previously suggested that peak BMD in the calcaneus in men is attained around 18 years (ECTS meeting 2002). The aim of this study was to evaluate this finding in a larger cohort of 4765 men. The present study is part of an ongoing study investigating relationships between anthropometric parameters, muscle strength, lifestyle factors including physical activity and calcium intake and genetic analysis and BMD in 12000 male military recruits. Since 1998, 4765 men (age 17.3-20.0 years) have now been recruited from the compulsory military service in Gothenburg, Sweden. Bone mineral density (BMD, g/cm2) of the calcaneus was measured with DEXA, Calscan. Isometric muscle strength of the total body was measured in Newton meters (Nm) using Isokai and physical capacity was estimated from a maximal stress test. Lifestyle factors including smoking, use of corticosteroids, training habits and calcium intake were evaluated by a questionnaire. Quadratic regression analysis revealed that peak BMD of the calcaneus(0.60±0.09 g/cm2) occurred at the age of 18.2 years. Stepwise regression analysis showed that years of regular physical activity were the strongest predictor of BMD, accounting for 8.8% of the variation in BMD. Other independent predictors were muscle strength (beta=0.12), body weight (beta=0.11), and age (beta= -0.06). All these factors totally explained 12.8% of the variation in BMD. To study the increase and decrease in BMD in more detail, we also performed two separately linear regression analyses, adjusting for all other variables except age. The analyses demonstrated a significant increase in BMD with 0.005 g/cm2 per month between the ages of 17.3-18.2 years (95% CI = 0.002-0.009), and a significant decrease in BMD with 0.002 g/cm2 per month between the ages of 18.3-20.0 years (95% CI = -0.003- -0.001). In conclusion, this study confirms our previous finding that peak bone mass in the calcaneus in men is attained around 18 years. However, once peak bone mass is reached, a small but significant bone loss seems to follow. The mechanism behind this decrease is presently not known, nor is its biological significance. DETERMINANTS OF BONE MINERAL MEASURES IN OLDER FEMALES WITH LOW, INTERMEDIATE AND HIGH BONE DENSITY: A TWIN STUDY J. D. Wark*, L. M. Paton School of Medicine, University of Melbourne, Australia Several anthropometric, health and lifestyle factors have been associated with variation in bone mineral density (BMD) in twin and non-twin adult females. Determinants of BMD in individuals with low bone mass are of particular clinical interest. Therefore, we examined risk factors for osteoporosis in 213 pairs of female twins (106 monozygotic, 107 dizygotic) aged more than 45 years (mean age 56 years), divided by tertiles of within-pair average total hip (TH) BMD Z-score. Tertile 1 (T1) was the lowest and T3 the highest tertile. Within-pair differences in risk factors were compared with within-pair percent differences in lumbar spine (LS) and TH BMD and with total body bone mineral content (TBBM), each adjusted for height or height and weight as appropriate. In the pooled data, lean mass (LM) and fat mass (FM) were significant determinants of height-adjusted LS and TH BMD and TBBM. Cigarette use (in total pack-years) was a significant determinant of height-weight-adjusted LS and THBMD and TBBM. Current sporting activity was associated significantly (R2 = 0.028, p=0.02) and calcium intake marginally (R2 = 0.017, P = 0.07) with height-weight- adjusted TBBMC. In the analysis by tertiles, LM remained strongly associated with all bone measures in all tertiles. In contrast, the association of FM with LS and TH BMD was non- significant or marginal in T3 (R2 = 0.028, p = 0.18; R2 = 0.006, p = 0.05, respectively), but persisted in T1 and T2. Current calcium intake was associated with height-weight-adjusted LS BMD (R2 = 0.075, p = 0.026) and TBBM (R2 = 0.099, p = 0.01) in T1 only. The association with smoking was similar across tertiles while the association with sporting activity was inconsistent. We conclude that (i) LM was associated strongly with bone mass at all levels of BMD, (ii) the association of FM with bone mass was diminished at high BMD, (iii) calcium intake was associated with bone mass only in subjects with low BMD and (iv) smoking was an adverse factor at all levels of BMD. The findings support efforts to increase calcium intake in individuals with low BMD. FARMACOGENETICS OF THE ESTROGEN RESPONSE IN A POPULATION OF DANISH POSTMENOPAUSAL WOMEN S. Silvestri1*, A. Gozzini2, A. B. Thomsen3, C. Christiansen3, A. Tanini2, G. Leoncini2, M. L. Brandi2 1Department of Clinical Phatophysiology, University of Florence, Florence, Italy 2Department of Internal Medicine, University of Florence, Florence, Italy 3Center for Clinical and Basic Research, Ballerup, Denmark Hormone Replacement Therapy (HRT) is used to treat climacteric symptoms and to prevent chronic diseases that often develop in women during this period of life. Common clinical practice shows how much variable it is the response to HRT in treated women. To analyse the genetic basis of such variability we studied the role of two intronic polymorphisms (PvuII and XbaI) of the Estrogen Receptor a (ERa) in 271 Danish postmenopausal women. We analysed the genotypes distribution in the population and the genotypes influence on the response to HRT in a subpopulation of 77 women treated for 3 years. The study population was very homogeneous as far as baseline characteristics are concerned: healthy postmenopausal women (48-60 years old), within 1-6 years after the menopause. The study was a double-blind placebo-controlled monocentric trial. Women were randomly assigned to one of five groups (17b-estradiol sequentially combined with gestodene respectively: I: 2mg and 25mg; II: 2 mg and 50 mg; III: 1 mg and 25 mg; IV: 1 mg estradiol continuously combined with 25mg gestodene and V: Placebo). PvuII and XbaI genotypes distribution followed the Hardy-Weinberg equilibrium. No association between baseline lumbar spine and hip BMD and different genotypes was found, while a significant association was found between the genotype ppxx and higher basal high density lipoprotein levels versus the genotype PPXX which showed lower HDL basal levels (P<0.001). No significant correlation was seen between genotypes and basal levels of total cholesterol, low density lipoprotein, tryglicerides, and glucose. In the subgroup of treated women we did not find any associations between genotypes and the change in both BMD and lipid profile after 3 years of HRT. Since a significant association was present at the baseline and it disappeared after 3 years of treatment, we can conclude that the absolute value of the response to HRT was greater in PPXX women. These data emphasize the role of ERa genotype in the modulation of the responses to HRT. The analysis will be extended to a larger number (a total of 6000) of postmenopausal women who have been treated with HRT from 2 to 6 years. OSTEOPOROSIS IN MEN S. KarthikShankar*, K. M. Prasannakumar, M. Dharmalingam, J. Patil M.S.Ramaiah Hospital, New Bel Road, Bangalore, Karnataka, 560 054, India Introduction: Osteoporosis is one of the most common problem faced in geriatric clinics nowadays. In next 15 yrs all hip fractures will occur in men.Several recent population based studies report that the prevalence of spinal fractures is similar or only slightly less in men compared with women.Vertebral fractures accur earlier in men & in middle aged men the prevalence of vertebral fractures is higher than in women.Mortality after hip & spine fractures is also higher in men than in women. Aim: To assess the prevalence of osteoporosis in South Indian men & to study the bone mineral density(BMD),bone mineral content(BMC) in men above 40 years. Method and Materials:120 apparently healthy South Indian men in 40 yrs - 80 yrs age group were studied. Age, Height, Weight and Body Mass Index(BMI) were recorded Serum testosterone values were estimated by RIA methods for all the individuals. The bone mineral density (BMD) and Bone Mineral Content(BMC) of spine (L1- L4) & hip were measured using Hologic Dual Energy X-ray Absorptionometry (DEXA). Results: The variation of bone mineral density (BMD) in different age groups of South Indian men are tabulated below. (Table 1) The serum testosterone values according to the age groups and the percentage of osteoporotic patients among the South Indian men are tabulated below. (Table 2) Conclusion: There is no significant difference in the Bone Mineral Density(BMD) among the different age groups of South Indian men. There is no correlation between the serum testosterone and osteoporosis in South Indian men. PARATHYROID HORMONE LEVELS PREDICT NON-VERTEBRAL, BUT NOT VERTEBRAL FRACTURES J. Finigan1*, D. M. Greenfield2, A. Blumsohn1, R. A. Hannon1, R. Eastell1 1Bone Metabolism Group, University of Sheffield, Sheffield, UK 2University of Sheffield, Sheffield, UK High levels of parathyroid hormone (PTH) have been associated with an increased risk of hip fracture; it is unclear whether they are associated with other fractures or with vertebral fractures. We have carried out a 10-year prospective study of a population-based group of women who were ages 50 to 85 years at baseline. We measured intact parathyroid hormone (PTH) by IRMA (Nichols Inc.) at baseline on 353 subjects. Incident vertebral fractures were determined by visual reading by a single radiologist, and non-vertebral fractures were confirmed by radiologist reports. Spinal radiographs were carried out at 2,5,7 and 10 years. The table shows the relative risks of non-vertebral and vertebral fracture using a Cox Regression Model, based on time to first fracture. Risks are given per standard deviation for PTH (with log transformation), and relative to the age group 50-59 for age at baseline. The 95% confidence intervals are shown in parentheses. The relative risk of a non-vertebral fracture approximately doubles with each decade of age, whereas the risk of a vertebral fracture increases almost fourfold. Baseline PTH measurements significantly predict non-vertebral fractures (p=0.017) yet do not predict vertebral fractures. This suggests that high PTH levels are more strongly associated with fractures of the appendicular than of the axial skeleton.
BONE MINERAL DENSITY IN CHILDREN WITH DELAYED PUBERTY G. Triantafyllidis, D. Karakaidos, P. Zosi, G. Kafalidis, G. Ouzouni, S. Pizanias, C. Karis* Pediatric Clinic of General Hospital of Nikea, Piraeus, Greece The timing of sexual maturation is an important determinant of adult bone mineral density. The aim of this study was to assess BMD in children with delayed puberty by comparing them to those with a history of delayed puberty (D.P) and a normal control group. SUBJECTS AND METHODS: We studied 25 children (10 boys and 4 girls with delayed puberty-group I and 11 children with a history of delayed puberty-group II.) Age ranged from 13-18 years. BMD was measured by dual energy X-ray absorptiometry at the lumbar spine L1-L4 levels. Our control group consisted of 25 age and BMI matched children. Calcium intake and physical activity were similar in both control and study groups. RESULTS: One way analysis of variance (one way ANOVA) was used to evaluate the results. BMD of children with delayed puberty as well as of those with a history of delayed puberty did not significantly differ from controls (sig. 0.297 and 0.604 respectively). In contrast children with delayed puberty (group I) had BMD values significantly different from those with a history of delayed puberty (group II, sig.0.041). When the results were separately analyzed no difference in the BMD of boys between any group was found in contrast with group I girls which significantly differed from group II girls (sig.0005) CONCLUSION: Our evidence of decreased BMD in children with delayed puberty compared to those with a history of delayed puberty indicate that the timing of puberty plays a major role in determining peak bone mass and D.P. probably affects BMD values in a reversible manner. It appears that adults with a history of delayed puberty will finally achieve normal bone mass.
BONE MINERAL DENSITY IN CHILDREN WITH CONGENITAL ADRENAL HYPERPLASIA P. Zosi, G. Ouzouni, G. Triantafyllidis, D. Karakaidos, S. Pizanias, F. Papadelis, C. Karis* Pediatric Clinic of General Hospital of Nikea, Piraeus, Greece Congenital adrenal hyperplasia (CAH) is due to group of enzymatic defects in cortisol synthesis from cholesterol. It is an inherited disease that requires corticosteroid replacement therapy. It is well known that during steroid therapy many complications can occur. The aim of the present study was to observe whether there is a difference in bone mineral density (BMD) between children treated with replacement corticosteroids and the normal pediatric population. PATIENTS AND METHODS : Twelve patients with congenital adrenal hyperplasia (CAH) who had been receiving oral cortisol (10-15 milligrams/m2/day ) in two or three doses for 4.5-11 years were enrolled in the study. At the time of the study the children ranged in age from 7 to 25 years old (mean age 12.41 ± 4.87 yrs). The bone mineral density (BMD-g/cm2) was assessed by Dual Energy X-ray absorptiometry (DEXA) at the lumbar spine (L1-L4) levels. Our control group comprised 25 healthy children of the same age. RESULTS: There was no significant difference in the bone mineral density between the patient (0.769 ±0.180 g/cm2) and the control (0.790 ±0.126 g/cm2) group .No statistically significant correlation was found between BMD values and the corticosteroid dose administered. CONCLUSION: We concluded that long term corticosteroid replacement therapy does not reduce BMD in CAH patients and has no deleterious effect on skeletal mineralization. Therefore it should not contribute to adult osteoporosis. Further studies over a longer follow up period and with a larger cohort of patients are required in order to reach a safe conclusion. BONE MINERAL DENSITY IN CHILDREN RECEIVING ANTICONVULSANTS D. Karakaidos, P. Zosi, N. Milioni, G. Triantafyllidis, G. Kafalidis, S. Pizanias, C. Karis* Pediatric Clinic of General Hospital of Nikea, Piraeus, Greece It is known that anticonvulsants generally affect vit D metabolism and that phenytoin, primidone and phenobarbital in particular are established contributors to the development of osteomalacia and rickets. The aim of the present study was to examine the probable effect of carbamazepine and valproic acid therapy on the bone mineral density (BMD) of children with epilepsy. SUBJECTS AND METHODS: 23 children with epilepsy without neurologic impairment treated with either carbamazepine (n=8) or valproic acid (n=15) for more than 28 months were included in the study. Mean age was 12.8±3.15 yrs. And mean duration of treatment 73.4 ±54.8 months. Duration of therapy was similar for both carbamazepine and valproic acid treated subjects. BMD was measured at the lumbar spine level (L1-L4) using dual energy X-ray absorptiometry. 25 healthy children of the same age range served as the control group. Calcium intake and physical activity were similar in both control and study groups. Mean serum levels of valproic acid and carbamazepine levels were 69.9 ±19.4 and 7.08 ±2.91 microg/dl , respectively. Serum Ca, ALP, phosphorus, aminotransferase and drug levels were normal in all groups. RESULTS: a) Bone mineral density values of children receiving both valproic acid and carbamazepine were not significantly different from controls (0.756 ±0.212- 0.746 ±0.132, respectively p=0.895). b) When the results were analyzed according to the treatment type, BMD was significantly lower in the valproic acid receiving group (0.546 ±0.057) than in controls (0.746 ±0.132, p=0.0005), whereas it was found to be significantly higher compared to controls in those receiving carbamazepine (0.869 ±0.174, p=0.0001). CONCLUSION: Our data suggest that children with epilepsy treated with valproic acid seem to have reduced BMD in contrast with those receiving carbamazepine. Consequently, the lowered bone mineral density associated with the use of valproic acid might be expected to increase the risk of future fractures.
DISTRACTION OSTEOGENESIS STIMULATES INCREASED NUMBERS OF CIRCULATING ENDOTHELIAL PROGENITOR CELLS D. Lewinson1*, S. Bisharat1, A. Rachmiel2 1Dept of Anatomy and Cell Biology, Faculty of Medicine, Technion, Haifa, Israel 2Dept of Oral and Maxillofacial Surgery, Rambam Medical Center, Haifa, Israel Endothelial progenitor cells (EPC) have recently been shown to circulate in increased numbers in several ischaemic experimental animal models and in similar clinical situations as well. The purpose of this study was to look for EPC in a large animal model in which an osteotomoized bone undergoes distraction osteogenesis. We have isolated EPC from the periphral blood of two sheep in which the maxillary bone was osteotomized and the fracture gap was distracted daily for about 2 weeks. The kinetics of the appearance of the EPC in the peripheral blood of the distracted sheep was compared to that of EPC in sham-operated osteotomized, but not distracted sheep. The increase in the percent of EPC from the total white blood cell counts (WC) was 2.5 and 4.5 fold in the two distracted sheep in contrary to no increase in the control sham-operated sheep. Fluorescentically labeled isolated EPC where re-injected to the auotologous animals. They were shown to localize to the callus tissue in ditracted sheep, but not to the callus of control sheep. Their endothelial nature was verified by Tie-2 immunohistochemistry. We suggest to consider ex-vivo expanded autologous EPC as vectors for introducing bone forming genes into bone regeneration sites created by distraction osteogenesis especially in compromized clinical situations. COLLAGEN AS BONE MORPHOGENETIC PROTEIN-1:ITS ROLE IN SKELETAL TISSUES AND ITS FUNCTION UPON ABNORMALITIES M. Tzaphlidou School of Medicine,University of Ioannina, Ioannina, Greece The characteristics of different extracellular matrices derive in large part from the synthesis, assembly, and deposition of collagen molecules and their organization into unique macromolecular structures. These collagen assemblies confer specialized properties to the extracellular matrix in which they are found. The synthesis of collagen molecules and their association to form fibrils requires a number of sequential post-translational events. These include intracellular processes such as hydroxylation and glycosylation, and extracellular ones such as procollagen processing and cross-linking. In bones, it is the procollagen C-proteinase that processes the major fibrillar collagen types I and III, and it may process prolysyl oxidase to the mature enzyme necessary to the formation of covalent cross-links. Type V collagen is a fibrillar collagen of low abundance that is incorporated into and helps regulate the shape and diameter of type I collagen fibrils. As a result a coarse network is formed in the extracellular matrix with thin fibrils. Bone collagen fibrils are much smaller than fibrils from other connective tissues, such as skin, in which collagen is composed by the same types. This difference in fibril diameters may account for the different mechanical properties of the two tissues. Upon skeletal abnormalities, such as osteoporosis, the overall collagen fibril architecture is altered. Such fibrils have a random arrangement in contrast to normal in which fibrils have a characteristic parallel arrangement. In addition, fibril diameter is dramatically affected. In ovariectomized rats, sacrificed 6 weeks after ovariectomy, mean trabecular bone collagen fibril diameter is 47.1 ±3.8 nm while in fibrils from age-matched normal rats is 51.2 ±6.1 nm. The mean values for ovariectomized rats, sacrificed 12 months after ovariectomy, and for the corresponding normals, are 46.1 ±5.5 nm and 53.2 ±4.9 nm respectively. Upon ovariectomy, cortical bone is also suffered of such significant (p<0.001) reduction in collagen fibril diameter. The question of whether skeletal abnormalities are an intrinsic collagen disorder remains to be demonstrated. TWIST INACTIVATION REDUCES CBFA1/RUNX2 EXPRESSION AND DNA BINDING TO THE OSTEOCALCIN PROMOTER IN OSTEOBLASTS M. Yousfi1, F. Lasmoles1, B. Kern2, P. J. Marie1* 1Inserm U349, Paris, France 2Dept Human Molecular Genetics, Baylor College of Medicine, Houston, TX, USA The Saethre-Chotzen (SC) syndrome is characterized by increased osteogenesis and premature fusion of cranial sutures resulting from mutations in TWIST, a basic Helix Loop Helix transcription factor. We previously showed that Twist is an important modulator of human osteoblast proliferation and differentiation. We found that Twist inactivation induced by genetic mutations modulates the expression of osteoblast specific genes such as osteocalcin (OC) and alpha 1(I) collagen (COLIA1). We therefore hypothesized that Cbfa1/Runx2 that controls OC and COLIA1 expression may be a molecular target gene for Twist in human osteoblasts. We tested this hypothesis in Twist mutant (M-Tw) calvarial osteoblasts obtained from a subject bearing the Y103X Twist mutation that introduces a stop codon, leading to a truncated protein without functional bHLH domain. We found that Twist haploinsufficiency in mutant osteoblasts reduced mRNA and protein levels for Cbfa1/Runx2 both during cell growth as well as during in vitro osteogenesis. Moreover, this effect was associated with altered expression of major osteoblast specific genes such as OC, COLIA1, osteopontin (OP) and bone sialoprotein (BSP) whereas osteonectin (ON) was not affected. In addition, electrophoretic mobility shift assay (EMSA) showed reduced-binding ability of Cbfa1 to its target OSE2 element in the OC promoter in mutant osteoblasts. By contrast, Twist inactivation did not hamper Cbfa1 binding on a similar upstream element present in the COLIA1 promoter in mutant osteoblasts. These results indicate that Twist inactivation may control human osteoblast differentiation by altering Cbfa1/Runx2 expression and Cbfa1 binding ability to the osteocalcin promoter, and possibly to OP and BSP promoters. This study provides the first evidence that Cbfa1/Runx2 is a target gene for TWIST in human osteoblasts. A NEW BONE HEALING MATERIAL: A HYALURONIC- CHONDROITIN-HEPARIN-LIKE BACTERIAL EXOPOLYSACCHARIDE Ph. Zanchetta1*, G. Godeau2, K. Senni2, S. Igondjo-Tchen2, N. Lagarde1, J. Guezennec3 1Service Anatomo-pathologie, CHU Morvan, 29200 Brest, France 2Laboratoire de Physiopathologie des Tissus non minéralisés, 1 rue Maurice Arnoux, 92100 Montrouge, France 3IFREMER Dept DRV/VP/BMM. BP 70 29280 Plouzane, France Critical Size Defect (CSD) technique was used to evaluate the bone regeneration capacity of a newly discovered hyaluronic acid like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent. A 5 mm diameter hole was made on each parietal bone of male rats. The right hole was filled with either 1 mg of a new bacterial exopolysaccharide referenced HE 800 or with collagen used as negative control, while the left hole remained free of any treatment. After 15 days, the holes and surrounding tissues were examined by direct examination, X-rays, and histological staining. Using HE 800, bone healing was almost complete after only 15 days with osteoblasts lying external bone surfaces and enhancing osteocyte inclusion. Neovascularization was also observed along with an organized trabecular bone. No abnormal bone growth or conjunctival abnormalities were noticed. At the end of the experiment, 95.9 % (SD6.2) bone healing (n=20) was observed (Graphic). Conversely, the collagen treated animals did not demonstrate significant healing 17.8 % (SD18.1). Chemical composition similar to heparin, chondroitin and hyaluronic acid of this polysaccharide could explain the results obtained. Adhesivness and chelation properties may play also an important role.
SYSTEMIC EFFECTS ON BONE HEALING OF AN HYALURONIC ACID-CHONDROITIN-HEPARIN LIKE BACTERIAL EXOPOLYSACCHARIDE Ph. Zanchetta1*, N. Lagarde1, G. Godeau2, K. Senni2, S. Igondjo-Tchen2, J. Guezennec3 1Service d Anatomo-pathologie, CHU Morvan, 29200 Brest, France 2Laboratoire de Physiopathologie des Tissus non minéralisés, 1 rue Maurice Arnoux, 92100, France 3IFREMER Dept DRV/VP/BMM. BP 70 29280 Plouzané, France Critical Size Defect (CSD) technique was used to evaluate the systemic activities on bone regeneration capacity of a newly discovered hyaluronic acid-chondroitin- heparin like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent. Some systemic effects were previously detected on earlier experiments. A 5 mm diameter hole was made on each parietal bone of male rats. The right hole was filled with 0.5 mg of a new bacterial exopolysaccharide referenced HE 800, while the left hole remained free of any treatment. After 21 days, the holes and surrounding tissues were examined by direct examination, X-rays, and histological staining. Using HE 800, bone healing was almost complete after only 21 days in the treated hole and always complete in the control side by a supposed systemic effect. Neovascularization was also observed along with an organized trabecular bone on both sides. No abnormal bone growth or conjunctival abnormalities were noticed. At the end of the experiment, 90.1 % (SD 5.2) bone healing (n=20) was observed on the treated side; conversely, the control side animals demonstrate an amazing healing 100% SD 0.5) by a systemic effect.
WITHDRAWN EXPRESSION OF SEMAPHORIN-3A AND ITS RECEPTORS IN BONE: POSSIBLE ROLE IN SKELETAL DEVELOPMENT C. Gomez*, B. Burt-Pichat, L. Malaval, C. Chenu INSERM Unit 403, Hôpital E. Herriot, Pavillon F, 69437 Lyon Cedex 03, France Recent studies have shown evidence for a neural regulation of bone development and remodeling. However, the molecules and mechanisms involved in the development and dynamic maintenance of bone innervation are unknown. During the development of the central nervous system, extending axons are oriented to their targets through the actions of different families of guidance molecules. We investigated the expression of Semaphorin 3A (Sema 3A) and its receptors in in vitro models of bone cell differentiation and in developing bone. RT-PCR analysis was performed at successive stages of osteoblast differentiation in newborn rat calvaria cell cultures. These experiments, completed by immunolabelling, showed the expression by osteoblastic cells of Sema 3A, as well as its receptors, Neuropilin-1 (NP-1) and -2, Plexins A1 and A2. Sema 3A and NP-1 were expressed throughout the osteogenic differentiation sequence, with little regulation except for an increase of NP-1 mRNA in differentiated stages. Only Plexins and Neuropilins were present in osteoclasts differentiated from RAW 264.7 cells. We studied the expression of Sema 3A and NP-1 in vivo using immunocytochemistry on rat long bone sections at gestational days (GD) 17 to 21, and 2 and 3 weeks post natal. At early stages of endochondral ossification (GD 17-19), both proteins were present in the periosteum and in chondrocytes of the mid-diaphysis before (GD17) and after vascular penetration (starting on GD18) and early primary ossification. The same labelling was observed in epiphyseal chondrocytes before and after the onset of secondary ossification. At day 21, NP-1 was still expressed in the periosteum, contrarily to Sema 3A. Both molecules were later detected in pre-hypertrophic and hypertrophic chondrocytes of the growth plate and in osteoblasts lining bone trabeculae. RT-PCR analysis was performed on differentiating cultures of the chondrocytic cell line MC615, confirming the expression of Sema 3A and NP-1 by this cell type. Our results demonstrate the expression of Sema 3A and its receptors in bone cells in vitro and in vivo during bone growth. Their distribution pattern, temporally and spatially preceding the onset of bone tissue formation, suggests that Sema 3A signalling may play a role in the regulation of skeletal development. EFFECTS OF TCDD ON METAPHYSEAL BONE IN A TCDD- SENSITIVE AND A TCDD-RESISTANT RAT STRAIN N. Stern1*, M. Lind1, M. Viluksela2, J. T. Tuomisto2, J. Tuomisto2, H. Håkansson1 1Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 2Department of Environmental Health, National Public Health Institute, Kuopio, Finland Introduction Bone development and maintenance are strictly controlled by hormonal interactions. Dioxins are known to disturb several hormonal systems, inter alia to have antiestrogenic effects. Therefore dioxin exposure may cause trabecular bone loss and decrease of bone strength at sites containing both trabecular and cortical bone such as long bone metaphysis. Sensitivity to dioxin-induced toxicity is highly variable. An animal model based on different sensitivity to various endpoints of dioxin toxicity has previously been developed. Long-Evans (L-E) is the most TCDD-sensitive rat strain, Han/Wistar (H/W) is the most resistant. In a recent study L-E rats demonstrated higher sensitivity than H/W rats to TCDD-induced changes in diaphyseal geometry and strength of tibia. In this study we used L-E and H/W rats to examine the effects of TCDD on cortical and trabecular bone of femur metaphysis. Material and Methods Female L-E and H/W rats were given a total dose of 0, 0.17, 1.7 and 170 (H/W only) mg/kg TCDD s.c. weekly during 20 weeks. The right femur was dissected and the length was measured. The femoral metaphysis was scanned at a point distanced 20% of the bone length from the distal end of femur with a pQCT system (Stratec XCT 960A, Birkenfeld, Germany) with a voxel size of 0.148 mm3. A 0.400 cm-1 attenuation threshold was set as a lower limit to define the trabecular bone region. The trabecular bone mineral density and area, as well as cortical/subcortical bone density and area were analysed. The Mann-Whitney Rank Sum test was used with a significance level of p<0.05. Results and Discussion Cortical/subcortical area of distal femur metaphysis was smaller in L-E rats at 1.7 and 17 microg/kg and in H/W rats at 17 and 170 microg/kg dose levels (Fig 1). Trabecular bone mineral density was decreased in LE rats at 17 microg/kg vs. corresponding controls (Fig 2), but not in H/W rats. The data show that TCDD exposure causes trabecular bone loss and confirms previous findings that dioxins interfere with bone remodelling.
BIOPHYSIOLOGY OF AUTOGENIC BONE GRAFT. ALP, QCT AND HISTOLOGIC SHORT-TERM STUDY A. Smailagic1*, A. Redji 2, B. Hadjihasanovic3, S. Lappalainen4 1University Clinic Center Sarajevo Clinic for Maxillofacial Surgery, Bosnia and Herzegovina 2Faculty of Medicine, Institute for Human Genetic and Biology, University of Sarajevo, Bosnia and Herzegovina 3University Clinic Center Sarajevo Radiologic and Diagnostic Center, Bosnia and Herzegovina 4 BonAlyse Oy, Jyväskylä, Finland Autogenic bone grafts are currently the most widely used method in skeletal surgery for reconstructive purpose. Certain problem with extensive resorbtion (up 50%), sequestration, inadequate integration with host bone can compromise success. Factors as rigid fixation, embriologic origin of transplant, blood supply and presence of growth factors are some of reason transplantation procedure success. White New Zealand rabbit underwent reconstruction of unilateral created non critical size defect of mandible, fixed with mini plate and bridged with autogenic bone graft from spine scapule. Result were evaluated measurement of ALP activity, QCT GEANIE 2.1 (BonAlyse Oy, Jyväskylä, Finland) program of bone analysing and clinical and histologic examinations.Result: ALP activity was significancy higher after 30 days, density of transplant after 30 days was 555-696 HU (GEANIE 2.1 analysing), and clinical inspection and histologic analysing showed bridged defect and complete integation of graft with host bone and new osteoblast, osteocit and angiogenesis. Conclusions: Significant higher ALP activity after 30 days indicate on time beginning of process osteoinduction and osteoblast differentiation. Histologic and clinic inspection indicate that after 45 days bone graft are total revasculated and integrated with host bone. Autogenic graft showed no immune response and viral transmission. Different embriogenic origin (intramembranose) of recipient bone-mandible and enhondral origin of graft from spina scapule showed no implicacations on osteointegated process in short term study. OVERLOADING OF THE TEMPOROMANDIBULAR JOINT UPREGULATES THE JNK/AP-1 SIGNALING PATHWAY TRIGGERING CHONDROBLASTIC DIFFERENTIATION D. J. Papachristou1,2*, P. Perttiniemi3, T. Kantomaa3, N. Agnantis2, A. G. Papavassiliou1, E. K. Basdra4 1Department of Biochemistry, University of Patras School of Medicine, Patras, Greece 2Department of Pathology, University of Ioannina School of Medicine, Ioannina, Greece 3Institute of Dentistry, University of Oulu, Oulu, Finland 4Department of Orthodontics, University of Heidelberg, Heidelberg, Germany Mechanical loading has been long recognized as an important regulatory factor in bone and cartilage homoestasis and a determinant of skeletal morphology. However, the molecular mechanisms that govern the response of chondroblasts to mechanical stimulation remain elusive. The transcription factor c-Jun together with members of the Fos family proteins (c- Fos, FosB, Fra1/2) are major components of the AP-1 (activator protein-1) transcription complex. Recent in vitro studies have indicated that AP-1 plays an important role in chondrogenic gene regulation as well as chondroblastic differentiation and proliferation. The aim of the present study was to explore the effect of increased temporomandibular joint loading on the proliferation and differentiation of condylar cartilage chondrocytes, in vivo. To this end, 100 rats were assigned to two groups: the first group was fed soft diet (which simulates normal masticatory movements), while the second group was fed hard diet that causes increased temporomandibular joint loading. Biopsy sections from temporomandibular joint of both groups were obtained at 2, 6, 12, 24, and 48 hours after the experiment initiation. We employed immunohistochemical staining analysis to investigate the in situ expression of c-Fos, in correlation to cellular levels of pc-Jun, the phosphorylated (hence activated) form of c-Jun. Moreover, the expression and activation profile of JNK2 (c-Jun N-terminal kinase 2), the principal kinase targeting c-Jun, was examined. The articular cartilage of the first group displayed nuclear immunoreactivity for c- Fos, that was gradually increased as the experimental procedure evolved. The expression levels of c-Fos were accompanied by a co-localized enhancement of pc- Jun. Augmented levels of JNK2 and its phosphorylated/activated form, p-JNK, were observed at 24 and 48 hours after experiment initiation. In the second group, immunoexpression of the aforementioned proteins was also increased as the experiment progressed. However, expression levels of the examined proteins were significantly higher in the second, compared to the first group, at 12, 24, and 48 hours after the experiment initiation. Our results suggest that mechanical loading potentiates the JNK/AP-1 signaling pathway and pose a novel mechanism that might be implicated in the chondroblastic differentiation process. THE EXTRACELLULAR MATRIX 1(ECM1) GENE IS ESSENTIAL FOR MOUSE EMBRYOGENESIS J. Liekens1*, J. Merregaert1, P. Smits1, L. Umans2, D. Huylebroeck2, A. Zwijsen2 1Lab of Molecular Biotechnology, Dept. of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium 2Department of Cell Growth, Differentiation and Development, (VIB), University of Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium The Extracellular matrix gene 1 encodes for a glycoprotein of 559 amino acid residues with a typical cystein CC-(X7-10 )-C distribution. This cystein arrangement forms 'double loop' domains similar to those o | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
