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Poster Presentations
Abstracts P-95 to P-140
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows:
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HISTIOCYTOSIS X - LCH ( HAND-SCHUELLER-CHRISTIAN DISEASE): CLINICAL CASE REPORT A. S. Smailagic*, H. P. Piranic, F. F. Foco, A. M. AlHalil University Clinic Center, Sarajevo Clinic for Maxillofacial Surgery, Bosnia, Herzegovina Histiocytosis X or Langerhans cell histiocytosis LCH, rare childhood disease in which there is an overgrowth of a type of tissue cell called a histiocyte. Annual incidence of 4 per million Etiology is unknown typified by bone involvement. Case History: The patient was born 1978, male with half-year history of pain and swelling submandibular left area, seen by a dentist and ortodontist. In July 1994, was seen by Maxillofacial surgeon and hospitalized. X-Ray showed destructive bone lesions corpus of mandible with aplasion ramus. Skeletal X-ray showed oval radiolucent lesion surrounded by thick margin of reactive bone affected vertebrae C5 C6, hand bone , rib r.5,7, l. 9. CT confirm. Biopsy of mandible lesion showed granulation areas with specific histiocyte cells for Hand-Schueller-Christian disease. Treatment:Aimed was to check progression of disease and treatments included chemotherapy and radiotherapy 1994-1996. After regression 1996, therapy has included surgical excision lesion of mandible and restore defect with autolog rib graft. On last control March 2001 progresion of disease torticollis with limitation of motions and long therm osteolisis and resorption of autolog rib graft. Discusion: Long time of period disease was not discovered. Clinic symptom was not convincing. Biopsy and skeletal X-ray imagining are recommended for definitive diagnostics. Chemotherapy and radiotherapy stopped spreading of disease for 4 years. Is surgery treatment recommended ?. Conclusions: Clinical LCH have broad spectar of symptoms and difficulty for diagnostics. Biopsy and characteristic skeleton X-Ray imagining is recommended for certain diagnostic. Radiotherapy and chemotherapy checked progression of disease for 4 years. Surgery is recommended in case with extensive bone lesion accompanied with painless disfunction, pathology fractures pseudoarthtosis and esthetic defects particularly in young person . Gold standard with autolog bone graft is not recommended because of systems character of bone disease. Allograft's accompanied with some osteoinductive factors as BMP's are promising. DYNAMOMETRY OF HAND IN MEDICAL PROGNOSIS OF WORKING CAPABILITY IN CHRONIC REGIONAL PAIN SYNDROME L. Krapac1*, A. Delija1, F. Skreb1, B. Rozman1, B. Hranjec2 1University Hospital Dubrava, School of Medicine, University of Zagreb, Croatia 2Department of Physical Medicine and Rehabilitation, University Hospital Dubrava, Zagreb, Croatia As epidemiological indicators are concerned the chronic regional pain syndromes (CRPS) are mostly connected with injuries, they are more prominent in women. In Croatia, in a sample of middle-aged patients (35 - 55 years of age) injuries are observed in 8.3%. Injuries of the hand are registered in 2% and of lower leg in 1% of the examined patients. CRPS of hand is diagnosed in Outpatient department for prevention of bone-joint system diseases in the Dubrava University Hospital on basis of medical examination, x-ray and /or scintigraphic, densitometric and electrodynamometric treatment. The MWPC is being observed in relation to disease severity, previous occupation, dominant hand as well as comorbidity. At the end of medical treatment as part of functional treatment the grip of the hand is measured by dynamometer. On basis of this finding medical prognosis of working capability is made. By electrodynamometry (EDM) not only grip force of the dominant and non-dominant hand, but endurance to bimanual work is obtained. By multiple check of strength and endurance during treatment a positive retroactive effect to patients motivation concerning restoration to health is achieved. During 2-year observation of patients with CRPS no difference between 'white' and 'blue' collar workers was registered. The age ranged from 30 to 64 years. The diagnosis of CRPS on the hand was significantly more frequent in women (30:3 men) while on the foot Sudeck-Leriche's syndrome is equally represented in women (34) and men (30). Significantly less CRPS was observed on dominant hand (31:2), although epidemiological data point to comparable injuries caused by traumas in right-hand patients. Only in one female patient CRPS was manifested on upper and lower extremity. At the end of treatment force of contraction in unhealthy hand measured by electrodynamometer was by 45% lower than expected. The case of a commonplace hand injury that results in severe CRPAS (III grade) was presented, the condition of symptom aggravation after surgical treatment of carpal tunnel in a female worker as well as dissimulation of discomfort difficulties in the right hand of a professional guitarist. The necessity of non-aggressive functional diagnostics of the hand in CRPS, testing of sympathetic nervous system, damaging effect of long-lasting immobilization as well as benefit of working therapy are being emphasized. VALIDITY OF DENSITOMETRY IN DIAGNOSIS AND TREATEMENT OF SUDECK'S DISEASE A. Delija*, F. Skreb, L. Krapac, B. Rozman, Z. Kuster University Hospital Dubrava, School of Medicine, Univeristy of Zagreb, Croatia Aim of the study : - prove osteoporotic component by measuring BMC - administering medicine (Alendronate) and calcium, physical therapy with special accent on magnetotherapy - remove painful component and get full function of extremity movement - follow the recovery of patient in the period of three, six and nine months after the first therapy session. Introduction: The lower value of BMC (Bone Mineral Component) as reaction to the soft tissue and hard particles with injury of ephiphyses and soft particles was measured in patients with complex regional syndrome (CRPSy - Sudeck syndrome ) by densitometric method. Materials and methods: In this study 23 patients aged between 38 and 65 with characteristic symptoms of CRPSy and radiological report showing the state after fracture with spotted atrophic bone and osteoporotic changes in distal and proximal place of the fracture were included. Pain and painful reduction of movement for more than 50% of functional measurement (FOP) are prominent syndrome and after the imobilisation removal. Densitometric measurements are performed on Hologic QDR - 45 000W (S/N 48111) Whole Body V8.26a:5 and in all patients it has been proved the deficiency of mineral content in bone structure from 35 to 85 gr in whole extremity in relation to collaterals. In control group the biggest difference in assymetry was 4 to 16 grams. After inclusion in therapy process patients subjectivly had pain reduction in more then 35% to 50%, functional recovery for more then 50% and measured BMC gained from 5 gr to 22.5 gr in first three months. After 11 months 3 patients were on intermittent treatment, 7 only on Alendronate and active exercise at home. Conclusion: Following the dynamics of treatment by densitometry and inclusion of Alendronate in treatment with already known physical procedures we managed acceleration of improvement and recovery in patients with CRPSy. At the end reaction of BMC is positively related to biphosphonate and low frequency magnetic field. DISTRACTION OSTEOGENESIS BY ILIZAROV AND UNILATERAL EXTERNAL FIXATORS IN DOGS V. Kusec1*, M. Jelic2, F. Borovecki2, S. Vukicevic2, K. Korzinek2 1Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 2Department of Anatomy and Department of Orthopaedic Surgery, Zagreb School of Medicine, Zagreb, Croatia Bone tissue's inherent capability for regeneration has been used in limb lengthening procedures after osteotomy/corticotomy and slow progressive distraction by an external fixation device. In this study the canine experimental model of distraction osteogenesis by two types of external fixators was evaluated. Following corticotomy either Ilizarov (N=6) or AO unilateral (N=9) external fixator was applied. Distraction started 1 week after surgery (2 x 0.5 mm/day) and lasted 3 weeks. Specimens were harvested from week 7-12. The outcome was assessed by x-ray, histology, histomorphometry and microradiographs. Bone regeneration as observed by X-rays was satisfactory and similar in both groups during and at the end of the experiment. Both endochondral ossification, emerging from the cortices, and intramembranous were found simultaneously in both fixator groups. Some parameters OS/BS, O.Th, MAR, BFR were significantly higher in the regenerate area than in other bone areas for both groups, and no difference in histomorphometric parameters existed between the two fixator groups. Percentage of mineralised bone evaluated on microradiographs varied between 9-49 percent for Ilizarov and 18-44 percent for the unilateral fixator group in the regenerate area. The outcome of the two external fixators was similar by x-ray, histology, histomorphometry and microradiographs. In this study period of 7-12 weeks postoperatively the bone formation was enhanced and prevailed in the distraction area. This study demonstrated the utility of the canine distraction osteogenesis experimental model for the investigation of many aspects of this corrective procedure. ALVEOLAR RIDGE RESUMPTION IN COMPLETE DENTURE WEARERS: ONE YEAR STUDY A. Celebic1*, F. Kovacic2, V. Carek1, I. Baucic1, J. Stipetic1, D. Knezovic1 1School of Dental Medicine, University of Zagreb, Croatia 2Dental Medicine Polyclinic, Split, Croatia Residual alveolar ridges show continual resumption after the loss of the natural dentition resulting in reduction of the morphologic face height and a counterclockwise rotation of the mandible. The aim of this study was to analyze the residual ridge resorption (RRR) in different regions of both jaws on teleroentgenograms of 50 complete denture wearers and to correlate such changes with age, sex, number of years edentulous, nighttime wear of the dentures and the surface of denture bearing area. The height of residual ridges was measured on 5 different sites of each jaw on lateral telerontgenographs by using a callibrated grid. The results revealed that all the patients showed significant RRR in one year period (p less than 0.01), 2.5 times bigger in the mandibular bone compared to the maxilla. RRR was bigger in men than in women, in patients who had their last extraction within a period of one year before receiving their dentures than in patients who extracted their teeth earlier, in younger individuals and in patients with bigger denture bearing area (p less than 0.01). Nighttime denture wearing made no significant influence on the rate of RRR. The biggest rate of RRR was recorded at frontal sites of residual ridges compared to the lateral sites. CLINICAL BONE DENSITOMETRIC STUDY ON PATIENTS TREATED WITH ITI IMPLANT SYSTEM D. Knezovic Zlataric1*, I. Filipovic Zore1, T. Svajhler1, A. Celebic2 1School of Dental Medicine, University of Zagreb, Croatia 2Private Dental Office, Zagreb, Croatia The aim of the study was to measure the bone mineral density (BMD) on regions of interest (ROIs) on maxillary alveolar ridges close to the implant and to compare them with BMD ROIs on the opposite sites of the maxilla. In cases if there were other remaining teeth in the maxilla the aim was also to compare BMD of ROIs close to the implants with the ROIs close to the remaining teeth. For this reason periapical radiograms with a 10 steps copper stepwedge attached to each film were made in 10 patients with 23 ITI implants in maxilla. Films were processed together and were digitised. Grey levels of each step were transformed to optical density values and using the third degree polynomial the regression formula was calculated for each image. All the measured values (ROIs) were expressed in actual copper stepwedge thickness equivalents using the calculated regression formula. Comparison of BMD close to the implant in comparison to BMD close to the remaining teeth shows the rate of success of osseointegration. This method may help dental surgeon to assess the success of the osseointegration of the implants and even to assess the quality of bone prior to the treatment. FAILURE OF BULK STRUCTURAL GRAFTS USED FOR ACETABULUM RECONSTRUCTION D. Delimar*, K. Korzinek, H. Klobucar, N. Cicak Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Croatia Background: Bulk structural grafts are routinely used for reconstruction of acetabulum roof during total hip replacement in patients with dysplastic hips. Materials and methods: Retrograde analysis (from the year 1985) was performed on two groups of patients. For the reconstruction of the acetabulum during total hip replacement in one group of patients was used autograft (40 patients) and in another allograft (20 patients). Inclination of the acetabulum cup coverage was measured from the sequential roentgenograms in one year intervals. Original questionnaire was designed and used by four different investigators for the evaluation of graft remodeling and incorporation. Results: Despite the excellent primary position and stability of the grafts used for the reconstruction of the acetabulum roof mid term results show that there is no adequate incorporation and remodeling under load of grafts causing instability and migration of the acetabulum cup. Conclusion: Reconstruction of acetabulum roof with bulk structural grafts is usually performed in younger patients with dysplastic hips. Such reconstructions show poor mid term results urging for early reoperations. RADIOLOGICAL DIAGNOSIS OF FIBROUS DYSPLASIA F. Kovacevic*, M. VukelicMarkovic, L. J. Vojnic, M. Jaksic, B. Brkljacic Clinical Hospital Dubrava, Zagreb, Croatia Fibrous dysplasia is a congenital malformation caused by postzygotic somatic mutation of the gene GNAS1 responsible for Gs-alpha regulatory proteins. The severity of the disorder is dependent on moment when mutation takes place. Its severe form , known as McCune-Albright syndrome, evolves with an early gene mutation. We present three patients, one with McCune-Albright syndrome, and the other two with monostotic form of fibrous dysplasia. The importance of various radiological diagnostic methods is discussed. THE VALUE OF PARATHYROID SONOGRAPHY IN SECONDARY HYPERPARATHYROIDISM H. Tomic-Brzac*, D. Pavlovic University Hospital Zagreb, Sveti Duh General Hospital, Zagreb, Croatia Parathyroid sonography is used in detection of enlarged parathyroid glands (PTG) for more than 20 years. Hyperparathyroidisms, i.e. hypertrophy and hyperplasia of PTG, is a well known complication in uremic patients. During these 20 years the knowledge of parathyroid gland function has improved, as has sonographic equipment. Today we use a 2-D high resolution sonography with a 10 MHz probe and colour Doppler. Up to now, parathyroid sonography was performed in more than 500 patients. In this study we investigated the sonographic changes of PTG in dialysis patients and a possible correlation between PTH level and PTG enlargement. In 120 patients (63 males and 57 females), mean age 45 years (14-79), dialysed for 7.8 (0.5- 22) years, PTG sonography (10 MHz colour Doppler probe) was performed. PTH was determined with Allegro intact PTH kit (normal range 1-6 picomol/l). In 58 (48 percent) patients uniform echoes of PTG was found, in 34 (28 percent) nodular pattern of PTG and in 28 (23 percent) degenerative changes of PTG were detected. By colour Doppler sonography paranodular vascularity was found in 38 percent of PTG, hypervascular capsule in 22 percent , internal vascularity in 28 percent and no vascularity in 12 percent of PTG. The highest PTH level was in patients with nodular hyperplasia and paranodular vascularity of PTG. Suspected nodular changes were more often found in male patients, but nodular changes of PTG were more prominent in female patients with higher level of PTH. In our experience PTH sonography with high-resolution equipment is very useful in detection of PTG hyperplasia, i.e. size and shape of parathyroid glands, and is more important in the management of dialysis patients than localisation of PTG. PARATHYROID CARCINOMA IN DIALYSIS PATIENT D. Pavlovic1*, H. Tomic-Brzac1, B. Sarcevic2, M. Radetic3 1Sveti Duh, General Hospital, Zagreb, Croatia 2University Hospital Zagreb, Zagreb, Croatia 3Univeristy Hospital for Tumors, Zagreb, Croatia Hyperplasia of the parathyroid glands (PTG) and increased secretion of parathyroid hormone (PTH) is an invariable consequence of chronic renal failure. There are two forms of PTG hyperplasia in patients with chronic renal failure: diffuse hyperplasia and nodular, more severe form of hyperplasia. Parathyroid carcinoma is seen very rarely in chronic renal failure patients, i.e. in patients with secondary hyperparathyroidism. We present a dialysis patient with parathyroid and thyroid carcinoma, and adrenal hyperplasia. The clinical investigation was done in 54 years old female patient who is being dialyzed for 12 years. The level of intact PTH was >150 picomol/l and alkaline phosphatase 389 U/L. Ultrasound of the neck (10 MHz probe and colour Doppler) showed nodular changes of the thyroid gland and four enlarged PTG, three suspected on diffuse and one on nodular hyperplasia. Needle aspiration biopsy of the most enlarged PTG was suspected of medullary carcinoma. Therefore additional investigation was performed. Calcitonin concentration was 91.6 picomol/l, and serum catecholamines concentration was increased: 34 microgram/l. CT showed enlarged left adrenal gland. During the first surgery left nephrectomy and adrenalectomy were performed. A month later, total thyroidectomy and parathyroidectomy were done. Carcinoma of one PTG ( suspected nodular hyperplasia by ultrasound) diffuse hyperplasia of three other PTG and follicular carcinoma of the thyroid gland were pathohistologically documented, as well as hyperplasia of the left adrenal gland. Two years after the surgery patient was in a good condition, PTH level was 3.2 picomol/l, and catecholamines level was 3.2 microgram/l. A dialysis patients present a unique group of patients with frequent impairment of endocrine functions. A careful clinical assessment and follow up is indispensable in prevention and treatment of complications in dialysis patients. CALCIPHYLAXIS: A RARE BUT SEVERE COMPLICATION IN DIALYSIS PATIENTS D. Pavlovic*, S. Cala, N. Jankovic Sveti Duh General Hospital, Zagreb, Croatia Calciphylaxis is a rare but serious complication in dialysis patients the pathogenesis of which is poorly understood. Dysregulation of calcium and phosphorus metabolism is thought to be a major pathogenic factor. In six hemodialysis patients (5F/1M), mean age 53 years on hemodialisys for 7.5 years, calciphylaxis was diagnosed. All were affected distally: 3 had leg ulcers, 1 had heel ulcers, 1 toe necrosis and in 1 fingers were affected. In 3 patients severe secondary hyperparathyroidism was present, and in 2 of them ulcers temporarily healed with the decline of PTH during pulse calcitriol therapy. In other 3 patients normal PTH values were detected. Calcium phosphate product over 6 micromol/L was only occasionally seen in 3 patients. No patients was remarkably overweight, but in all 6 patients remarkable loss of body weight (from minus 7 percent to 21 percent) 6-12 months prior or during manifestation of calciphylaxis occurred. In 4 patients non insulin depended diabetes mellitus was first noticed during the same period. Three deaths were caused by acral gangrene and in 1 patient death was cardial. Prevalence of calciphylaxis in our haemodialysis patients is 1.7 percent with predominance in the female gender, younger age and longer haemodialysis treatment. Whether this is the cause or consequence of calciphylaxis remains to be clarified. RENAL OSTEODYSTROPHY IN CROATIA BASED ON BONE HISTOMORPHOMETRY: FIFTEEN YEARS OF EXPERIENCE D. Krpan1*, Z. Lajtman2 1General Hospital Sveti Duh, University Medical School, Zagreb, Croatia 2General Hospital Merkur, University Medical School, Zagreb, Croatia The metabolic bone disease and mineral metabolism disturbances are frequent complications in renal failure, and still a great challenge of the modern medicine. Renal osteodystrophy is the generic term generally used to describe the skeletal complications of renal failure and it encompasses a wide spectrum of bone disorders. Based on the predominant histopathologic patterns, it is often classified as: Predominant Hyperparathyroid Bone Disease, Mixed Uremic Osteodystrophy, Low- Turnover Osteomalacia, Adynamic Uremic Bone Disease, Mild Uremic Bone Disease. But because of the great variability of histopathologic patterns no current classification is completely satisfactory so we could consider the classification of renal osteodystrophy as not final. Since the current therapeutic approach to the renal osteodystrophy is to normalize the defect in bone remodeling, histopathologic pattern is the best basis for the accurate diagnosis and planning the appropriate treatment. Thus, bone biopsy and histomorphometry are the crucial diagnostic procedures in the evaluation of renal osteodystrophy. This work provides a review of renal osteodystrophy in Croatia describing the histomorphometric characteristics of bone in the group of 1000 uremic patients on hemodialysis randomized among 2600 bone biopsies performed in the last fifteen years. PAGET'S DISEASE AS A CAUSE OF SEVERE PARAPLEGIA, WITH COMPLETE RECOVERING AFTER ALENDRONATE THERAPY: CASE REPORT D. Krpan1*, J. Buljan-Culej2, D. Dogan1, K. Orsolic1, Z. Lajtman3 1General Hospital Sveti Duh, University Medical School, Zagreb, Croatia 2Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia 3General Hospital Merkur, University Medical School, Zagreb, Croatia Paget's disease is a localized disorder of bone remodeling. Increased numbers of large osteoclasts initiate the process at affected skeletal sites, and the increase in bone resorption is followed by an increase in new bone formation, altering bone architecture. We report a clinical case of unusual clinical and radiological features of Paget's disease in a 42-year old, previously healthy Caucasian woman who suffered of severe paraplegia. Clinical symptoms appeared suddenly as a parastesia and weakening of the legs and developed to complete paraplegia during next two months. Extended diagnostic evaluation subsequently done, demonstrated serum alkaline phosphatase levels 50% higher then normal (145 mmol/l) gamma globulin values slightly higher and other biochemical parameters in normal range. NMR of the vertebrae showed compression myelopathy of vertebral bodies in region Th XII caused by the proliferation of vertebral body. X-ray of frontal bones, vertebrae and the proximal femur showed hyper mineralization and demineralization zones. Whole-body skeletal scintigraphy was atypical, and thus was no diagnostic asset. Ascendant lumbar myelography showed stop of the contrast flow in the region of Th XII and L I. Diagnostic evaluation including repeatable performed bone biopsy and pathology assessment did not prove neoplastic disease. Since diagnostic evaluation did not explain the cause of the disease, no therapy was applied and the patient was completely immobilized during a year and half, before she came in our clinic, where, transiliacal bone biopsy and histomorphometry has been performed and confirmed the diagnosis of morbus Paget. The patient was treated daily with 10 mg alendronate and 0.25 mcg calcitriol. Six months later the patient was able to stand up and after eight months complete recovery has been achieved. Subsequent analyses indicated: normalization of bone turnover, less osteoplastic foci on x-ray images of vertebrae and pelvis, regression of hyperosification in areas of vertebrae Th XI and Th XII on NMR images and improved mobility of the patient. In conclusion the 8-month therapy with alendronate and calcitriol was effective and successful. ABNORMALITIES OF THE FETAL SKELETON ASSESSED BY THREE-DIMENSIONAL ULTRASOUND A. Kurjak*, S. Kupesic, M. Kos Department of Obstetrics and Gynecology, Sveti Duh Hospital, Medical School University of Zagreb, Croatia Objective: To establish the effectiveness and advantages of three-dimensional ultrasound (3D US) in the evaluation of the abnormal fetal skeleton. Patients and methods: Since 1995 we examined a total of 1840 high-risk pregnant patients between 16 and 38 weeks of gestation examined by conventional two- dimensional (2D) and three-dimensional (3D) ultrasound (Combison 530 D and Voluson 730) manufactured by Kretztechnique, Austria. Results: Comparing 2D and 3D ultrasound we found that 3D ultrasound provided a significant diagnostic gain in 42 out of 58 (72.4 percent) fetuses affected by different forms of skeletal abnormalities. Viewing data in multiplanar image (coronal, frontal and axial planes) detection of 38 cases of skeletal abnormalities was enabled. Volume rendering of the entire volume permitted the continuity or abnormality of the fetal spine to be viewed in a single image. Three-dimensional ultrasound provided tomographic evaluation of the fetal skeleton and enabled us to present transparent images, which was not possible with 2D ultrasound. In each case we compared the prenatal transparent reconstruction of the fetal skeleton with the postnatal radiogram. Real-time 3D ultrasound imaging expanded our knowledge on fetal movements in fetuses affected with skeletal abnormalities. In patients with complex anatomic deformities and limited mobility (N=8), sonographer extracted detailed diagnostic information available using various 3D ultrasound modes. In 9 out of 58 cases limited quality of surface reconstruction aroused from oligohydramnios. Conclusions: Three-dimensional ultrasound can assist in the diagnosis of skeletal abnormalities because it offers the potential to better understand spatial relationships of normal and abnormal fetal anatomy. Improved visualization of fetal features has improved the skeletal anomaly identification and has been immediately appealing for clearly sharing development information with the family. Networked imaging allows problematic cases or cases discovered in remoted areas to be sent for consultation via networked computer imaging to the specialists with vast experience. BONE MARKERS REVEAL DECREASED COLLAGEN TYPE I SYNTHESIS AND CHANGE IN BONE TURNOVER DURING BISPHOSPHONATE TREATMENT IN OSTEOGENESIS IMPERFECTA V. Kusec1*, N. Huzjak2, I. Barisic2, A. Resic2, I. Baric3, D. Anticevic4 1Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 2Department of Pediatrics, Children's University Hospital, Zagreb, Croatia 3Department of Pediatrics, Clinical Hospital Centre, Zagreb, Croatia 4Orthopaedic Clinic, Clinical Hospital Centre, Zagreb, Croatia Continuous action of bone cells, i.e. remodeling of the skeleton, is reflected by measuring the products of their activity in serum or urine. Availability of methods for determination of bone markers has provided some data on bone turnover in osteogenesis imperfecta patients and the utility of bone markers in evaluation of bisphosphonate treatment. It has been demonstrated that procollagen type I propeptide was reduced in children with osteogenesis imperfecta in comparison to controls, but other bone markers were not considerably different. In severely affected adults with osteogenesis imperfecta bone resorption markers were increased, but also both normal and reduced concentrations were found. Treatment with bisphosphonates caused reduction of bone markers. Results obtained in a group of 20 osteogenesis imperfecta children and adults were similar. Levels of procollagen were below or within the reference range for premenopausal women in the majority of patients which were younger than 15 years, although increased concentrations would be expected due to intensive skeletal growth. Decreased procollagen in OI patients probably reflects less collagen type I formation and retarded skeletal growth. Effect of bisphosphonate therapy was observed as decrease in procollagen after commencement and as continuous decrease during one year therapy. Bone resorption in some children younger than 10 years was below the upper limit of reference range for premenopausal women, but increased in older patients. Similar to procollagen results, much higher levels of resorption markers characteristic of intensive growth and turnover would be expected, also suggesting growth impairment. It can be expected that clinical utility of bone markers in osteogenesis imperfecta patients will be validated after more experience is gathered generated by bisphosphonate treatment and biochemical assessment. In comparison to other metabolic bone diseases, a benefit of a choice of specific bone marker and a degree of change in monitoring can be expected. FIBRODYSPLASIA OSSIFICANS PROGRESSIVA: REPORT OF TWO FEMALE PATIENTS D. Anticevic1*, S. Grazio2, L. Bukovac3 1Department of Orthopaedic Surgery, Medical School, Zagreb, Croatia 2Department of Rheumatology, Medical School, Zagreb, Croatia 3Department of Paediatrics, Medical School, Zagreb, Croatia PURPOSE: To present two girls with fibrodysplasia ossificans progressiva (FOP) who represent maybe all patients with that rare disease in 4,5 million country. METHODS: A twelve year old girl present herself in orthopaedic out-patient clinic with typical features of FOP i.e. small great toes, painful soft tissue heterotopic bone formation around the neck and shoulder and scoliotic deformity of the spine. The diagnosis of FOP was established in another institution, previously. She was seeking treatment for her deformed spine. We did not recommend any surgical intervention due to well known fact that surgery will aggravate already existing deformity. She was followed-up for next thirteen years and she was given supportive therapy as well as advice how to avoid more flare-ups and heterotopic bone formations. In a course of the time, her spine become grossly deformed, still she is able to walk with the cane. A special hospital-based medical interdisciplinary board was established to help her in a case of an emergency. Second patient is one-year girl who presented herself with elbow painful swelling to the University Department of Paediatrics. Bilateral hallux valgus was noted but not connected with X-ray revealed calcifications in soft tissues. Paediatrician and orthopaedic surgeon specialist in oncology because of possible neoplasm considered biopsy. Due to experience with first FOP patient, second orthopaedic opinion was given, biopsy was cancelled and correct diagnosis was made. The girl is then followed-up for next five years and only two episodes of painful soft-tissue swelling were encountered. Currently, she is happy and playful child waiting to start school years. SIGNIFICANCE: Two clinical history of this rare and devastating disease (FOP) showed that long-term follow-up and experience with older patient helped to made correct and timely diagnosis in a younger patient. CONCLUSION: Every patient with congenital hallux valgus and ectopic ossification should be considered for diagnosis of FOP. Multidisciplinary approach is essential correct diagnosis and management. RECONSTRUCTION OF OSSEOUS DEFECTS WITH COMPRESSED ALLOGENIC CANCELLOUS BONE FRAGMENTS, AUTOLOGOUS RED MARROW AND BMP-7 IN ANIMAL EXPERIMENT T. Djapic1*, M. Jelic1,3, V. Kusec2, S. Vukicevic3, M. Pecina1 1Department of Orthopedic Surgery, School of Medicine, University of Zagreb, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 3Department of Anatomy, School of Medicine, University of Zagreb, Croatia Compressed cancellous bone is usual form of bone allogenic transplantation in orthopaedics surgery especially to fill a large cavity in tumor or cyst defects or acetabular defect cavity in revision hip surgery. The idea was to improve an osteointegration of compressed allogenic cancellous bone with addition of autologous red marrow or BMP-7. Material and methods: An ulnar segmental-defect model was used to evaluate bone healing in adult male New Zeland White rabbits. In six rabbits a defect was filled with compressed allogenic cancellous bone. In another six rabbits a defect was filled with compressed allogenic cancellous bone with addition of autologous red marrow and in third group with six rabbits a defect was filled with compressed cancellous bone with addition of 300 micrograms of recombinant human BMP-7 ( osteogenic protein -1). In four rabbits that served as a control group defect received no implant. The defects were examined radiographically periodically for two weeks. All animals were sacrificed 10 weeks after operation. Amputated limbs were examined radiographically and histologically. Results: In defects that filled with compressed cancellous bone and autologous red marrow and BMP-7 osteointegration was significant better. Conclusions and clinical relevance: Addition of autologous red marrow and BMP- 7 to compressed allogenic cancellous bone can improved osteointegration. OSTEOGENIC PROTEIN-1 (BMP-7) IN THE TREATMENT OF LONG BONE NON-UNIONS IN THE LOWER EXTREMITY M. Pecina Department of Orthopedic Surgery, School of Medicine, University of Zagreb, Croatia Osteogenic protein-1 was successfully used in treatment of 5 patients in long bone non-unions in the lower extremity. Two patients were treated because of femoral non- union, two because of tibial non-union and one after non-union of radius. In one patient, M.K., 44 years old, non-union of the femur occurred after war injury. After osteosynthesis was performed with condylar plate and autologous cancellous bone grafting, 2 doses of OP-1 were applied. Three months postoperatively bone healing was observed. In another patient, B.S., 32 years old, non-union has occurred after unsuccessful femoral elongation. After removal of Wagner apparatus, osteosynthesis with a condylar plate and screws was performed with addition of osteogenic protein-1 device and an autologous cancellous bone graft. Three months postoperatively bone defect bridging occurred. The next patient, D.S., 83 years old, had a distal tibial shaft non-union developed after osteosynthesis with plate and screws. After intramedullary nailing with OP-1 application, bone healing occurred after 5 months. Another patient, Z.I., 64 years old, had unsuccessfully been operated 14 times during the time period of 12 years due to distal tibial shaft non-union. After application of external fixator by Ilizarov together with OP-1 with resection of fibula, 4.5 months postoperatively bone healing is observed. In another patient, K.V., 73 years old, after two unsuccessful surgical procedures, osteogenic protein-1 device has been applied to the radius during the third procedure of compressive osteosynthesis with plate and screws. Four months postoperatively bone healing was observed. Local application of OP-1 did not induce any local or systemic complications in any of our patients. Application of OP-1 after internal or external fixation has induced bone healing in all our patients treated for non-unions. THE FALL OF PHOSPHORYLATED METABOLITES LEVELS DURING PREGNANCY IS ASSOCIATED WITH A RISE IN ALKALINE PHOSPHATASE IN SUBJECTS WITH HYPOPHOSPHATASIA S. J. Iqbal*, T. Davies, R. Cole, A. Daudia, S. Holland Department of Chemical Pathology, Leicester Royal Infirmary, Leicester, UK Hypophosphatasia (HYPOS) is a rare bone disease characterised by low circulating levels of tissue non-specific alkaline phosphatase (TNSALP) and increased levels of phosphorylated metabolites (PHOS.MET), including serum pyridoxal phosphate (PP), and urine phosphoethanolamine (PE), the natural endogenous substrate for TNSALP. In normal pregnancy, placental alkaline phosphatase (PALP) level rises and is reflected by a raised level of total serum alkaline phosphatase activity (TALP). We measured TALP and the PHOS.MET in two female heterzygote carriers for hypophosphatasia; A (26 yrs) and B (31 yrs) around their pregnancy. TALP was measured by an automated method. AMP buffer, PNPP substrate, 30 deg C (RR 40-130 IU/L) PP by HPLC (RR 12-97 µmol/L) and PE by HPLC (per mmol to urine creatinine RR<10 µmol/mmol Cr). Results before, during 3rd trimester of pregnancy, and one week after delivery, were as follows. In A; TALP was 20, 203, 94 and PP 134, 7, 16. In B; TALP was 13, 46, 19 and PE 19, <5, and 5. The rise in TALP was due to rise in PALP component. The reciprocal changes in TALP and PHOS.MET suggests that PALP can hydrolyse PHOS.MET in HYPOS. CADMIUM AND CALCIUM INTERACTIONS: EXPERIMENTAL DATA M. Piasek*, M. Blanusa, M. M. Saric, K. Kostial Institute for Medical Research and Occupational Health, Zagreb, Croatia Due to increasing environmental pollution with toxic metal cadmium (food, cigarette smoke) in the last decades, potential cadmium effects with regard to the so called 'Itai-Itai Byo' (ouch-ouch disease) have attracted considerable attention. It is a syndrome with extensive bone demineralisation and renal tubular damage identified in Japan after World War II in multiparous women above 45 years of age who were exposed to cadmium-contaminated water and food. Toxic effects of orally administered cadmium on bone metabolism in experimental animals fed on calcium- deficient diets have been frequently reported during seventies. Results of experimental studies in vivo and in vitro started in eighties by Bhattacharyya and co-workers on mice and dogs, support the view that cadmium exposure in conjunction with calcium deficiency and pregnancy/lactation are key etiological factors for the Itai-Itai-like syndrome. Chronic exposure to cadmium has been directly linked to bone loss, low bone mass, and increased incidence of fracture independent of cadmium-induced renal toxicity. Our results in rats have shown that lactating animals present a group with higher susceptibility to cadmium-induced bone mass loss compared to non- pregnant rats. Our results together with other authors' data have provided evidence that cadmium causes increased calcium bone loss in midlactating animals. We also found that adolescent females have increased susceptibility to cadmium-induced reduction of bone mass, especially under condition of lower (0.3%) calcium intake in the diet. We investigated another type of cadmium-calcium interaction in evaluation of the effect(s) of calcium supplementation during suckling period on toxic metal retention. In suckling rats supplemented with calcium (1%, 3% and 6% in cow's milk) and concomitantly exposed to cadmium per os, concentrations of cadmium were significantly decreased in organs and carcass (skeleton). The effect was dose-related. No calcium-effect on tissue cadmium was found in pups exposed to cadmium prior to calcium supplementation. Calcium supplementation per se significantly increased calcium concentration in the carcass. It was concluded that calcium supplementation during the suckling period could be an efficient way of reducing oral cadmium absorption and retention without affecting tissue essential trace elements. Further research on cadmium-calcium interactions is necessary in both humans and experimental models. LONG BONE BOWING IN HUMAN CAMPOMELIC DYSPLASIA RESULTS FROM AN ECTOPIC PRIMARY OSSIFICATION CENTER WHERE CARTILAGE GROWS PERPENDICULAR TO THE AXIS OF CARTILAGE ANLAGEN A. Corsi1,2*, M. Riminucci1,2, M. Roggini3, P. Bianco2 1Department of Experimental Medicine, Laboratory of Pathology, University of L'Aquila, Italy 2Department of Experimental Medicine and Pathology, La Sapienza University, Rome, Italy 3Department of Pediatrics, Section of Radiology, La Sapienza University, Rome, Italy Campomelic dysplasia (CD) is a severe skeletal dysplasia caused by heterozygous mutations of Sox9 in which most of the endochondrally formed bones are affected. Although not pathognomonic of CD, symmetric and anterior congenital bowing of the long bones (CBLB, campomelia) of the lower limbs, especially the tibiae, is the most striking skeletal feature. Many mechanisms, including abnormalities in lower limb vascularization, imbalanced muscular development and/or faulty fetal positioning in uterus, have been proposed to explain CBLB. We investigated the radiologic and morphological changes observed in the tibiae of four second trimester fetuses with CBLB. In the younger fetus (15 weeks), the cartilaginous, unossified cartilage anlagen of the tibiae were hypoplastic and markedly bowed, and showed an ectopic center of ossification at the apex of the incurvation. Here, the axis along which chondrocytes matured to hypertrophy was perpendicular to the long axis of the cartilage anlagen, with the equivalent of the proliferative zone at the periosteal aspect, and hypertrophy at the opposite end. In three older fetuses (18, 22, 23 weeks), the tibiae were proportionally different in length and extensively ossified at the diaphysis, but still hypoplastic and similarly bowed; proximal and distal epiphysis and growth plates were recognizable. The incurvation was included into the fully ossified region of the shaft. A complex array of secondary bone trabeculae and vascular channels oriented perpendicularly to the normal longitudinal axis of the bone rudiment was obvious. Our data show that campomelia in humans may result from a primary bending of the cartilage anlagen which precedes their ossification and is associated with the formation of a misaligned and ectopic primary ossification center. Subsequent modeling and remodeling of this aberrant center restores proximal and distal growth plates, but the ortogonal growth momentum in the rudiment generates an abnormal direction of secondary bone formation at the midshaft. It remains to be determined whether these growth plate dysmorphisms that are upstream of the abnormal bone growth observed in CD are directly dictated by Sox9 mutations, or rather reflect the interplay of mechanical, muscular and/or vascular influences on a simple hypoplasia of cartilage anlagen caused by Sox9 haploinsufficiency. BISPHOSPHONATE THERAPY IN PATIENTS WITH OSTEOGENESIS IMPERFECTA N. Huzjak1*, I. Barisic1, A. Resic1, V. Kusec2, D. Anticevic2, D. Dodig2, D. Primorac3 1Children's Hospital, Zagreb, Croatia 2University Medical Center, Zagreb, Croatia 3University Medical Center, Split, Croatia Background: Severe osteogenesis imperfecta (OI) is a hereditary disorder characterised by increased bone fragility and progressive bone deformity. Secondary osteoporosis is an important feature of OI. So far, no effective medical treatment is available. Antiresorptive activity of the aminobisphosphonates may improve clinical outcome in children. Aim: to assess the clinical impact of the administration of bisphosphonates in Croatian OI patients. Methods: We introduced therapy in 1998 encouraged by parents from Croatian society of OI (HUOI). Here we report results of 1-3 years treatment with intravenous pamidronate (APD) in seven children with severe OI, 4 female and 3 male with age distribution ranging from 3 months to 11 years at the entry. Pamidronate was administered either in the form of monthly infusions at a daily dose of 1- 1.5 mg/kg during a period of 6 months followed by a pause of three months, or in the form of three daily infusions every four months, the administered dose being the same. All patients received 500-1000 mg of calcium and 1000 IU of vitamin D daily. Results: During treatment, DEXA measurements showed a gradual increase of bone density. Number of confirmed fractures decreased in all patients. The reduction in pain as well as improvement in well-being and ability were impressive in two male patients who had been confined to a wheelchair, but now they are able to walk using crutches. Well-known acute phase reactions were noted in two children during first infusion cycle and asymptomatic hypocalcemia was noted in three children. During the treatment body mass index of three children was significantly increased. Conclusion: Although the bisphosphonates do not correct basic abnormalities in OI, they significantly alter the natural course of the disease and improve patients' quality of life. For the time being they seem not only effective but also devoid of any adverse effects on bone growth and remodelling. CO-EXISTENCE OF OSTEOGENESIS IMPERFECTA AND KLINEFELTER SYNDROME I. Atanasova*, A. M. Borisova, B. Lozanov, N. Sestrimska, P. Kumanov, R. Ivanova, L. Diankov Clinical Centre of Endocrinology and Gerontology, Medical University, Sofia, Bulgaria Osteogenesis imperfecta (OI) and Klinefelter syndrome (KS) are separate genetically determined disorders affecting the skeletal system. OI encompasses phenotypically and genotypically a heterogeneous group of autosomal, dominantly inherited diseases due to mutation(s) in the genes encoding procollagen-I with a great variability of gene expression which suggests the role of yet not identified factors segregating independently that act to modulate the final phenotype. The X chromosome includes genes that are known to determine the skeletone structure. Impaired bone mineral density (BMD) and osteoporosis are common in hypogonadotropic hypogonadism with extra X chromosome (KS). We report a case in which both OI and KS are present. Our male proband with OI type I presented with 48 bone fractures since 2 years of age, blue sclerae, hypermobile joints, dentinogenesis imperfecta, but normal hearing, normal height-175 cm, eunoichoidal proportions of the skeleton without deformities. His family is affected with OI type I, mild form and typical autosomal dominant mode of inheritance. The combination of both diseases in our patient can explain the difference in OI phenotype in comparison with other affected family members and suggests the probable role of genes in gonosomes in the phenotype variability of OI. Our case also requires a special treatment approach including biphosphonate and testosterone substitution which improved the clinical course of both diseases. The occasional combination of OI with KS in our patient provokes a set of pathophysiologic, diagnostic and therapeutic questions. TREATMENT OF IDIOPATHIC OSTEOPOROSIS IN MALE PATIENTS USING ALENDRONATE M. Cokolic1, R. Hren2* 1Teaching Hospital Maribor, Maribor, Slovenia 2University of Ljubljana, Ljubljana, Slovenia In men with osteoporosis, but no obvious cause, the treatment choice remains to be clearly established. The aim of this study was to assess the effectiveness of alendronate therapy in treatment of male patients with idiopathic osteoporosis. Ten men with idiopathic osteoporosis (T-score at lumbar spine or hip < -2.5 SD, serum concentrations of free testosterone, Ca, P, Mg, and alkaline phosphatase (ALP) within normal limits) were enrolled in a prospective study between March 1997 and September 2000. Patients were 48 to 65 years old (mean: 59 years). They were treated with alendronate sodium (10 mg/d) in combination with 500-mg elemental calcium. The BMD in the lumbar spine (L2 - L4) and left hip was measured in all patients using dual energy X-ray densitometry (Hologic QDR2000+) at the start of the treatment and at 12 to 48 months after initiation of the treatment. The serum levels of Hgb, Hct, WBC, Plt, testosterone, Ca, P, Mg, Na, K, Cl, ALP, AST, ALT, BUN, and creatinine were measured every 12 months. Average baseline BMD was 0.762 g/cm2 (n=10, range 0.686 to 0.816 g/cm2) at the lumbar spine and 0.721 g/ cm2 (n=10, range 0.697 to 0.742 g/cm2) at the hip. After treating patients on average for 28 months, the average BMD was 0.808 g/cm2 (n=10, range 0.729 to 0.888 g/cm2) at the lumbar spine and 0.775 g/ cm2 (n=10, range 0.751 to 0.795 g/cm2) at the hip. BMD thus increased on average by 6% at the lumbar spine and 7.5% at the hip. Serum levels remained within normal limits throughout the treatment, with no adverse events observed during the study. Results of our on-going study suggest that alendronate sodium can provide clinically relevant benefits in male patients with idiopathic osteoporosis. TREATMENT INTERVENTIONS IN POSTMENOPAUSAL WOMEN WITH OSTEOPOROSIS IN SLOVENIA R. Hren1*, M. Cokolic2 1University of Ljubljana, Ljubljana, Slovenia 2Teaching Hospital Maribor, Maribor, Slovenia Balancing the treatment goals of the physician with those of the patient has become an important focus in the clinical management of osteoporosis. It is important that clinical measures of therapeutic management (such as, bone mineral density increase, fracture reduction) are correlated with the treatment choice. In this study, we examined the treatment status of postmenopausal women with osteoporosis in Slovenia. Women who participated in the study were required to have been previously diagnosed with osteoporosis. In the survey, we collected the fracture status, age, body mass index, lumbar spine (L2-L4) bone mineral density, and treatment choice. The survey was conducted at 10 DEXA centers. We prospectively enrolled 945 women with postmenopausal osteoporosis, with a mean age (±SD) of 64 (±9) years, mean body mass index of 26 (±4), and mean T-score at lumbar spine of -3.3 (±0.8). The fracture status of the study group was as follows: 18% of women suffered the wrist fracture, 3% the hip fracture, 18% had some degree of kyphosis, and 44% experienced the height loss >3 cm. Women in the study were treated with alendronate sodium (71%), calcitonin or hormone replacement therapy or etidronate sodium (10%), and calcium and vitamin D (5%); 14% were not treated. Results of our survey suggest that in Slovenia, more than 70% of postmenopausal women diagnosed with osteoporosis are treated with bisphosphonate alendronate. REPEATED INGESTION OF CALCIUM-FORTIFIED WATER DECREASES SERUM CONCENTRATIONS OF NTX BUT NOT OF ICTP J. Guillemant1, C. Accarie2, V. de la Guéronnière3, S. Guillemant1,2* 1Faculté de Médecine Pitié-Salpêtrière, Paris, France 2EPHE, Nutrition Hydrominérale, Paris, France 3Pôle Expertise Eau, Bourg-la-Reine, France In a previous study (Am J Clin Nutr 2000) we showed that a single oral intake of calcium-rich water could significantly increase the serum concentration of ionized calcium (iCa) and suppress serum concentrations of CTX for 4 hours demonstrating that calcium-rich mineral water could be considered as an efficient source of dietary calcium to prevent osteoporosis. Until now 3 immunoassays detecting N- or C- terminal telopeptides fragments of typeI collagen in serum are available. Since it has been suggested that these fragments could be generated through distinct collagenolytic pathways we decided to compare the responses of serum NTX and ICTP to the ingestion of calcium-fortified water. Ten young adult males (21-26 y) ingested at three times (at 08.00, 11.00 and 14.00) 660 ml of either calcium-fortified (as bicarbonate, chloride and sulfate) spring water (300 mg/L of elemental calcium) or calcium-poor (<10 mg/L) mineral water. Blood was collected at 08.00, 11.00, 14.00 and 17.00, referred to as P0, P3h, P6h and P9h, immediately before every intake of water for measurement of iCa, NTX and ICTP. Oral intake of calcium-fortified water resulted in a progessive increase in serum iCa (from 1.252 to 1.319 mmol/L) and decrease in serum NTX (by 19.3% at P3h, 24.7% at P6h and 23.1% at P9h) while serum ICTP concentrations were not affected. Since ingestion of calcium-poor mineral water induced a modest (-10.6%) but significant (P<0.01) decrease in NTX we compared the two sets of assays with repeated-measures two-factor analysis of variance with interaction. Ingestion of calcium-fortified water resulted in a significant decrease in serum NTX (time, P<0.0001; treatment, P=0.006; time-by-treatment, P=0.09). The present findings show that NTX and ICTP are not equivalent as indices of the osteoclastic response to the intake of calcium. THE INFLUENCE OF DEHYDROEPIANDOSTERONE THERAPY ON THE SKELETAL METABOLISM OF MICE WITH SYSTEMIC LUPUS ERYTHEMATOSUS D. Schapira1*, A. Weiss2, A. Kabala2, Z. Blumenfeld3 1The B. Shine Department of Rheumatology, Rambam Medical Center, Haifa, Israel 2Laboratory for Musculoskeletal Research, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel 3Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, Israel Introduction: Systemic lupus erythematosus (SLE) is an inflammatory disease with multisystemic damage. Osteoporosis occurs in patients with SLE. The etiology is multifactorial and includes prolonged corticosteroid treatment, the effect of immunological factors on bone resorption, relative immobility, amenorrhea, avoidance of sunlight, renal failure, etc. Laboratory mice are satisfactory models for human osteoporosis. In previous studies we have shown that MRL mice (inbred mice which spontaneously develop SLE) may serve as a model for the skeletal metabolism in SLE. Dehydroepiandosterone (DHEA) is an adrenal steroid with androgenic activity. Some studies have shown a beneficial effect of DHEA on disease activity in SLE and on bone density. Aim of the study: To elucidate the influence of prolonged DHEA treatment on the skeletal metabolism of SLE mice. Materials and Methods: MRL mice were divided into two groups: controls (fed with routine rodent chow) and treated animals (fed with DHEA enriched food) and were followed from 1.5 to 6 months of life. Measurements at 1.5, 3, 4.5 and 6 months included: animal and femoral body weights, femoral bone biochemistry (calcium, phosphorus, magnesium, protein); alkaline and acid phosphatase enzymatic activities, bone histology and histomorphometry (femoral head and neck). Results: No intergroup differences in the animal and femoral body weights and in the bone mineral and protein content; similar alkaline phosphatase enzymatic activity yet, reduced (-29%) acid phosphatase and higher (+41%) alkaline/acid phosphatase ratio in treated animals; minimal age-related decreases in the trabecular bone volume in the treated group as compared to losses of up to 31% in controls, resulting at the end of the trial in higher values (+37.6% femoral head; +36% femoral neck) in the DHEA fed mice. Conclusions: DHEA increases skeletal mass in SLE mice but does not seem to influence the bone mineral content. Further studies using combinations of different doses of DHEA and calcium and vitamin D are required. EFFECT OF DUPLICATE URINE COLLECTION ON RESPONSE RATE TO RISEDRONATE: DATA FROM THE IMPACT STUDY R. Eastell1*, R. A. Hannon1, P. Garnero2, P. D. Delmas2 1Clinical Sciences Center, Northern General Hospital, Sheffield, UK 2University Claude Bernard, Lyon, France Risedronate (RIS) treatment produces decreases in the levels of biochemical markers of bone resorption, with maximal effects observed at about 3 months. The effect of treatment in individuals can be monitored by using the least significant change (LSC) in the biochemical markers. We compared the percentage of RIS- treated women in the IMPACT study who were responders (i.e., whose change in marker level exceeded the LSC) based on the mean of duplicate measurements of urinary N-telopeptide of type I collagen (NTX) with the percentage of responders based on single measurements. The IMPACT study included 2386 women with osteoporosis (T-score at -2.5 or lower at the spine or hip or a T-score between -2.5 and -1 with a peripheral fragility fracture). This preliminary report is based on 1030 women who had urinary NTX measurements at baseline, 3 months, and 6 months, using the Ortho Clinical Vitros analyzer. At each time point, 2 samples were taken 1 day apart (second morning void). The LSC for double measurements was set at 30percent (equivalent to a P-value of 0.1); the LSC for single measurements was 42 per cent (30 times 1.414). With double measurements, the response rates at 3 and 6 months were 70 per cent and 72 per cent, respectively. With single measurements, the rates at 3 and 6 months were 55 per cent and 61 per cent, respectively. These response rates are conservative as the subjects were treated with calcium supplements (1000 mg Ca/400 IU Vitamin D daily) for 2 to 4 weeks prior to obtaining the baseline urine samples. Also, no adjustments were made with regard to compliance and persistence of the subjects while on treatment. We conclude that the reduction in variability achieved by taking the mean of two measurements results in a substantial improvement in the ability to identify responders to RIS treatment. IN VIVO EFFECTS OF PDE-IV INHIBITORS ON MURINE BONE M. Kometani1, K. Toriyama1, M. Kneissel2, L. Widler2, M. Glatt2* 1Novartis Pharma Research, Tsukuba, Japan 2Novartis Pharma Research, Basel, Switzerland Introduction: Inhibitors of phosphodiesterase type 4 (PDE-4) have been shown in vitro to increase the number of mineralized bone nodules and to reduce the number of TRAP-positive multinuclated cells in rat bone marrow cultures [1]. We have tested rolipram and XT-44, two selective PDE-4 inhibitors, for their potential positive effects on bone of intact mice and ovariectomized rats with established osteopenia. Methods: 12-week-old female ICR mice were once daily treated with XT-44 (1, 3, or 10 mg/kg, p.o.) or rolipram (5, 10 or 20 mg/kg, i.m.) for 8 weeks (5 d/w). At the end of the experiments, mice were killed, tibiae and 5th vertebrae were isolated for DEXA analysis with a Piximus apparatus (Lunar) or compression testing with a mechanical strength test apparatus (TK-252, Unicom, Tokyo). 6-month-old female Wistar rats were ovariectomized (OVX). 3 months later treatment was initiated: the animals received XT-44 (1, 3, or 10 mg/kg, p.o. on alternating days) or rat PTH (1-34) (5 microg/kg, s.c. once daily) for two months. Changes in bone mass and geometry were monitored by DEXA and pQCT. At necropsy lumbar vertebra 5 was excised and subjected to mechanical loading. Results: In intact mice, rolipram or XT-44 did not show significant and dose- dependent, positive effects on bone mineral density or bone mineral content. The maximum compression load determined on LV5 was not changed either. In OVX rats, DEXA did not reveal a positive effects of XT-44 treatment, whereas a low dose of rat PTH (1-34) significantly enhanced BMD. pQCT analysis of the proximal tibia metaphysis showed a marginal positive effect of XT-44 on cancellous BMD. In comparison, rat PTH (1-34) significantly increased total and cancellous BMD and cortical thickness. Compression testing of LV5 showed no effects of XT-44 treatment on maximum load, however rat PTH (1-34) exerted a significant positive effect. Conclusion: The selective PDE-4 inhibitors rolipram and XT-44 do not increase bone mass in intact mice. In osteopenic, estrogen-depleted rats, both compounds are ineffective in restoring tibial bone mass and lost mechanical load resistance in vertebrae. [1] Waki et al., Jpn J Pharmacol 79:477 (1999) THE EFFECT OF HORMONE REPLACEMENT THERAPY OR ALENDRONATE TREATMENT ON MARKERS OF BONE TURNOVER AND BONE MINERAL DENSITY IN POSTMENOPAUSAL WOMEN J. Jelcic1*, V. Kusec2, M. Kifer1, M. Korsic1 1Department of Endocrinology, Internal Medicine Clinic, Clinical Hospital Centre, Zagreb, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia Hormone replacement therapy (HRT) and alendronate are well established antiresorptive agents used for prevention and therapy of osteoporosis. In this study data for 287 postmenopausal women (132 osteopenic and 155 osteoporotic) were analysed. Bone mineral density (BMD) was measured for diagnostic and follow-up purposes by DEXA (dual-energy x-ray absorptiometry). Bone turnover was estimated by measuring serum osteocalcin, procollagen type-1 propeptide, telopeptide of collagen type I and urine telopeptide, free deoxypyridinoline and pyridinoline. HRT received 48 percent of osteopenic and 22 percent of osteoporotic patients, and alendronate received 36 percent of osteopenic and 65 percent of osteoporotic patients. Patients with established osteoporosis were older (66±7.9 yr) than those with osteopenia (59.8±8.7 yr). Repeated BMD showed an average increase of 2.18 percent for both the lumbar spine and the hip. The results did not differ significantly with respect to age, condition (osteopenia or osteoporosis) or treatment. BMD change during treatment correlated significantly and positively with the period of follow-up densitometry (4-24 months) only for the lumbar spine. The average concentrations of bone turnover markers before the start of therapy were mostly below the reference range for postmenopausal women. After initiation of either HRT or alendronate bone markers decreased and continued with this trend or remained decreased. Telopeptide measured in urine show the highest decrease. The telopeptide values were also significantly higher in osteoporotic than in osteopenic patients prior to treatment, and the decrement after therapy was also significantly greater in osteoporotic (75 percent) than in osteopenic patients (26 percent). These results showed beneficial effect of HRT and alendronate therapy on bone density, and a more pronounced effect on markers of bone turnover in osteopenic and osteoporotic patients. Bone marker(s) with the greatest decrement after therapy should be recommended as a rational course of treatment monitoring. Monitoring treatment by densitometry should be performed at intervals greater than 1 year to enable observation of clinically significant changes. TOTAL SERUM CALCIUM IN PERIODONTITIS-AFFECTED PATIENTS D. Plancak1*, A. Bosnjak1, K. Jorgic-Srdjak1, D. Bozic1, B. Vizner2 1Department of Periodontology, School of Dentistry, University of Zagreb, Croatia 2Department of Endocrinology, School of Medicine, University of Zagreb, Croatia Periodontal disease is characterized by resorption of the alveolar bone and loss of attachment, which eventually leads to tooth loss. Although it is not actually evident to which extent calcium is related to periodontal disease, if skeletal bone mass is related to mineral bone density and periodontal disease, it is plausible that calcium concentration, as it affects alveolar bone mineral density, will affect periodontal disease. In order to establish a relationship between periodontal disease and serum calcium levels we performed laboratory tests on serum and total serum calcium in a randomly collected sample. The total of 77 subjects was collected randomly from patients visiting the Department of Periodontology of the School of Dentistry in Zagreb. Patients were divided in two groups according to the clinical diagnostic procedures (mean attachment loss, gingival bleeding) and radiographic status which determined the extent and severity of the periodontal disease. All total serum analyses were performed on venous blood serum samples collected at the Laboratory for Endocrinology of the Clinical Hospital 'Sestre Milosrdnice' in Zagreb, together with the antropometric variables. The results show significantly lower serum calcium concentration in subjects with greater attachment loss and more gingival bleeding (p<0.1 percent). There is also a lower serum calcium concentration in subjects older than 35 years of age and males. The study demonstrates that there might be a correlation between total serum calcium levels and more severe periodontal disease. However, this study did not cover the dietary calcium uptake, and further work should be directed to correlating the level of serum calcium with the dietary uptake and possible dysfunctions of its resorption in the gastrointestinal system. ACETABULAR FRACTURES IN THE ELDERLY PATIENTS WITH OSTEOPENIA D. Pericic*, D. Djurdjevic, D. Hudetz Traumatology Hospital, Zagreb, Croatia Having the opportunity of treating elderly patients with acetabular fractures, we tried to evaluate risk, efficiency and complications of open reduction and internal fixation. In the period between 1994 and 1998 we performed open reduction and internal fixation in 42 patients. Age of the patients was between 60 and 82 years with mean age of 69 years. We treated 31 men and 11 women who all suffered from a certain degree of osteopenia. 20 patients out of 42 had a traffic accident and 22 patients have fractured their acetabulum after falling. Preoperatively we controlled pain with continuous epidural analgesia, performed X-ray and CT evaluation, assessed degree of osteopenia and performed antithrombotic and antibiotic prophylaxis. Postoperatively all the patients were soon mobilized and continued stationary rehabilitation. As far as complications are concerned we noted pulmonary embolism, bleeding from the GI tract, sciatic nerve lesion, infection, loosening of the implanted material, aseptic necrosis of the femoral head and heterotopic bone ossification. Primary open reduction is preferable to total hip replacement due to difficulties in reconstruction of the bone stock and insertion of the prosthesis. The decision to perform primary open reduction should be based on medical status of the patient, degree of osteopenia, fracture pattern and experience of the operating team. STATINS DECREASE BONE TURNOVER IN POSTMENOPAUSAL WOMEN: A CROSS-SECTIONAL STUDY L. Rejnmark1,2*, N. H. Buus2, P. Vestergaard1, F. Andreasen2, M. L. Larsen3, L. Mosekilde1 1Department of Endocrinology and Metabolism C, Aarhus Amtssygehus, Aarhus University Hospital, Denmark 2Department of Clinical Pharmacology, Aarhus University, Denmark 3Department of Medicine and Cardiology, Aarhus Amtssygehus, Aarhus University Hospital, Denmark Background: Statins have been suggested as potential agents in the management of osteoporosis. Reviews of medical records have shown an increased bone mass and some studies have shown a reduced occurrence of fractures in subjects on long term treatment with statins. We studied the effects of treatment with statins on calcium homeostasis, bone turnover, and bone mineral density. Design: In a cross-sectional design, plasma levels of parathyroid hormone (PTH) and biochemical bone markers, bone mineral density (BMD), and body composition (fat- and lean tissue-mass) were measured in 140 postmenopausal women who had been treated with a statin for more than two years (median 4 years) and compared to 140 age- and gender-matched population based controls. Results: Plasma levels of biochemical bone markers were lower in the statin treated subjects than in the controls: osteocalcin (-9%, p=0.03), bone-specific alkaline phosphatase (-14%, p<0.01), and C-terminal telopeptide of type I collagen (-11%, p<0.01). On the other hand, plasma PTH levels were 16% higher in the statin treated subjects than in the controls (p<0.01). However, body composition and BMD at the lumbar spine, hip, forearm, and whole body did not differ between the two groups. No correlation could be demonstrated between changes in biochemical quantities and dose or duration of statin use. Conclusion: Our data show that statins affect the function of bone cells. Most likely, the effect is antiresorptive. IBANDRONATE: SERUM KINETICS, TISSUE DISTRIBUTION AND BINDING TO BONE FOLLOWING INTRAVENOUS BOLUS INJECTION F. Bauss1,2*, R. Endele1, E. Besenfelder1, J-P. Hoelck1 1Roche Diagnostics GmbH, Pharma Research, D-82372 Penzberg, Germany 2Institute of Pharmacology & Toxicology, Heidelberg University, D-68169 Mannheim, Germany Bisphosphonates (BPs) when given intravenously are traditionally administered by slow infusion. Ibandronate is a highly potent, nitrogen-containing BP that can be administered by i.v. bolus injection in clinical trials, a convenient alternative to infusion. Here, a series of studies was conducted in rats to characterise the pharmacokinetics of ibandronate using this approach. Male and female Sprague-Dawley rats were administered 14C-ibandronate 0.1mg/kg as a single i.v. bolus injection. Radioactivity was analysed in serum, urine, whole animal sections (autoradiography), soft tissue and bone. As ibandronate is not metabolised, radioactivity accounts for the intact drug. Ibandronate was rapidly cleared from plasma and eliminated predominantly by renal excretion. Total body clearance was 13.7ml/min/kg, renal clearance for the entire collection period (0-96 hours) was 3.25ml/min/kg, terminal serum half-life was 7.1 hours and phase of distribution was <2 hours. As demonstrated by serial whole-body autoradiography after 24-96 hours, ibandronate deposits predominantly in bone, with negligible amounts in kidney, liver and spleen. When analysed for detailed tissue distribution, approximately 40-50% of the dose was found in bone, predominantly in trabecular bone, with <2% in all non-calcified tissues, even after just 2 hours. Following long- term distribution analysis up to 365 days after dosing, approximately 18% of the administered 14C-ibandronate was found in total. Detailed analyses of remnants (% of dose) and elimination half-life (days) after 1 year showed deposition (in order of decreasing concentration) in carcass (16.1%, 380 days), femoral metaphyses (1.3%, 440 days), femoral diaphyses (0.9%, 500 days), kidney (0.005%, 24 days), liver (bql, 22 days) and spleen (0.01%, 77 days). Thus, as with other bisphosphonates, ibandronate is rapidly cleared from serum, with renal elimination being the predominant route, and binds selectively to bone, where it has a long elimination half-life. The potency and high concentration of ibandronate in bone are consistent with recent data supporting its long-term activity in preventing bone loss and maintaining bone quality in aged ovariectomised rats with treatment intervals of up to 6 weeks. The clinical role of ibandronate i.v. bolus injections with extended drug-free intervals is currently being defined in postmenopausal osteoporosis. STRONGER EFFECT OF ALENDRONATE THAN ETIDRONATE ON LUMBAR SPINE BMD GAIN AFTER ONE YEAR OF TREATMENT D. Kastelan*, Z. Giljevic, V. Plavsic, I. Aganovic, M. Korsic Division of Endocrinology, Department of Internal Medicine, Clinical Hospital Centre Zagreb, Croatia Background: Bisphosphonates are powerful inhibitors of osteoclasts induced bone tissue resorption. Their chemical structure is characterised by two P-C bonds. The purpose of this study was to asses and compare efficacy of alendronate and etidronate in the treatment of osteoporosis. Methods: 146 women with osteopenia/osteoporosis were divided into two groups. Group 1 (n=91, mean age = 63.9±1.1, lumbar spine BMD = 0.726±0.01, Tsc. = - 2.9±0.1, total hip BMD = 0.726±0.01, Tsc. = -2.9±0.1) received alendronate 10 mg daily and group 2 (n=55, mean age = 64.3±1.0, lumbar spine BMD = 0.758±0.02, Tsc. = -2.5±0.2, total hip BMD = 0.786±0.02, Tsc. = -1.4±0.2) received etidronate 400 mg for 14 days in three months intervals. All patients received calcium and vitamin D at a dose depending on a level of urinary calcium. Bone mineral density was assessed using a dual X-ray absorptiometry at baseline and after 12 and 24 months. Student's T-test was used for data analysis. Results: After one year of treatment the mean percent of BMD increase in alendronate group was 7.4±1.4 percent in lumbar spine (p<0.00001) and 4.8±1.2 percent in total hip (p<0.00001). In etidronate group BMD increased 3±0.9 percent in lumbar spine (p<0.01) and 3±1.1 percent in total hip (p<0.005). Average lumbar spine BMD gain induced by alendronate was significantly higher comparing to etidronate (p<0.01). After 2 years of therapy, alendronate induced 9.6±2.1 percent increase of lumbar spine BMD and 11±4.5 percent increase of total hip BMD while in etidronate group 5.4±1.5 percent increase in lumbar spine BMD and 4.6±3.1 percent increase in total hip BMD was observed. Differences between groups were not statistically significant. Conclusion: Study confirmed both agents to be effective in treatment of osteoporosis with significantly stronger effect of alendronate in lumbar spine after one year of treatment. After 2 years of treatment although BMD gain in alendronate group was higher in all measured area the differences between groups were not statistically significant, probably because of low number of women being treated for two years. INHIBITION OF THE OSTEOBLASTIC MATRIX METALLOPROTEASE ACTIVITY INDUCES INCREASED BONE MASS IN VIVO V. Geoffroy1*, N. Legoupil1, P. Clément-Lacroix2, C. Morieux1, S. Roux1, I. Terraz3, J. Rossert3, M-C. de Vernejoul1 1INSERM U349, Hôpital Lariboisière, Paris, France 2Aventis Pharma, Romainville, France 3INSERM U489, Hôpital Tenon, Paris, France Matrix metalloproteases (MMPs) play an essential role in the physiological process of matrix remodelling especially in bone. Recently, osteoblastic MMPs have been suggested to be involved in the osteoclastic process of bone resorption as coupling agents and therefore to be regulators of bone mass. Mice overexpressing the Tissue Inhibitor of Metalloprotease 1 (TIMP-1) under control of the osteoblast specific elements of the collagen type 1 promoter were generated and analysed. Transgenic mice overexpressed TIMP-1 at physiological level, specifically in the osteoblasts. Bone mineral density (BMD) at the total body, femur and caudal vertebrae was measured by Dual-energy X-ray absorptiometry (DEXA) using a Piximus (Lunar) in 1-, 2.5- and 4-month-old mice. BMD was identical in males at any site and at any time. By contrast, in females, BMD was higher in 1-month-old transgenic mice compared to wild type's, at the caudal vertebrae, a site rich in trabecular bone. We therefore analysed more in detail 1-month-old transgenic and wild type mice. Peripheral quantitative computed tomography (pQCT) showed that in the tibia of females transgenic mice, total and trabecular BMD were increased whereas cortical bone was unaffected. In addition, micro computed tomography (microCT) and histomorphometry showed that trabecular bone volume was increased in tibia and trabecular separation, a hallmark of osteoclastic bone resorption, was markedly decreased. These results favour the hypothesis that osteoblastic metalloproteases are required for resorption of trabecular bone in vivo. The reason why the phenotype was present only in females is not yet understood. CP-533,536, A NOVEL NON-PROSTANOID EP2 RECEPTOR SELECTIVE PROSTAGLANDIN E2 (PGE2) AGONIST, STIMULATES LOCAL NEW BONE FORMATION IN RATS H. Z. Ke*, H. Qi, D. T. Crawford, M. Li, V. M. Paralkar, T. A. Owen, L. C. Pan, K. O. Cameron, B. A. Lefker, B. Lu, W. A. Grasser, L. J. Yu, P. DaSilva-Jardine, D. D. Thompson Pfizer Global Research and Development, Groton Labs., Groton, Connecticut, USA PGE2 significantly increases bone mass and bone strength when administered systemically or locally to the skeleton. PGE2 binds to all four receptor subtypes, EP1- EP4. We have found that the EP2 receptor subtype plays an important role in the local bone anabolic activity of PGE2. CP-533,536 is a newly discovered, non-prostanoid EP2 receptor selective agonist. CP-533,536 binds selectively to the EP2 receptor with an IC50 of 11 nM (binding IC50 is greater than 2800 nM for EP1, EP3, and EP4). Similarly, CP-533,536 was selective as an EP2 receptor agonist when measured against other prostanoid receptors including the prostaglandin D2 (DP), prostaglandin F2a (FP), prostacyclin (IP), and thromboxane receptors (TP) in the prostanoid receptor family. CP-533,536 stimulated the EP2 receptor to initiate signaling by the cAMP pathway with an EC50 value of 5 nM. To determine the local anabolic effects of CP-533,536, we locally injected this compound into the bone marrow of the proximal tibial metaphysis in 6-week-old male rats. Single injection was given on day 1 at doses of 0, 0.3, 1 or 3 mg/kg (n=10 per group), and the rats were necropsied on day 7. The injected tibia was analyzed using PQCT, bone histomorphometry and biomechanical testing. The PQCT and histomorphometric analysis showed that CP- 533,536 dose-dependently increased new bone formation in the injected site. Total mineral content and density significantly increased at 1 mg/kg or 3 mg/kg of the CP- 533,536-treated groups as compared with vehicle controls. Area of new bone induction increased significantly by 606% and 1280% at 1 or 3 mg/kg of the CP- 533,536-treated groups as compared with vehicle controls, respectively. The initial maximal load increased significantly by 172% and 181%, respectively, at 1 or 3 mg/kg of the CP-533,536-treated groups as compared with vehicle controls. These results showed that CP-533,536, a novel, non-prostanoid EP2 receptor agonist stimulated local bone formation, increased bone mass and bone strength when delivered locally to bone marrow cavity in rats. These results indicated that EP2 receptor plays an important role in PGE2's local bone anabolism, thus agonizing the EP2 receptor provide therapeutic potentials for local bone augmentation and bone healing. COMPARISON OF THE EFFECT OF ALENDRONATE AND ALENDRONATE WITH HRT ON BONE MINERAL DENSITY Z. Giljevic*, D. Kastelan, M. Kralik, I. Hruskar, M. Korsic Division of Endocrinology, Internal Medicine, Clinical Hospital Centre, Zagreb, Croatia Estrogens and bisphosphonates are well-established antiresorptive agents in treatment of osteoporosis. This study evaluated effect of hormone replacement therapy (HRT) and alendronate combination therapy in comparison to alendronate alone. In the study we administered a combination of alendronate 10 mg daily and HRT to 24 osteoporotic/osteopenic women, mean age 56.3±1.72 years (x±SE), average lumbar spine BMD 0.747±0.03 g/cm 2 (Tsc.-2.6±0.23), average femoral neck BMD 0.622±0.02 g/cm 2 (Tsc.-2.7±0.24) and average total hip BMD 0.743±0.02 g/cm 2 (Tsc.-1.9±0.19). Control group (n=57) with mean age 59.8±1.38 years, average lumbar spine BMD 0.732±0.02 g/cm 2 (Tsc.-2.8±0.15), average femoral neck BMD 0.615±0.01 g/cm 2 (Tsc.-2.7±0.12) and average total hip BMD 0.718±0.02 g/cm 2 (Tsc.-2.2±0.17) received only alendronate 10 mg daily. All patients received calcium in daily dose between 800 and 1500 mg, depending on calcium serum and urinary level. Bone mineral density of lumbar spine, femoral neck and total hip was measured by DEXA, at baseline and after 12 and 24 months of therapy. After 12 months of treatment BMD increased at almost all measured sites in both groups. In alendronate plus HRT group, rate of BMD increase was 4.1 percent in lumbar spine, 0.6 percent in femoral neck and 3.6 percent in total hip. In the control group 6.2 percent increase of BMD was observed in lumbar spine and 3.6 percent in femoral neck. The cumulative change from baseline BMD, after 24 months of follow up, was 7.78 percent in lumbar spine, 1 percent in femoral neck and 6.1 percent in total hip in alendronate plus HRT group and 10.77 percent in lumbar spine, 1.4 percent in femoral neck and 2 percent in total hip in the control group. Combination of alendronate and HRT is an effective therapy for osteoporosis, especially in non-responders to HRT. Effect on total hip bone density was significantly greater in women who received alendronate plus HRT than in women who received alendronate alone. EFFECT OF EGG-SHELL CALCIUM AND COMBINATION THERAPY WITH COLLAGEN ON THE OVARIECTOMIZED RATS K. Svik*, R. Istok, M. Stancikova, P. Masaryk Institute of Rheumatic Diseases, Piestany, Slovak Republic Objective. The effect of egg-shell calcium and combination therapy with collagen type I in preventing OVX-induced bone loss was studied in an animal model of postmenopausal osteoporosis. The aim of the study was to determine the influence of egg-shell as a source of calcium and the additive effect of collagen on ovariectomy induced osteoporosis in the rats. Methods. Adult female Sprague-Dawley rats 250±10 g body weight were subjected to bilateral ovariectomy (OVX) and sham operation (SHAM). Forty animals were divided into four groups: (1) sham-operated controls, (2) OVX controls, (3) OVX rats treated with egg-shell calcium 30 mg/kg, (4) OVX rats treated with combination of collagen type I (0.36 mg/kg, isolated from bovine Achilles tendon) and egg-shell calcium (30 mg/kg). For a period of 9 weeks the control animals were on a calculated diet containing 7.5 g Ca/kg, 6.5 g P/kg and 1000 IU vitamin D3/kg diet of the rats usual dietary requirements. Bone mineral density (BMD) and bone mineral content (BMC) of the whole body and femur was determined using DEXA. Pyridinoline (Pyr), deoxypyridinoline (Dpyr) and creatinine were assessed by HPLC method in the urine. Results. BMD, BMC of the whole body and the femur were markedly decreased in OVX rats in comparison with SHAM controls. These densitometric parameters were significantly increased by egg-shell calcium and even more manifestly in combination therapy - egg-shell calcium plus collagen. The beneficial effect of egg-shell calcium and the combination therapy egg-shell calcium plus collagen was demonstrated also with urinary markers of osteoresorption Pyr and Dpyr. Conclusion. Our data suggests that preventive treatment of ovariectomized rats with egg-shell calcium and combination therapy of egg-shell calcium plus collagen type I markedly inhibit loss of bone mineral density and content as well as increase of biochemical markers of bone resorption Pyr and Dpyr in urine. CANCELLOUS BONE MASS AND COMPRESSIVE STRENGTH ARE INCREASED IN ADULT RATS AFTER STATIN TREATMENT H. Oxlund*, T. T. Andreassen Dept of Connective Tissue Biology, Inst of Anatomy, University of Aarhus, Aarhus, Denmark Statins are widely used as cholesterol-lowering agents. Some of the statins possess bone anabolic properties: statins increased expression of the bone morphogenetic protein-2 gene in osteoblasts resulting in increased bone formation of mice calvariae, increased the cancellous bone volume in 3-month-old rats and induced a minor decrease in osteoclast number (Mundy et al., Science 286, 1946-1949, 1999). Simvastatin increased the femoral periosteal bone apposition rate, but did not change the body weight or femoral length of 12-month old rats (Oxlund, J Bone Miner Res 14(S1), S313, 1999), and increased the vertebral bone mass and strength (Oxlund et al., J Bone Miner Res 15(S1):S549). In the present study the effects of statin on cancellous bone were examined further. Twenty-one female Wistar rats, 12 months old, was given Simvastatin MSD (10 mg/kg/day) or placebo by a gastric tube for 3 months. Two mm high specimens was cut transversely from the distal femoral metaphysis and from the 5th lumbar vertebral body. The specimens were composed of cancellous bone surrounded by a shell of cortical bone which were analysed by histomorphometry and by a micro-CT-scanner. The cancellous bone within the cortical and endocortical shell of each specimen was then submitted to a biomechanical compression test in a materials testing machine between a set of upper and lower platens with diameters corresponding to the diameter of the cancellous bone of each specimen. The cancellous bone volume and compressive stress of the cancellous bone in the distal femoral metaphysis of the statin group (19.1±2.5 % and 21.3±7.5 millinewton/mm2, mean values±SEM) were increased (2p<0.001 and 2p=0.02) compared with the placebo group (10.5±1.6 % and 9.9±3.1 millinewton/mm2). The cancellous bone volume and compressive stress of the vertebral bodies from the statin group (52.7±1.6 % and 31.8±2.7 newton/mm2) was increased (2p<0.001 and 2p<0.04) compared with the placebo group (42.8±1.7 % and 24.1±1.9 newton/mm2). In conclusion, statin given per orally to adult rats increased the cancellous bone mass and compressive strength in both distal femoral metaphyses and vertebral bodies. BONE MORPHOGENETIC PROTEIN-6 INCREASES BONE MINERAL DENSITY OF OVARIECTOMIZED RATS J. Buljan-Culej*, P. Simic, G. Grahovac, A. Grskovic, M. Habek, S. Vukicevic Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia We have discovered that human recombinant bone morphogenetic protein-6 (BMP- 6), given systematically, can completely restore lost bone in aged ovariectomized rats. To determine whether discontinuation of BMP-6 therapy results in rapid bone loss we performed the following set of experiments. SD female rats were ovariectomized at 6 months of age and therapy started 5 months later. The animals were divided into three groups: (1) sham (n=20), (2) ovariectomized (OVX) plus acetate buffer as a vehicle (n=20) and (3) OVX plus BMP-6 (n=40) (3x/week, 10 microg/kg iv). At twelve weeks hind limbs of BMP-6 treated rats reached the values of sham animals confirming that BMP-6 can effectively restore lost bone. We next divided the BMP-6 treated rats into three subgroups as follows: (1) OVX plus BMP-6 (n=15) (3x/week, 10 microg/kg iv), (2) OVX plus 17alpha-estradiol (n=15) (3x/week, 50 plus 50 plus 75 microg/kg sc) and (3) OVX with no therapy (n=10). Bone mineral density (BMD) of total body, lumbar spine and hind limbs was measured 6 weeks later and no significant differences of BMD was found in animals previously treated with BMP-6. However, animals which continued to receive BMP-6 had significantly higher hind limb BMD as compared to sham animals. In conclusion these results suggest that the bone restored in ovariectomized BMP-6 treated rats was not lost after discontinuation of the BMP-6 therapy. BONE MORPHOGENETIC PROTEIN-6 RESTORES LOST BONE IN OVARIECTOMIZED RATS J. Buljan-Culej1*, V. Kusec2, P. Simic1, G. Grahovac1, A. Grskovic1, M. Habek1, K. Sampath3, S. Vukicevic1 1Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia 2Central Laboratory, University Hospital Zagreb, Zagreb, Croatia 3Creative BioMolecules, Hopkinton, USA Since current therapy for osteoporosis is based only on antiresorptive drugs, there is a great need for agents that could restore lost bone and prevent fractures. Good candidates are bone morphogenetic proteins (BMPs), which can, when applied locally, induce formation of new tissues like bone, cartilage and ligament. There is no evidence that a human recombinant BMP, given systematically, can restore bone in an osteoporotic animal model. SD female rats were ovariectomized at 4 months of age and therapy started 12 months later. Bone mineral density (BMD) of total body, lumbar spine and hind limbs was measured at 6 and 12 weeks period. Animals were divided into six groups: (1) sham (n=30), (2) ovariectomized (OVX, n=20), (3) OVX plus 17beta-estradiol (n=20) (3x/week, 50+50+75 microg/kg sc), (4) OVX plus BMP-6 (n=20) (3x/week, 10 microg/kg iv), (5) OVX plus BMP-6 (n=20) (3x/week, 50 microg/kg iv) (6) OVX plus 17beta-estradiol+BMP-6 - 10 microg/kg (n=20). BMD values of hind limbs in BMP-6 rats treated for three months reached the values of sham rats. The effect of the lumbar spine showed less efficacy. 17beta-estradiol alone was significantly less effective at both sites. Ex vivo BMD values of excised femur, tibia and lumbar spine, were similar to the in vivo measurements. Results of pQCT analyses showed an increase in the cortical thickness of BMP-6 treated animals. Since the size of bones was not change the increased cortical thickness in BMP-6 treated rats was the result of endocortical bone formation. Histomorphometry in aged animals showed significant enhancement in bone volume at the sites where at least some bone was present at the beginning of therapy, since 1.7 years old animals did not have any permanent trabecular bone in the mid methaphysis at the beginning of therapy. These results show, for the first time that systemically administered BMP-6 has a significant anabolic effect in osteoporotic rats. INTERMITTENT HPTH ADMINISTRATION PREVENTS THE REDUCTION OF TRABECULAR BONE VOLUME DUE TO SKELETAL UNLOADING S. Tanaka*, A. Sakai, H. Tsurukami, S. Ikeda, S. Uchida, T. Nakamura Dept. of Orthopaedic Surgery, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan To clarify the effects of intermittent hPTH(1-34) administration on trabecular bone turnover and bone marrow cell development in skeletal unloaded limbs, we performed experiments with tail-suspended mice. Eighty C57BL/6J male mice, 8 weeks of age, were assigned to 4 weight-matched groups (Groups 1, 2, 3 and 4; n=20 each). Mice of Groups 3 and 4 were tail suspended. Mice were given subcutaneous injections of hPTH five times a week at the respective dose of 0 (vehicle) for Groups 1 and 3, 40 micro gram /kg body weigh for Groups 2 and 4. Bilateral tibia were harvested at 8 days and 15 days after the start of the experiment. We performed histomorphometric analyses on the proximal metaphyses and bone marrow cell cultures. Bone histiomorphometry: At 8days, the value of trabecular bone volume (BV/TV) and bone formation rate (BFR/BS) of Group 3 was significantly reduced compared to that of Group 1. But, the values of Group 4 was larger than that of Group 3, maintaining at the similar level as that of Group 1. At 15 days, the value of BV/TV of Group 3 was significantly reduced. BV/TV value of Group 4 reduced to the level of Group 3. While BFR/BS values of Group 4 maintained larger compared to Group 3, the value of Oc.N/BS significantly increased compared to the values of Groups 2 and 3. Bone marrow cell: At 8 days. The number of ALP positive CFU-f colonies of Group 3 reduced, and that of Group 2 increased compared to Group 1. In Group 4, the value also increased to Group 1. The number of TRAP positive cell developed from bone marrow cell culture of Group 3 did not differ, and that of Group 2 increased compared to that of Group 1. In Group 4, however, the value further increased compared to that of Group 2. These data clearly indicated that the bone mass increasing effect of intermittent hPTH administration reduced during the unloading of the skeleton. Intermittent hPTH injections increased osteogenic and osteoclastogenic activities in bone marrow cells, consequently leading to bone mass increase in the ground condition. But, during unloading, the increase in osteoclastogenic activity seemed to further increase with passage of time, leading to alleviate the bone mass increasing action of the agent. PREVENTION, EARLY DIAGNOSTIC AND MANAGEMENT OF OSTEOPOROSIS IN PERIMENOPAUSAL WOMAN IN FOUR MUNICIPALITIES OF SARAJEVO CANTON I. Gavrankapetanovic1*, F. Gavrankapetanovic1, E. Kucukalic-Selimovic1, J. Dizdarevic2, D. Ivanisevic1, B. Hadjihasanovic1, I. Hrizar2 1University Clinic Center Sarajevo, Sarajevo, Bosnia and Herzegovina 2Ministry of Health Sarajevo Canton F. BiH, Sarajevo, Bosnia and Herzegovina Goals: Evaluation of menopausal-dependent metabolic disorders in woman, determination of bone density in selected groups, therapeutic measures where is necessary, follow up of treated and borderline cases, development of prevention measures and early diagnostic procedures in prevention of complications and decreasing risk of fractures in topic group. Patients and methods: We are targeting women of four Sarajevo Canton municipalities. Each of them will be announced to visit her gynecologist 3-6 months after spontaneous break of menstruation cycles (age 44-45). With each of them we plan to make a detail introductory conversation, clinical examination, and evaluation of factor of risk; in selected cases - bone densitometry and measuring of bone markers (osteocalcin and telopeptid), scintigrafy if necessary. Densitometry and bone markers will be realized in every case of two or more factors of risk, earlier fractures, and in cases where women can't or don't want to use hormonal therapy even if it is necessary (contraindications, lose of faith etc.). Results (what we expect): Osteoporosis was not problem of great interest in our Canton in last years. Ordinary, it was mainly surgical problem, without cooperation of other specialists. We didn't develop measures of early diagnostics and prevention. We expect bad results in all groups in the beginning of research, and good results with continually improvement in field of understanding and continually struggle in field of prevention of osteoporosis. EFFECT OF CHOLINE STABILIZED ORTHOSILICIC ACID ON BONE DENSITY IN CHICKS. M. R. Calomme1*, P. Wijnen3, J. B. Sindambiwe1, P. Cos1, J. Mertens2, P. Geusens2,4, D. A. Vanden Berghe1 1Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium 2BIOMED, Limburg University Center, Diepenbeek, Belgium 3Poultry Practice De Achterhoek, Ruurlo, The Netherlands 4Department of Rheumatology, University Hospital, Maastricht, The Netherlands Broiler chicks on a normal diet (1.4mg Si/g) were supplemented with choline stabilized orthosilicic acid (Ch-OSA) to investigate the effect of silicon on the serum calcium concentration and bone mineral content (BMC) density (BMD) in the femur. A group of 42,500 chicks was administered Ch-OSA (13.5mg Si/100kg bodyweight/2days) in their drinking water for 6 weeks which increased the total dietary Si intake with less than 0.5%. A control group of 42,600 chicks of the same age was started in parallel with identical feeding but without Ch-OSA supplementation. Samples of 30 randomly chosen chicks were taken in each group at the age of six weeks to analyse the serum calcium concentration and femura. Femoral BMC and BMD were analyzed by Dual Energy X-ray Absorptiometry. Scans were recorded for both total femur and 5 regions of interest in the femur. Differences between means were evaluated with a one-tailed Student t-test. The serum Ca concentration was significantly higher (p<0.05) in supplemented chicks (74.85±13.82mg/ml, n=60) compared to controls (69.47±15.99 mg/ml, n=60). The BMC was significantly higher for supplemented chicks compared to the controls in all the scanned areas of the femur. Total BMC was also significantly higher (+8.4%, p=0.016) for supplemented chicks compared to controls. The BMD was significantly higher at the midshaft (+4.25%, p=0.0209), the distal metaphysis (+4.88%, p=0.0102), and the hip region(+5.6%, p=0.014) for supplemented chicks compared to controls. In conclusion, increasing the total dietary intake of broiler chicks with less than 0.5% in the form of Ch-OSA resulted in a significant higher serum calcium concentration and higher bone mass and density in cortical and trabecular bone of the femur. These results confirm earlier studies suggesting an essential role of silicon in bone (1) and collagen (2) metabolism. (1) Seaborn et al. (1994). J. Trace Elem Exp Med, 7, 11. (2) Calomme et al. (1997), Biol Trace Elem Res, 56, 153. RISEDRONATE BUT NOT ALENDRONATE SLOWS DISEASE PROGRESSION IN THE GUINEA PIG MODEL OF PRIMARY OSTEOARTHRITIS J. M. Meyer*, R. W. Farmer, M. C. Prenger Procter & Gamble Pharmaceuticals, Mason, Ohio, USA Most animal models of osteoarthritis involve chemical or surgical initiation of the disease, although the majority of human OA is considered primary. The Duncan- Hartley guinea pig is a model of primary OA and mimics human disease in many aspects. Cartilage lesions begin at about 3 months of age or 750g weight. They are bilateral and begin primarily on the medial tibial plateau (MTP). Chondrocyte cloning, osteophytes and tidemark duplication can also be seen. Disease severity progresses as the animals age and gain weight. This may be a useful model for testing potential structure modifying OA drugs. Animals were randomized into the treatment groups shown in Table 1. The bisphosphonates risedronate (Actonel(r)) and alendronate (Fosamax(r)) or sterile isotonic saline (vehicle control) were administered via subcutaneous injection 5 consecutive days/week for 12 months. At sacrifice, the left tibia of the stifle joint was disarticulated and stained with Evan's blue dye. Joints were then placed in 10% neutral buffered formalin and digitally photographed. These images were analyzed using a BDS Image Analysis System (Oncor Image, Gaithersburg, MD). Lesion, MTP and osteophyte areas were manually outlined by a single blinded grader, and area calculated by the software program. A non-parametric Friedman's rank sum test was used to compare treatment groups to control. Median scores are reported. Risedronate but not alendronate had a statistically significant effect on
cartilage lesion and osteophyte size in the guinea pig model of primary OA. This is
consistent with previous data (ASBMR:SA472, 2001) suggesting not all bisphosphonates are
effective at slowing disease progression in this model. These data suggest risedronate may
be efficacious as a structure modifying drug in OA.
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