Cancer and bone
P-1
BONE MARKERS IN DETECTION ON BONE METASTASES IN PROSTATE CANCER
C. De la Piedra1*, N. A. Castro-Errecaborde2, C.
Mendez-Davila1, C. Garcia- Moreno1, L. Rodriguez de Acuńa1,
M. L. Traba1
1Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid,
Spain
2Family Medicine, Hospital Clinico San Carlos, Madrid, Spain
Early detection of bone metastases in prostatic carcinoma (PC) is very
useful in treatment and prognosis of the disease. Although these metastases are mainly of
esclerotic type, both osteoblastic and osteoclastic activities are affected.
Sixty six non-treated patients with: benign prostate hyperplasia (n=21),
PC in several stages without bone metastases (TXMO) (n=31), and PC
in several stages with bone metastases (TXM1) (n=14) were studied.
The aim of this work was to evaluate the sensitivity and specificity of a group of bone
markers in order to discriminate between patients MO and M1. The
following markers were studied: A) Bone formation: 1) Serum Bone Alkaline Phosphatase,
IRMA (Tandem Ostase, Beckman); 2) Serum Procollagen I Aminoterminal Propeptide (PINP), RIA
(Orion Diagnostica); B) Bone resorption: 1) Urinary Collagen I Aminoterminal Telopeptide
(NTX), ELISA (Ostex); 2) Collagen I Carboxyterminal Telopeptide (CTX) : 2-A) urinary
alfa-CTX, RIA (Osteometer), 2-B) serum beta-CTX, Elecsys (Roche); Collagen I
Carboxyterminal Telopeptides (ICTP), RIA (Orion Diagnostica).
In all cases, the levels of bone markers were significantly higher in
group M1 than in group MO. A complete separation of groups MO
and M1 was achieved with serum PINP and serum beta-CTX, with a sensitivity and
specificity of 100%. (Grant FIS 98/0967).
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P-2
EVIDENCE OF INCREASED BONE TURNOVER IN BREAST CANCER PATIENTS WITH BONE
METASTASES IN A RETROSPECTIVE STUDY
I. Martinovic1*, D. Vrbanec2, M. Jurcic2,
B. Belev2, V. Kusec3
1Department of Oncology and Radiotherapy, Clinical Hospital
Centre, Zagreb, Croatia
2Department of Pathophysiology, Clinical Hospital Centre,
Zagreb, Croatia
3Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
This retrospective study involved an analysis of available data on tumor
markers and bone markers of 28 patients with clinical evidence of breast cancer
metastases. This included one or more sets of data on tumor markers (CEA, CA 15-3, MCA),
bone formation markers (total and bone alkaline phosphatase, osteocalcin, procollagen),
and bone resorption markers (telopeptide, pyridinolines). The data on bone markers were
compared with those obtained from breast cancer patients without metastases and normal
women of the same age range. In patients with bone metastases approximately 80 percent of
the values on tumor markers (CEA, CA 15-3) and between 16-96 percent of the values on bone
markers were elevated, predominantly bone resorption markers. Bone resorption was also
significantly higher than in breast cancer patients without metastases than in normal
women, as assessed by telopeptide and both telopeptide and pyridinolines, respectively.
Statistically significant positive correlations between tumour markers (CEA, CA 15-3) and
bone resorption markers indicated that bone destruction was the main bone action in the
metastatic disease. Correlations between bone markers were positive, revealing involvement
of both formation and resorption processes in the metastatic spread of breast carcinoma to
the skeleton. Correspondingly, in a small sample a positive correlation of CEA and MCA
with bone formation indicator procollagen was found. These results showed increased bone
turnover with prevailing bone resorption in breast cancer patients with bone metastases.
Bone resorption was higher than in breast cancer patients without metastases and normal
women of the same age range. In addition, bone destruction might be related to the extent
of the malignant disease.
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P-3
COMBINED USE OF HYDROXYLAPATITE CERAMICS AND DEMINERALIZED BONE MATRIX
FOR SUBSTITUTION OF BONE DEFECTS (AN EXPERIMENTAL STUDY)
V. V. Golovchenko1*, V. I. Luzin2, E. P. Berezhnoy2
1Department of Traumatology and Orthopedy, State Medical
University, Lugansk, Ukraine
2Department of Normal Anatomy, State Medical University,
Lugansk, Ukraine
The experimental research was carried out on 72 white rats with initial
mass 130- 150 grams. The purpose of the study was to determine the influence of a
demineralized bone matrix in a combination with powdery hydroxylapatite ceramics
(CERHAP-M) on reparative osteogenesis. A 2.2-millimeter circular tibial bony defect was
made in proximal metaphysis and combination of demineralized bone matrix with powdery
hydroxylapatite ceramics was placed on the defect. Were used control groups where defects
were empty (the group 1) or where defects was filled by a only demineralized bone matrix
(the group 2) or by a only CERHAP-M (the group 3). Six rats from each group were
sacrificed on 15, 30, 60, and 90 days after grafting.
On sections of a zone of the bone defects, painted by hematoxylin-eosine,
were carried out morphometric analysis of biodegradation of particles of hydroxylapatite,
and also counted an index of osteointegration. Besides, defined a volumetric contents of a
trabecular bone in a zone near to zone of a defect.
Histomorphometric research has shown, that at filling of a defect of
CERHAP-M have detected, that this material has mainly osteoconductive properties. The
demineralized bone matrix exhibited mainly osteoinductive activity. In both cases the
osteogenesis did not pass cartilagenous stage of a bone formation.
If at filling of defects of the demineralized bone matrix in combination
from a hydroxyapatite ceramics the degree of a biodegradation of particles of ceramics
increased (the square of particles of CERHAP-M was decreased). Together with it, in
comparison with group 3 the index of osteointegration was enlarged, and the volume size of
a trabecular bone to 15 and 30 days was more check on 21.76 % and 14.69 %.
Thus, the combined application of demineralized bone matrix with
hydroxylapatite ceramics for filling of defects is accompanied by improvement of tissue
composition of a regenerate and optimizes processes of reparative regeneration of a bone.
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P-4
ENDOGLIN (CD105) EXPRESSION AND DISTRIBUTION ON HUMAN SARCOMA CELLS
L. Postiglione1*, G. Di Domenico1, L. Del Vecchio2,
S. Montagnani3, M. Macrģ2, G. Rossi1
1Dipartimento di Biologia e Patologia Cellulare e Molecolare
'L. Califano', Un. 'Federico II', Napoli, Italy
2Servizio di Immunoematologia, AORN 'A. Cardarelli', Napoli,
Italy
3Dipartimento di Scienze Biomorfologiche e Funzionali, Un.
'Federico II', Napoli, Italy
Endoglin (CD105) is a homodimeric transmembrane glycoprotein, primarly
expressed on endothelial cells, and represents an accessory component of the TGF- beta
receptor complex. Endoglin binds TGF-beta1 and beta3 with high affinity and recent studies
have shown that it interacts with other components of the TGF-beta superfamily, including
activin-A, BMP-2 and BMP-7. There are some evidences supporting the idea that Endoglin is
involved in angiogenesis and represents a powerful marker of neovascularization. It has
been demonstrated that CD105 is expressed in hemopoietic tumors, such as mielogenous
leukemias (AML) and lymphoblastic leukemias (B-ALL), and the analysis of CD105 expression
in the various subtypes indicates that it is always expressed by most immature subtypes
and that it is absent on more differentiated ones. These data suggest that Endoglin
expression might be modulated during the cancerogenesis and they lead to study CD105
expression in human solid tumors of different histotype.
The aim of our study was to evaluate Endoglin expression and distribution
on different human tumoral cells of connectival origin, by Western blotting, Flow
cytometric analysis and Immunofluorescence microscopy, using a specific anti- CD105 mAb
(mouse, SN-6, Valter Occhiena, Torino). The cells used in our study were: human
undifferenziated osteosarcoma cells MG-63 and human differenziated osteosarcoma cells
SaOS-2; human chondrosarcoma cells SW1353; human rhabdomiosarcoma cells A204, human
fibrosarcoma cells Hs913T; human epitheloid sarcoma cells VA-ES-BJ. Human skin fibroblasts
and human endothelial cells were used as a normal control.
Results showed a different expression pattern of Endoglin on the
different tumoral cell lines. CD105 protein was strongly expressed on several human
sarcoma cells studied as MG-63 SW1353, Hs913T and VA-ES-BJ.
Whereas, CD105 protein was almost absent on A204 and it showed a
heterogeneous pattern expression on SaOS-2 cells, characterized by a double peak, by Flow
cytometric analysis. These results indicate that Endoglin is differently expressed in
sarcoma cells, showing a high level of expression on almost all human sarcoma cells,
except the human rhabdomiosarcoma cells A204.
These data strongly suggest that CD105 is a promising molecular target to
set up innovative diagnostic and bioimmunotherapeutic approaches in human malignancies of
connectival origin.
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Cell biology: osteoblasts
P-5
VALIDITY OF BIOCHEMICAL BONE MARKER IN PATIENTS WITH MYELOMA MULTIPLEX
M. S. Prebilic, Z. C. Orlic*, A. N. Duletic, I. Host, I. Prebilic, D.
Petranovic
Internal Clinic, University of Rijeka, Croatia
Osteolysis, hypercalcaemia and pathologic fractures are common
complications in patients with multiple myeloma.
In 64 multiple myeloma patients we performed bone densitometry (DEXA) of
lumbal spine and hip to analyse bone mass. Homeostasis of bone metabolism was evaluated by
electrolytes (calcium, phosphorus, magnesium in serum and 24 h urine) creatinin, whole
alkaline phosphatase, bone specific alkaline phosphatase and osteocalcine in serum and
pyridinoline and deoxypyridinoline in urine.
We compared those data with the same kind of analyses of 50 patients with
osteoporosis type I of same age and sex.
Patients with multiple myeloma had significantly higher level of collagen
degradation products in urine compared with osteoporotic patients (p<0.05).
The difference between of those two groups was statisticaly highest in
patients with stage III multiple myeloma. We conclude that biochemical markers of bone
destruction and hypercalcaemia are significant prognostic factors for bone loss which
needs to be prevented before fractures appear.
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P-6
IMMUNOLOCALIZATION OF OSTEOCALCIN IN CALCIFIED MICROSPHERES IN LAMB
VERTEBRAE AND MOUSE BONES USING PAP METHODS
S. M. Shahtaheri1*, J. E. Aaron2, B. A. Oakley2
1Hamadan Medical School, Hamadan, Iran
2Department of Human Biology, University of Leeds, Leeds, UK
Osteocalcin is a calcium binding protein with a high affinity for
hydroxyapatite and uncertain role in calcification. In order to better understand the role
of this bone specific protein in skeletal metabolism it is essential to determine its
precise distribution. Using rabbit anti-bovine polyclonal antibody and anti-mouse affinity
purified monoclonal antibody the distribution of the stain was examined in chick limbs, in
foetal lamb vertebrae and in 5-days-old mouse calvarium and long bone respectively.
Controls incubated without primary antibody were negative. In undecalcified preparations
the antibody was located in osteoblasts and in discrete bands at the calcification front.
In decalcified preparations the stain was also found in association with a proportion of
osteocytes and in the region of the golgi body and outside the cells as stained granules.
Further stain was distributed heterogenously throughout the mineral matrix (but not in the
osteoid tissue) and the nature of the staining was granular. At all locations staining was
associated with particles approximately 1 µm in diameter resembling the calcified
microspheres which constitute the inorganic phase of immature and mature bone. It was
concluded that osteocalcin was integral to these microspheres where it functions in the
containment of calcium phosphate, influencing its form and biomechanical properties.
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P-7
INVOLVEMENT OF BETA-ACTIN IN THE EFFECTS OF FETAL CALF SERUM ON THE
BIOSYNTHESIS AND SECRETION OF OSTEOPONTIN IN MG-63 CELLS IN CULTURE
D. W. Liu1*, B. B. Vandahl2, S. Birkelund2,
B. Melsen1
1Department of Orthodontics, Royal Dental College, Aarhus,
Denmark
2Department of Medical Microbiology and Immunology, University
of Aarhus, Aarhus, Denmark
Living condition of cells in vitro determines their functional responses
to mechanical stimuli. To look into the impacts of serum supplementation on the
biosynthesis and secretion of osteopontin in relation to beta-actin filaments distribution
in MG-63 cells, different concentrations (0%, 2%, 5% and 10%) of Fetal Calf Serum (FCS)
supplemented MEM culture medium were approached. To pulse- label osteopontin, [35S]-methionine/cysterine
was incorporated in culture medium (147 mCi/ml). By autoradiography and
immunofluorenscence tests, it was revealed that both the 'intra-' and 'extra-' cellular
components of the newly synthesized (labeled) osteopontin were drastically reduced when
the serum supplementation was lowered from 10% as normally used down to 5% and below
(p<0.05). Meanwhile, beta-actin staining exhibited an obvious change of the shape and
size of the cells. As a conclusion, the shape and size of the cells (reflected by
beta-actin expression and orientation) are actively involved in the biosynthesis and
secretion of osteopontin in MG-63 cells. It therefore is inferred that a proper serum
concentration in medium (not less than 5%) be chosen in in vitro mechanoconduction
experiments. Since the lower serum supplementation simulates the reduction or occlusion of
blood circulation in periodontal ligament when heavier (either orthodontic or traumatic)
force is loaded to teeth, the findings in this study do suggest lighter force be used to
move teeth in a way of protecting the PDL cells from being starved from blood supply in
clinical orthodontics.
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P-8
EFFECTS PRODUCED BY DIHYDROTESTOSTERONE AND BICALUTAMIDE ON INTERLEUKIN-6
AND AMINOTERMINAL PROPEPTIDE OF PROCOLLAGEN I RELEASE BY HUMAN OSTEOBLASTS IN CULTURE.
M. Lefort1*, M. Dķaz-Curiel2, C. De la Piedra1
1Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid,
Spain
2Internal Medicine, Fundacion Jimenez Diaz, Madrid, Spain
It is known the protective effect of androgens on bone mass, although the
mecanisms involved are not fully understood. On the other hand, new non-steroidal
antiandrogens like Bicalutamide (Casodex) have been developed, that exert an inhibitory
action on prostatic cell growth, but they have the same positive effects than androgens on
bone.
The aim of this work was to investigate if the protective effect of
dihydrotestosterone (DHT) and Bicalutamide (Bic) on bone mass is produced through the
inhibition of interleukin-6 (IL-6) synthesis, known osteoclastic activator, by the
osteoblast, both at constitutive and IL-1 stimulated level. The possible effects of DHT
and Bic on aminoterminal propeptide of procollagen I (PINP) has also been studied.
Osteoblasts were obtained from trabecular bone of male patients
undergoing orthopedic surgery. The cells were cultured as described by Marie et al. (In
Vitro Cell & Developm Biol 1989; 25:373-380) with some modifications. Osteoblasts at
confluence were cultured in serum and phenol red-free DMEM containing 0.1 % BSA for 24 h
and 10-7 M DHT (Fluka), 8 µg/ml Bic (Astra-Zeneca) or vehicle (ethanol)
were added with or without IL-1beta (1ng/ml). After 24 h, levels of IL-6 (RIA, Medgenix)
and PINP (RIA, Orion Diagnostica) were determined in culture medium.
As expected, IL-1 induced an increase in IL-6 secretion. However, the
constitutive or IL-1 stimulated IL-6 production were not altered by treatment with DHT or
Bic. We did not find any significant effect of Bic or DHT on PINP levels.
These findings suggest that the beneficial effects of DHT and Bic on bone
mass may not be mediated by a decrease in osteoblastic IL-6 secretion or an increase in
collagen production. Other mechanisms may be implicated in the effects of DHT and Bic on
bone mass.
Supported by ASTRA-ZENECA.
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P-9
DISORDERS BONE FORMATIONS IN IMPAIRMENT HEMOPOIESIS
V. I. Rugal*, V. Gonchar, V. N. Kravets
Russian Research Institute of Hematology and Transfusiology, St.
Petersburg, Russia
AIM. Earlier we demonstrated the importance of bone cells in regulation
development hemopoietic precursors in normal hemopoiesis. The aim of this work was study
of bone tissue in impairment hemopoietic function of bone marrow.
METHODS. Functional and structural features cells of iliac bone have been
characterized in 50 patients with hypoplasia hemopoiesis connected with defects of
hemopoietic microenvironment. Cultural, morphometric and ultracytochemical methods were
used.
RESULTS. The organ culture of bone tissue showed high proliferative
activity of cells precursors of bone marrow stroma. The morphometric investigation of the
whole bone fragments showed the increase of value of bone trabeculs in hypoplastic
conditions 1.4 volume increase, in aplasia 1.7 volume increase. The number of osteogenic
cells per unit of square in histological preparation of iliac bone in hypoplasia was 1.5
volume increase, and in aplasia was 2.0 volume increase. The ultracytochemical analysis
demonstrated the increase functional activity of the intramedullar and endosteal cells of
bone fragments. In osteogenic and stromal cells were found pathological intranuclear
bodies.
CONCLUSIONS. The cells of bone tissue are the main part of hemopoietic
microenvironment. Disorders of the structure and function osteogenic cells can be cause
impairment both bone and development of hemopoietic precursors. Close relationship between
bone cells and hemopoietic cells confirms the thesis about the existence of a common cell
progenitor of the hemopoietic and bone tissue in adult.
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P-10
EFFECTS OF 17BETA-ESTRADIOL, TAMOXIFEN AND RALOXIFENE ON INSULIN GROWTH
FACTOR I AND TRANSFORMING GROWTH FACTOR I EXPRESSION IN HUMAN OSTEOBLAST CULTURE
C. Garcia-Moreno*, C. De la Piedra
Biochemistry Laboratory, Fundacion Jimenez Diaz, Madrid, Spain
It is known that estrogens exert a protective action on bone mass and
that postmenopausal lack of estradiol leads to an accelerated bone loss, but the
physiological mechanisms implicated in the estrogen actions on bone are not fully
understood. Insulin-like growth factor (IGF-I), an anabolic protein, and transforming
growth factor I (TGF-I), an inhibitor of osteoclast activity and recruitment, are two
local factors synthesized by the osteoblasts, which play an important role in the
remodeling coupling. The aim of this work was to investigate if the actions of 17beta-
estradiol and raloxifene and tamoxifen (two selective estrogen receptor modulators) on
bone were mediated through the induction of changes in the expression of IGF-I and/or
TGF-I. For that purpose, we investigate the effects of 17beta-estradiol, raloxifene and
tamoxifen on IGF-I and TGFbeta-I mRNA levels in a primary human osteoblast culture.
Osteoblasts were obtained from seven different postmenopausal women undergoing orthopedic
surgery. The cells were cultured as described by Marie et al. (In Vitro Cell &
Developm Biol 1989; 25:373-380). Osteoblasts were preincubated for 24h in serum-free DMEM
supplemented with 0.1% BSA. After preincubation, the culture medium was changed by DMEM
supplemented with 0.1% BSA (control samples), and 17beta-estradiol, tamoxifen or
raloxifene at 10-8M concentration were added to the culture. After 1, 3, 6, 24
or 30h, medium was removed and total RNA was isolated. After RNA extraction, IGF-I and
TGFbeta-I mRNA levels were assessed by reverse transcription followed by polymerase chain-
reaction (RT-PCR). Incubation with 10-8M 17beta-estradiol or 10-8M
raloxifene, but not with 10-8M tamoxifen, increased significantly the
constitutive levels of IGF-I mRNA after 24 hours and 30 hours of incubation. However, we
did not find any significant effect of these three compounds on TGFbeta-I mRNA at any of
the studied incubation times. These findings suggest that the effects of estradiol and
raloxifene on bone mass may be mediated by an increase in IGF-I expression, but not by
actions on TGFbeta-I. Tamoxifen effects on bone appear not be mediated by these cytokines.
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P-11
BMP-1 EXPRESSION IS DEVELOPMENTALLY REGULATED DURING IN VIVO OSTEOGENESIS
IN THE RAT TIBIA: A STUDY BY IN SITU HYBRIDIZATION
P. Manduca1*, S. Zanotti1, F. Galmozzi1,
A. Favre2, O. Barbieri1, D. Di Martino1
1Dobig, University of Genova, Italy
2Ist.G.Gaslini, Genova, Italy
We have investigated the expression of BMP-1 (procollagenase I), a
proteinase involved in fibrillar collagen processing, which removes the carboxyl-terminal
fragment from pro-collagen trimers of type I, II and III collagens. In osteogenic cells in
culture BMP-1 produces the carboxyl-trimer (C3) from pro-collagen type I, a molecule
recently proven to be chemoattractant for human endothelial and tumor cells.
We here present evidence, obtained by in situ hybridization, that the
expression of BMP-1 mRNA is developmentally regulated in the bone, cartilage and bone
marrow compartments of the developing long bone (tibia) in fetal, new born and young
animals.
In bone cells, the expression of BMP-1 is maximal in lining osteoblasts
in active phase of bone deposition and is associated also to osteoclasts. BMP-1 is
expressed in the periosteum in modulated fashion during bone growth. The expression in the
bone marrow is limited to areas (possibly clones of stromal cells) and some of these are
specifically localized within the bone shaft microenvironment. Both proliferating and
mature-hyperthrophic chondrocytes express BMP-1, although the location and kind of cells
expressing it varies during development of the bone. Moreover, the expression of BMP-1 in
the epiphysis shows marked asymmetries, being most expressed in the proximal epiphysis.
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P-12
NEW WAY OF BONE REGENERATION IN EXPERIMENT
V. A. Gonchar*, V. I. Rugal, V. N. Kravets, F. Baranovsky, V. Michailov
Institute of Haematology and Transfusiology, St. Petersburg, Russia
AIM: To study peculiarities the regeneration of long tube-bones fractures
by treatment Osteolin in experiment at rats.
METHODS: Adult rats caused combine mechanic fractures of tibiae and
fibiae. Experimental rats got 0.1 mg/kg immunoglobulins (Ig) sera of rabbits immunized by
femur epiphyses from adult rats. Control rats got analogically same dose Ig sera nonimmune
rabbits. Course of treatment included 3 injections with interval 48 hours. Hystological
investigation made once a week from day of fracture.
RESULTS: Control rats showed usual bone regeneration: to 3d week was
formed callus with tipical osteo structure (OS) on all fracture zone (FZ) without bone
marrow canal (BMC). Periosteum was presented several layers of fibroblastlike cells.
Experimental rats already 1-st week showed total growth of cartilage tissue (CT)
apparently bone marrow genesis and forming periostal muff. Simultaneously was formed
central BMC which determined by osseal model without OS but with extracellular
basophillia. In CT arised niduses of concellous bone (NCB). At 3rd week restored
thickening FZ by CT layer and wide periostal zone. To 4th week compact osseal tissue (COT)
with typical OS became predominated on sites where persisted NCB arised zones. Atypical
cell were not found.
CONCLUSION: Formation COT from CT is phylogeny ancient. Used Osteolin
allowed to reactivate this genetic program in precurser cells common for haemopoetic,
cartilage and osseous tissues.
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P-13
CHANGES IN GENE EXPRESSION IN HUMAN OSTEOBLASTS FROM YOUNG AND OLD DONORS
IN DIFFERENT CULTURE CONDITIONS
D. C. Ireland*, S. R. Beavan, S. Bord, J. E. Compston
School of Clinical Medicine, University of Cambridge, Cambridge, UK
Osteoporosis is a common disease of the elderly. Loss of biosynthetic
ability by osteoblasts could contribute to the alterations in bone resorption and
formation seen with ageing. We have examined the growth of osteoblasts from female donors
aged one year (young donor, YD-hOBs) and 66 years (old donor, OD-hOBs) in different
culture conditions. Markers of proliferation, differentiation, mineralization, senescence
and apoptosis were measured in cells grown on plastic or collagen in medium with human
serum, without (proliferation medium) and with (differentiation medium) hydrocortisone and
beta-glycerophosphate. mRNAs for ER-alpha, ER-beta, COL1, ALP, OC, RANKL and OPG were
measured by real-time RT-PCR. Mineralization was detected using von Kossa reagent,
senescence using detection of beta-galactosidase activity at pH 6 and apoptosis using
TUNEL and antibody against active caspase 3.
YD-hOBs grew well in proliferation medium on both substrates. OD-hOBs
grew more slowly than YD-hOBs in proliferation medium on plastic and remained in clumps.
OD-hOBs cultured in proliferation or differentiation medium on collagen grew more quickly
than on plastic and formed monolayers. OD-hOBS on collagen in differentiation medium
formed mineralized nodules five days after reaching confluence whereas YD-hOBs did not.
Cultures of OD-hOBs on plastic and collagen in both proliferation and growth medium showed
very few positive cells when stained for apoptosis but a high proportion of positive cells
when stained for senescence.
Changes from days two to nine in mRNA levels in hOBs cultured on collagen
in differentiation medium reflected the different rates of differentiation of the cell
lines. ER-beta mRNA increased 18.5-fold (p<0.001) in OD-hOBs but decreased slightly in
YD-hOBs. COL1 and ALP mRNA levels increased in YD-hOBs (5-fold and 9-fold (p<0.01)) but
decreased 3.5-fold (p<0.001) and 1.3-fold (ns) in OD-hOBs. When 17- beta estradiol was
added, COL1, ALP and OPG mRNAs were higher at both two and nine days in YD-hOBs than in
controls (2-fold, 2-fold and 2.5-fold (p<0.05) at two days with saturating estradiol)
but were not increased in OD-hOBs. The limited synthesis of COL1 mRNA by OD-hOBs and their
altered sensitivity to estradiol may be attributable to their increased rate of
differentiation and mineralization
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P-14
DO STEM CELLS AGE?
H-V. Leskelä1,2*, S. Niskanen1, J. Risteli3,
P. Lehenkari1,2
1Department of Anatomy and Cell Biology, University of Oulu,
Finland
2Department of Surgery, University of Oulu, Finland
3Department of Clinical Chemistry, University of Oulu, Finland
Human bone marrow contains both hematopoietic stem cells and non-
hematopoietic stem cells. The latter are often referred to as mesenchymal stem cells or
marrow stromal cells (MSCs) (1). MSCs are multipotent cells that can differentiate into
multiple lineages including osteoblasts, adipocytes, chondrocytes, myocytes, astrocytes,
oligodendrocytes and neurons (1). Alkaline phosphatase and type I collagen are associated
with the bone cell phenotype and are actively expressed during the maturation of the
osteoblast (2). During mineralization, calcium deposits are formed into the mature organic
matrix and develop into bone-like nodules (3). In this study we investigated the ability
of MSCs, from female patients at different ages, to differentiate into osteoblasts and the
ability of these cells to mineralize extracellular matrix in vitro.
Human MSCs were obtained from 18 women ranging in age from 43 to 90 years
from the femur neck. For osteogenic differentation MSCs were cultured in the basal medium
in the presence and absence of dexamethasone (100nM). Alkaline phosphatase activity and
the amino-terminal propeptide of type I procollagen (PINP) concentration were determined
at days 7, 14 and 21. For extracellular matrix mineralization MSCs were cultured in basal
medium in the presence and absence of b- glycerophosphate (10mM). Matrix mineralization,
associated with the synthesis of calcium, was demonstrated at days 21, 28, 35, 42 and 49
by determining calcium concentration.
Dexamethasone treatment increased levels of alkaline phosphatase 2.3-fold
and PINP 6.8-fold, when MSCs were cultured for 21 days. In addition, b- glycerophosphate
stimulated calcium deposition 3.0-fold when cultured for 49 days. In our material the
activity of alkaline phosphatase increased with age for women
when MSCs cultured 21 days. Our data suggest that the MSCs osteogenic
potential does not decrease in aging woman. This observation might partly explain why
osteoporotic bone has clinically good healing capacity.
(1) Colter et al., 2001; (2) Beresford et al., 1986; (3) Dippolito et
al., 1999
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P-15
INFLUENCE OF SOME NON-IONIZING ELECTROMAGNETIC WAVES ON MORPHOFUNCTIONAL
STATE OF EPIPHYSEAL CARTILAGES OF PREADOLESCENT WHITE RATS
V. I. Luzin
Department of Normal Anatomy, State Medical University, Lugansk, Ukraine
The research was carried out on 280 preadolescent white rats with initial
mass 65- 70 grams. The animals were irradiated by electromagnetic (EM) waves with
frequency 61.5 GigaHaertz in modes of impulse and continuous generation, Helium- Neon
laser in modes 0.5 milliWatts/cm2 during 10 minutes and 15 milliWatts/cm2
during 2 minutes, and also by volume-combinative impulse EM fields with amplitude
0.04/0.01 Teslas ('direct' configuration) and 0.04/0.05 Teslas ('transversal'
configuration).
Morphometry of epiphyseal cartilages of a tibial bone carried out with
usage of classification of V. Koveshnikov.
It was established, that at long (30 and 90 days) exposure under
experimental animals by EM waves of the highest frequency in all series marked the
increase of common width of an epiphyseal cartilage with decrease of its osteogenic
functions (first of all decrease the volumetric contents of a primary spongiosa in a zone
of primary osteogenesis), the most expressive at impulsed regimes.
Enlargement of an proximal tibial epiphyseal cartilage and all of its
zones with disturbance of boneformed function accompanied influence of He-Ne laser
begining from the 30th day of exposure (first of all decrease volumetric contents of an
osteoblasts in a zone of primary osteogenesis). During the readaptation after 30-day's
exposure the revealed faults were softed in a large degree; amplitude of deviations was
directly dependent on power of radiation.
At last, at irradiation by volum-combinative EM impulse fields was also
detected the enlargement of an proximal tibial epiphyseal cartilage and all of its zones
with decrease of volum contents of a primary spongiosa in a zone of osteogenesis, which is
restored during the period of readaptation. In the group with 'direct' configuration of
impulses the expressiveness of deviations during a readaptation was larger.
The received data allow to suppose, that investigated low-intensive
non-ionizing radiations have the similar mechanism of action on morphofunctional
properties of epiphyseal cartilages of long tubular bones of preadolescent rats.
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P-16
MICROARRAY ANALYSIS OF LATE PHASE GENES DURING BMP2- INDUCED OSTEOBLAST
DIFFERENTIATION
B. L. T. Vaes1*, K. J. Dechering3, D. S. de Jong1,
J. M. A. Hendriks1, E. J. J. van Zoelen2, W. Olijve1,3,
W. T. Steegenga1
1Department of Applied Biology, University of Nijmegen,
Nijmegen, The Netherlands
2Department of Cell Biology, University of Nijmegen, Nijmegen,
The Netherlands
3Target Discovery Unit, N.V. Organon, Oss, The Netherlands
Osteoblast differentiation can be described by three phases. Firstly a
proliferation phase, followed by a phase in which extracellular matrix is deposited, and
in the last phase this matrix becomes mineralized. The three phases are characterized by
specific gene expression patterns, but only the transcription factor cbfa1 and the late
phase gene osteocalcin are known to be osteoblast specific. For a more accurate
description of osteoblast differentiation and the identification of novel osteoblast
specific genes, microarrays were employed to analyze gene expression on a broad scale. To
study osteoblast differentiation, the murine mesenchymal progenitor cell line C2C12 was
used as a model system. When treated with bone morphogenetic protein-2 (BMP2), these cells
differentiated into osteoblastic cells within six days, with up-regulation at the mRNA
level of osteoblast markers such as alkaline phosphatase and osteocalcin.
Microarray analysis was done with mouse cDNA arrays with which expression
regulation of approximately 9500 genes can be measured. During a time course experiment,
343 genes were over twofold modulated on microarray. These genes, (180 up- and 163
down-regulated) were used for cluster analysis to order the genes according to similarity
in expression profiles. Cluster analysis divided the genes into groups of distinct kinetic
patterns, showing early, intermediate or late phase responses. Description of the dataset
in terms of protein function elucidated subsequent transient regulation of sets of
proteins involved in intracellular signalling (e.g. transcription factors), extracellular
matrix formation (e.g. collagens), and other secreted proteins (e.g. extracellular
messengers and proteases).
A subset of 54 late phase genes was further studied to determine
osteoblast specific induction by BMP2. Expression analysis in different cell types,
revealed four novel genes and four ESTs that were preferentially induced in
osteoblast-like cell types, and were identified as new late phase markers for in vitro
osteoblast differentiation.
The knowledge about temporal expression of large sets of genes and their
function leads to a better understanding of osteoblast differentiation, function and bone
development. In combination with specificity of expression regulation, this facilitates
the search for key regulators of osteoblast development that in future might serve as drug
targets to treat bone related disorders.
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P-17
CLONING AND CHARACTERIZATION OF THE 5' FLANKING REGION OF THE MURINE
OSTEOPROTEGERIN GENE
P. Nemeth*, F. Varga, K. Klaushofer
Ludwig Boltzmann Institute of Osteology,4th Medical Dept.,
Hanusch-Hospital, Vienna, Austria
Osteoprotegerin (OPG), as a decoy receptor, is well known to inhibit
osteoclastogenesis and osteoclast function by preventing receptor activator of NF- kappaB
ligand (RANKL) from binding to receptor activator of NF-kappaB (RANK). To gain more
insight into the regulatory process of OPG we cloned and characterized the murine
osteoprotegerin promoter.
Cloning of the OPG 5' flanking region was done by genome walking. The 3'
primers were located within the 5' untranslated transcript of the murine OPG mRNA. 5'
primers were provided by Clontech, sequencing was done automatically. The correctness of
the sequence was confirmed by reamplification of genomic mouse DNA. The primers were
derived from the cloned promoter sequence.
Electro mobility shift assay (EMSA) was performed with chemically
synthesized oligo nucleotides labeled with polynucleotide kinase (Pharmacia) and in vitro
translated Cbfa1 (ivtCbfa1) using an erythrocyte translation system (Promega).
For transfection assays we cloned the putative promoter into SEAP2-basic
reporter vector (Clontech). Transfections were done into both MC3T3-E1 and ST2 cell lines
using liposomal transfer (Dosper, Roche).
We isolated 1761 base pairs of the 5'flanking region next to the putative
transcription start for the murine OPG mRNA (GenBank AB013899S1). Comparison with the
human OPG promoter revealed only weak homology. Compared with the same part of the humane
promoter we found three Cbfa1 binding sites. Referring to the transcription start of the
OPG mRNA, the most distal Cbfa1 binding site was found to be homologue to the sequence of
the collagen alpha (I) promoter. It bound ivtCbfa1 only very weak. The proximal Cbfa1
binding site was homologue to the osteopontin Cbfa1-binding site with a higher affinity to
ivtCbfa1. The middle Cbfa1 binding site showed the highest affinity in the EMSA and
presented a sequence that has not been described until now. Transfection of the murine OPG
promoter construct into MC3T3-E1 and ST2 cells, showed a weak increase of the reporter
gene expression, but was only significant in ST2 cells.
In summary we isolated a 5'flanking region of the OPG gene, which
contained 3 Cbfa1 binding sites capable of binding ivtCbfa1 and showed promoter activity
in transfection assays into osteoblast like cells.
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P-18
ISOLATION OF A CDNA WITH HOMOLOGY TO CARBOXYPEPTIDASE D FROM OSTEOBLASTS
M. Rumpler*, F. Varga, K. Klaushofer
Ludwig Boltzmann Insitute of Osteology, Hanusch Hospital, 4th Medical
Dept., Vienna, Austria
Cell lines can change their phenotype and their morphology with ongoing
culture time often accompanied by an increased formation of tightly sealed contacts. The
differentiation of bone marrow stromal cells into osteoblasts can be inhibited by
preventing cell adhesion. In ostoeblastic cells confluence is considered to be a
transition point in the differentiation process. This implicates that the formation of
cell-cell contacts, together with other regulatory influences, play an essential role in
the differentiation process. We used Differential Display to find genes, which are
regulated when osteoblast-like cells reach confluence.
Total RNA was isolated from MC3T3-E1 cells seeded at 5,000 and 50,000
cells/cm2 in aMEM supplemented with 5% FCS and cultured for 3 days. Total RNA
was reverse transcribed with an oligo-dT primer. Thereafter the cDNA was amplified using
an arbitrary primer and an oligo-dT primer in a PCR. After gel electrophoresis
differentially expressed bands were isolated, cloned and used for further investigations.
RNA from mouse tissue was isolated using TRIZOL reagent, reverse transcribed and amplified
in a PCR.
One isolated cDNA fragment exhibited a length of 550 bp. Sequence
analysis exhibited a homology to the carboxypeptidase D from mouse, human and rat in a
distance of 149 bp. This homologue region belongs to the domain 3 of the carboxypeptidase
D. The presence of this cDNA was verified in MC3T3-E1 cells, ST2 and NIH3T3 cells by PCR
with gene specific primers. Treatment of the mouse stromal cell line ST2 with ascorbic
acid led to an increased expression of this cDNA. In MC3T3-E1 cells its expression was
increased by T3 treatment. The expression of this cDNA with homology to carboxypeptidase D
was found in brain, lung, kidney, ribs, calvariae, heart and liver, but not in thymus,
spleen and muscle. In summary, the finding of a new differentially expressed gene with a
partial homology to a carboxypeptidase points to a function of this enzyme in osteoblasts.
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Cell biology: osteoclasts
P-19
LOW CONCENTRATION OF CLODRONATE IN THE BONE RINSING SOLUTION CAUSES
APOPTOSIS OF CULTURED OSTEOCLASTS
M. I. Kellinsalmi1*, M. Hämäläinen1, P.
Lehenkari1,2
1Department of Orthopaedics, Universital Hospital of Oulu,
Finland
2Department of Anatomy and Cell Biology, University of Oulu,
Finland
Background of the study: Bisphosphonates are the most important group of
antiresorptive drugs when situations related to excess bone resorption are treated. Our
aim is to study the prevention of osteolysis in the border of the femoral stem and bone
and also in the bone-cement border using non-amino bisphosphonate, clodronate, to find out
the optimal concentration of clodronate in the rinsing solution and optimal rinsing time.
To study these parameters we made osteoclast cultures with rat bone
cells, which were grown in the bovine bone chips. Prior to culture the bone chips were
rinsed using different concentrations of clodronate-saline solution.
Results: We find out that concentrations 10 mM, 2 mM and 0.2 mM of
clodronate lowered equally effectively the number of osteoclasts and the resorbtion area.
The mean apoptosis index of the control group was 0.09. It was tripled in concentration
0.02 mM also the apoptosis index was increased to 0.49 when rinsing time was continued to
three minutes. With 0.2 mM concentration the apoptosis index was the same whether the
rinsing time was one or three minutes. Further more the apoptosis index increased only a
little with concentration 2 mM being 0.73.
Conclusion: The mechanism of action of clodronate found to be osteoclast
specific; no effect on stromal cells could be observed even at high concentrations. Our
data suggests that the decrease in osteoclast number was due to osteoclast apoptosis that
was dose- and timedependently increased. Clodronate attached very tightly to the bone and
already short rinsing using 0.02mM concentrations had a clear effect in the prevention of
the bone resorbtion. Finally, we conclude that the optimal concentration of clodronate,
when applied locally in the rinsing saline is 0.2 mM and the optimal rinsing time is one
minute.
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P-20
THE IDENTIFICATION OF GENES REGULATED IN OSTEOCLAST DIFFERENTIATION BY
ARRAY ANALYSIS
T. Vaughan1*, C. J. Aitken2, M. A. LeRoy1,
A. S. Mellick1, W. E. Simcock1, M. S. Kim1, G. C.
Nicholson2, N. A. Morrison1
1Genomics Research Centre, School of Health Science, Griffith
University, Gold Coast, Australia
2Department of Medicine, The University of Melbourne, Barwon
Health, Geelong Hospital, Geelong, Australia
Osteoclasteogenesis is regulated in vivo by the action of
osteoblast/stromal cells which express the membrane bound receptor activator of NF-kappa B
ligand (RANKL). In vitro the soluble form of RANKL can be used in combination with
macrophage colony stimulating factor (M-CSF) to induce the production of osteoclast like
cells from peripheral blood monocytes (PBMCs). PBMCs treated with M-CSF alone results in
the production of macrophage like cells that can be employed as a control to examine the
effects of RANKL on a range of gene targets. Total RNA from monocytes treated with M-CSF
alone and treated with RANKL and M-CSF together was isolated at 8 hours, 12 hours, 24
hours, 8 days and 21 days after treatment. Differential display (DD) was performed on RNA
(8 hours, 12 hours, 24 hours, 8 days treatment with RANKL) the DD products were pooled to
generate a probe for screening a gene array system derived from a human osteoclast cDNA
library. Hybridisation of cDNA against the array revealed additional regulated genes.
Gene clones that showed significant regulation in monocytes treated with
M-CSF and RANKL compared with monocytes treated with M-CSF represent genes that are
targets for RANKL-specific regulation. Osteopontin, creatine kinase B and cystatin B were
up regulated by the treatment of RANKL. Thymosin beta-4, proteasome ATPase and thioredoxin
binding protein were down regulated by the treatment of RANKL. Relative fold differences
observed in several genes identified from array analysis were confirmed using real time
PCR. These include the osteoclast acidic protease cathepsin K, and the protease inhibitors
cystatin B and cystatin C. Cathepsin K relative fold expression peaked at one week
treatment at 224±1.15 and subsequently decreased with increasing time of exposure to
RANKL. Cystatin C relative fold of expression peaked at two weeks RANKL treatment at
8.4±1.08 and cystatin B showed a steady increase in expression. Our hypothesis is that
gene clones, which showed significant regulation of M-CSF and RANKL when compared with
M-CSF alone, represent potential downstream targets of RANKL activation. Genes that are
regulated by the action of RANKL during osteoclastogenesis may provide potential targets
for novel anti-osteoporotic drugs.
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P-21
IMMUNOLOGICAL AND HISTOCHEMICAL DETERMINATION OF TRAP 5B TO MEASURE
OSTEOCLAST ACTIVITY UNDER ANTI- OSTEOLYTIC TREATMENT
A. T. Mehlhorn1*, H. Rechl1, A. W. Stemberger2,
R. Gradinger1
1Department of Orthopaedic Surgery of the Technische
Universität, Munich, Ismaningerstr. 22, 81675 Munich, Germany
2Department of Experimental Oncology of the Technische
Universität, Munich, Ismaningerstr. 22, 81675 Munich, Germany
Background: The tartrat resistant acid phosphatase 5b is regarded to be
the most sensitive parameter of osteoclast function. Detection in serum is performed by a
two- site enzyme linked immunoassay. We developed a method for localization and
quantification of TRAP 5b activities in bone and compared the results with histological
examinations of the corresponding bone areas.
Methods: A total of 12 femoral heads divided in two different test groups
were examined. The control group (Age: 70±7 y, m:f = 1:3, n=6) underwent a total hip
arthroplasty without preoperatively receiving bone affecting drugs. Bisphosphonate group
(Age: 63±11y, m:f = 1:3, n=6) received 10 mg/d Alendronate (n=5) or 5 mg/d Risedronate
(n=1) because of manifest osteoporosis at least 50 days prior to operation. Samples of
three defined areas were prepared from the specimen: the femoral head (A1), the femoral
neck (A2) and the intertrochanteric area (A3). Half the bone chips were grinded, incubated
in isotonic saline and the supernatant was analysed by Bone TRAP_
Assay (Medac Diagnostica, Germany). The remaining chips were fixed in buffered 2.4%
formalin, cold-embedded in methylmethacrylat and stained with a mixture of naphtol-AS-BI
phosphate substrate, L(+)-tartrat and Fast Red Violet LB. TRAP-positive areas and cells
were counted in all sections.
Results: The quantitative determination of TRAP 5b demonstrated in the
bisphosphonate group a decrease in enzyme activity compared with the controls: 18% in area
A3 (0.179±0.131 vs. 0.218±0.195 Unit/mg), 5% in A2 (0.210±0.140 vs. 0.218±0.083
Unit/mg) and 11% in A1 (0.318±0.158 vs. 0.359±0.297 Unit/mg). The histological
examination confirmed these results, it showed a lower number of TRAP- positive lacunae in
sections of the bisphosphonate group.
Conclusion: Bisphosphonate treatment decreases TRAP 5b activity as a
marker of osteoclast function in all areas of arthritic femoral heads. Thus, the described
histochemical method and ELISA are suitable tools to evaluate the local impact of
bisphosphonates on osteoclast activity.
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P-22
TARTRATE-RESISTANT ACID PHOSPHATASE 5B AND OSTEOCALCIN REFLECT THE BONE
RESORPTION RATE IN IN VITRO HUMAN OSTEOCLAST CULTURES
H. Ylipahkala*, K. K. Ivaska, T. A. Hentunen, H. K. Väänänen, J. M.
Halleen
Institute of Biomedicine, Department of Anatomy, University of Turku,
Turku, Finland
Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) 5b into
the circulation as an active enzyme, suggesting that serum TRAP 5b activity may be a
useful marker of bone resorption. In the serum, TRAP circulates in a high molecular weight
complex that contains alpha-2-macroglobulin. Osteocalcin is the most abundant
non-collagenous protein in bone matrix. Serum osteocalcin is generally considered as a
marker of bone formation, but some osteocalcin may also be released during bone
resorption.
We have studied the secretion of active TRAP 5b and osteocalcin from
resorbing osteoclasts using an in vitro human osteoclast culture where peripheral blood
mononuclear cells were cultured on bovine bone slices and allowed to differentiate into
bone-resorbing osteoclasts in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone.
TRAP 5b activity (BoneTRAP, SBA-Sciences, Oulu, Finland) and C- terminal cross-links of
type I collagen (CrossLaps, Osteometer Biotech, Herlev, Denmark) were measured using
commercial immunoassays, and total osteocalcin using an in-house immunoassay. TRAP complex
was purified using gel filtration chromatography.
Both medium TRAP 5b activity and total osteocalcin correlated
significantly with medium CrossLaps and osteoclast number (r=0.9, p<0.0001 in all
cases). These results suggest that TRAP 5b and bone matrix-derived osteocalcin are
secreted from resorbing osteoclasts. Purified TRAP complex contained also calcium,
suggesting that TRAP 5b may be secreted together with calcium released from bone matrix,
and they may circulate in blood in the same complex together with alpha-2-macroglobulin.
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P-23
THE SIZE OF IN VITRO CULTURED OSTEOCLASTS WAS INCREASED AND THE
OSTEOCLASTIC ACTIVITY DECREASED AFTER TREATMENT WITH MMP-INHIBITOR BB-94
V. V. Parikka*, H. K. Väänänen
Institution of Biomedicine, University of Turku, Turku, Finland
Matrix metalloproteinases (MMPs) are a group of enzymes capable of
degrading extracellular matrix components. The exact roles of MMPs in bone biology are not
understood, but they have been suggested to be required for the solubilization of bone
matrix and possibly for osteoclast migration and anchorage. At least two members of the
group, MMP-9 and MT1-MMP, have been demonstrated to be present in osteoclasts. In
addition, MMPs of osteoblast lineage cells probably also affect osteoclasts.
We have previously shown that bone resorption is disturbed in the
presence of MMP-inhibitor batimastat (BB-94). In the present study we further analysed the
effects of BB-94 on osteoclasts. A mixed rat bone cell population was cultured on bovine
bone slices for three days. After the culture period cells were stained for
tartrate-resistant acid phosphatase (TRAP) and with DNA-binding fluorochrome Hoechst 33258
to visualize nuclei. F-actin was stained with TRITC-conjugated phalloidin to detect actin
rings.
MMP-inhibitor BB-94 at 6microM concentration, did not significantly
affect the number of osteoclasts. Instead, the average number of nuclei per osteoclast was
significantly increased (7.41±0.36 versus 9.53±0.55). In addition, the number of mono-
and binuclear TRAP-positive cells was lower on BB-94 treated slices. Phalloidin staining
revealed that the formation of actin rings was dramatically decreased by BB-94. These
result were reproduced with another MMP-inhibitor BB- 2516. Another major group of
proteinases expressed in osteoclasts is cysteine proteinases. To confirm that the effect
on osteoclast size was specific for MMP- inhibition, we studied the effect of cysteine
proteinase inhibitor E-64 on osteoclasts. It had no effect on the size of osteoclasts even
at 50microM concentration.
To conclude, we demonstrated that the osteoclast size was increased and
the osteoclastic activity decreased by MMP-inhibitor BB-94. We suggest that the observed
phenomenon may be due to disturbed migration of osteoclasts.
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Genetics
P-24
COMPLICATIONS IN LEG LENGTHENING PROCEDURE BY WAGNER APPARATUS IN
CHILDREN: DIFFERING CONGENITAL AND ACQUIRED AETIOLOGY OF SHORTENING
M. Baretic
General Hospital 'Sveti Duh', University of Zagreb, Croatia
Leg lengthening procedure is often accompanied by many complications.
Connection between aetiology of abbreviation and complications was studied in 36 patients
(average age 11 years and 2 months). All the patients were younger than 18 yrs without
previous leg lengthening operative procedure made of any kind. They underwent 41 lower
limb leg lengthening operations (33 femoral and 8 tibial) by Wagner apparatus. To the one
of the children femur and tibia on the one leg were elongated and to the other one
together femur and tibia on both legs were lengthened. Their medical data were collected
retrospectively and analysed. Average preoperative leg length discrepancy was 5.4 cm:
average lengthening achieved was 5 cm. Wagner apparatus was placed in situ in average
during 5 months and 21 days. Children were divided by the aetiology of leg shortening in
two groups; in 25 of them aetiology was congenital (congenital idiopathic hyoplasia,
congenital hip dislocation consequences, coxa vara, mb. Ollier, mb. Klippel-Trenaunay,
achondroplasia) and 11 of them had acquired aetiology of abbreviation (tumours resection
effect, paresis et paraparesis consequences caused by polio, meningitis,
myelomeningocoela, tumours resection effect, posttraumatic status). 22 elongations were
followed by one or more complications (14 patients had joint contractures, 8 pin-track
infections, 4 fractures, 2 too early consolidations, 1 pseudarthrosis). Complications were
divided on heavier (an additional operative procedure was required) and easier (solved
with physical therapy and antibiotics). It was presumed that the frequency of
complications and their severity are influenced by aetiology of shortening. Statistical
data evaluated by Hi square test show no significant difference. It seems that the
aetiology does not play the role in prognosis of complication rate and severity during leg
lengthening procedure by Wagner apparatus. Reoperations could be avoided by continuous
physical therapy considering setting the operated leg under graduate pressure, rigorous
pin-track toilette with microbiological sample taking and AO fixation with spongioplasty.
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P-25
VITAMIN D RECEPTOR GENE POLYMORPHISMS AND BONE MINERAL DENSITY IN
POSTMENOPAUSAL WOMEN IN MALTA
C. Vidal1*, M. Brincat2, A. Xuereb1,3
1Institute of Health Care, University of Malta, Malta
2Department of Obstetrics and Gynaecology, University of Malta
Medical School, Malta
3Department of Pathology, University of Malta Medical School,
Malta
Osteoporosis is a disease with a strong genetic component. One of the
genes that has been implicated in osteoporosis is the Vitamin D receptor (VDR) gene,
although there have been conflicting results as to whether VDR gene polymorphisms
influence bone mineral density (BMD). The aim of this study was to determine whether
correlation exists between VDR gene polymorphisms and bone mineral density in
postmenopausal women in Malta.
104 postmenopausal women (aged 48-62 years) were recruited for this
study. Amplification of the desired target sequence of the VDR gene was carried out by
PCR, and the subjects were genotyped on the presence (b) or absence (B) of the BsmI
restriction enzyme cleavage site in the VDR gene. BMD at the lumbar spine and femur was
measured using a Norland DEXA 486. Serum concentrations of procollagen Type 1 and urinary
deoxypyridinoline crosslinks were used as biochemical markers of bone turnover.
The genotype frequencies observed (BB=16.4%, Bb=51.9% and bb=31.7%) were
similar to those observed in other Caucasian populations. BMD at both anatomical sites was
observed to be highest in BB homozygotes, although the difference between the genotypes
was not statistically significant (ANOVA p=0.768; 0.967 for lumbar spine and femur,
respectively). The biochemical marker of bone formation was observed to be highest in the
BB genotype, whereas the biochemical marker of bone resorption was found to be lowest in
the BB group, although both these results were not statistically significant (ANOVA
p=0.591; p=0.806 respectively). A non- statistically significant difference (Chi-square
test p=0.624) was observed in the frequencies of VDR genotypes between normal individuals
and a group of osteoporotic women at the lumbar spine (t-score<-2.5.)
In conclusion, there does not seem to be an association between
polymorphisms in the VDR gene and BMD in postmenopausal women in Malta.
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P-26
POLYMORPHISM IN THE VITAMIN D RECEPTOR GENE AND THEIR RELATIONSHIP WITH
BONE LOSS IN POSTMENOPAUSAL WOMEN
M. Dķaz-Curiel1*, J. Iborra2, M. Quesada3,
J. Farrerons4, M. Sosa5, R. Pérez-Cano6, J.
Gonzįlez-Macias7, A. Dķez-Pérez8, J. Cannata9
1Fundación Jimenez Dķaz, Madrid, Spain
2Novartis Farmacéutica, Spain
3Hospital Reina Sofia, Cordoba, Spain
4Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
5Hospital Universitario Insular de las Palmas, Gran Canarias,
Spain
6Hospital Universitario Virgen de la Macarena, Sevilla, Spain
7Hospital Central de Asturias, Asturias, Spain
8Hospital del Mar, Barcelona, Spain
9Hospital Marques de Valdecilla, Santander, Spain
Bone mineral density (BMD), the major determinant of fracture risk, is
under strong genetic control. Although polymorphisms of the vitamin D receptor (VDR) gene
have been suggested to account for some genetic variation in bone mass, the influence of
VDR genotypes on osteoporosis remains controversial. We examined the possible effect of
these genotypes on postmenopausal bone loss.
Patients and methods:We performed a multicenter trial in 211
postmenopausal women (mean age 54.3). VDR polymorphisms were determined by RFLP using Bsm
I (intron 8), Apa I (intron 8) and Taq I (T1055-C) after PCR. For the hypothesis contrast
and for every polymorphism, we have analysed the genotypes separately and after we have
analysed grouped in unfavorable genotypes and their relationship with bone loss rate. We
have also analysed the lumbar spine and femoral neck BMD. Bone mass was measured by DEXA.
The percent change in BMD at 2 yr was used to classify subjects as slow bone loss
(<OR=1.4%), intermediate (between 1.4 and 2.7%) and fast bone loss patients (estimated
annual bone loss >2.7%).
Results:The prevalence of genotypes were BB: 22.3%, Bb 40.2% and bb:
37.5%; AA 31.3%, Aa 41.1% and aa 27.7% and TT 48.2%, Tt 51.8% and tt 18.8 %. Distribution
of different genotypes, grouped and ungrouped, was similar between the three groups of
annual bone loss rate, neither did we find any significant association between the
differents genotypes and bone mass loss.
Conclusions:In this study, VDR genotype is not associated with the annual
bone loss in postmenopausal women
This study was supported by Novartis.
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P-27
INFLUENCE ALLELES OF VDR3, COL1A1 AND CALCR GENES ON SPEED OF LOSS OF BMD
IN POSTMENOPAUSAL WOMEN
M. V. Moskalenko1*, M. V. Aseev1, I. E. Zazerskaya2,
V. S. Baranov1
1Lab. Pren. Diag., Ott's Inst. of Obst. & Gynecol. RAMS,
St-Peterburg, Russia
2Pavlov's State Medical University, St-Peterburg, Russia
Osteoporosis is a common disease affecting the majority of older
postmenopausal women. This skeletal disease characterised by bone mineral density, which
leads to impaired skeletal strength and increased susceptibility to fracture. A genetic
contribution to osteoporosis is well documented, but genes and alleles variants conferring
osteoporotic risk are largely undefined.
The distribution of functionally uncompleted alleles of VDR, COL1A1 and
CALCR genes in Russian population and in the group of the postmenopausal women was
investigated. The first group was the control group (174 women from north-west region of
Russia without osteoporosis). The second group was the group of 119 postmenopausal women
(all of them had been in menopause for 4-5 years).
After 12 months observation, we have allocated two groups of the
postmenopausal women differing on speed of loss of BMD. T-criterion have decreased during
12 month in group of women with fast loss of BMD (10±6.38%). T-criterion have during 12
month in group of women with slow loss of BMD (2±8.23%).
Genomic DNA from peripheral blood was extracted by standards methods with
using proteinase K. The polymorphisms of VDR3, COL1A1 and CALCR genes were studied by
PCR-RFLP method (polymorphism VDR3/Taq I, COL1A1/Apa I and CALCR/Alu I).
The differences in allele combinations of VDR and Col1A1 genes between
group of control group and group of postmenopausal women with slow and fast loss of BMD
were proved. Frequencies of functionally impaired alleles of VDR gene (t allele) was 32.7%
(first group), 13.9% (group with slow loss of BMD), 46.8% (group with fast loss of BMD)
respectively. Frequencies of functionally impaired alleles of COL1A1 gene (s allele) was
17.9% (first group), 2.4% (group with slow loss of BMD), 30.8% (group with fast loss of
BMD) respectively. Frequencies of functionally impaired alleles CALCR (T allele) of gene
was 73.8% (first group), 66.7% (group with slow loss of BMD), 81.3% (group with fast loss
of BMD) respectively. Analysis of VDR, Col1A1 and CALCR alleles frequencies in two groups
of postmenopausal women proved significant association between BMD loss values (speed) and
particular alleles of the corresponding genes in these patients (p<0.001).
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P-28
POLYMORPHISMS IN THE PROMOTER OF THE INTERLEUKIN-6 GENE ARE NOT
ASSOCIATED WITH OSTEOPOROTIC FRACTURES OR BONE MASS
B. L. Langdahl*, M. Carstens, L. Stenkjęr, E. F. Eriksen
Dept. of Endocrinology, Aarhus University Hospital, Aarhus, Denmark
Interleukin-6 (IL-6) is a potent stimulator of bone resorption and IL-6
levels are elevated in postmenopausal women compared to premenopausal or HRT treated
women. It has previously been shown that an AT rich VNTR polymorphism in the 3' UTR of the
IL-6 gene is associated with bone mineral density in both pre- and postmenopausal women.
Three polymorphisms have been identified in the promoter region:G-597-A, G-572-C
and G-174-C. G-174-C has been demonstrated to be associated with
production and serum levels of IL-6. We therefore wanted to investigate if these
polymorphisms affect the prevalence of osteoporotic fractures and bone mass in 342
osteoporotic patients and 369 normal controls. The polymorphisms were examined by RFLP
using Mnl I, Aci I and Nla III after PCR. BMD was examined by DXA.
The G-597-A polymorphism was not found in our population. The
genotype distribution of G-572-C was 0% CC, 6.3% GC and 93.7% GG in
osteoporotic patients and 0.8% CC, 7.7% GC and 91.5% GG in normal controls (chi-sq.=2.69,
p=0.26). The genotype distribution of G-174-C was 26.5% GG, 54.4% GC and 19.2%
CC in osteoporotic patients and 29.4% GG, 49.2% GC and 21.4% CC in normal controls
(chi-sq.=1.43, p=0.49). Both polymorphisms were in Hardy-Weinberg equilibrium and in
linkage equilibrium. Combining the genotypes resulted in 6 different haplotypes which were
distributed similarly in osteoporotic patients and normal controls (chi-sq.=4.63, p=0.46).
BMD of the lumbar spine (ls), femoral neck (fn) and total hip (th) in the
different genotypes are presented below (mean±SD). BMD was not different between
individuals with the different genotypes or combined genotypes at any site.
In conclusion: Three polymorphisms in the promoter of the IL-6 gene are
not associated with osteoporotic fractures or BMD at the spine or hip.
Genotype |
No |
BMD(ls) |
BMD(fn) |
BMD(th) |
-572:CC |
3 |
0.870±0.077 |
0.724±0.086 |
0.851±0.124 |
-572:GC |
43 |
0.898±0.164 |
0.734±0.099 |
0.828±0.130 |
-572:GG |
547 |
0.868±0.184 |
0.702±0.123 |
0.817±0.153 |
ANOVA |
|
0.59 |
0.25 |
0.85 |
-174:GG |
169 |
0.883±0.180 |
0.710±0.116 |
0.829±0.150 |
-174:GC |
304 |
0.859±0.182 |
0.698±0.124 |
0.811±0.158 |
-174:CC |
130 |
0.885±0.180 |
0.713±0.121 |
0.825±0.143 |
ANOVA |
|
0.25 |
0.38 |
0.51 |
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P-29
INTERLEUKIN-6, TRANSFORMING GROWTH FACTOR BETA AND BONE TURNOVER DECREASE
WITH POST-TRANSPLANT PERIOD
V. Kusec1*, R. Smalcelj2, D. Rogic1, Z.
Puretic2
1Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
2Dialysis unit, Clinical Hospital Centre, Zagreb, Croatia
Disturbance of bone metabolism and renal osteodystrophy which develop in
the course of chronic renal failure and continue after kidney transplantation are a major
risk factor for the skeletal integrity. There is considerable evidence that bone turnover
is increased and bone density decreased in kidney transplant recipients. Several cytokines
involved in bone turnover, e.g. interleukin 6 (IL-6) and transforming growth factor beta
(TGF beta) are also influenced by PTH, mostly secreted in excess in kidney transplant
recipients. Involvement of IL-6 and TGF beta has not been demonstrated in bone disorders
after kidney transplantation. The aim of this study was investigation of IL-6 and TGF beta
concentrations in relationship to post- transplantation time and biochemical markers of
bone turnover. This study comprised 19 patients within the first post-transplant year and
45 with post-transplant period of 15-175 months. None of the patients had rejection crisis
at least three months before the commencement of the study. IL-6, TGB beta, intact PTH,
bone alkaline phosphatase (BALP), osteocalcin (OC) and procollagen type I propeptide
(P1CP) were measured in serum and deoxypyridinoline crosslinks (DPD) in urine by
commercial ELISA kits. In 16 kidney transplant recipients within the first post-
transplant year biochemical analyses were performed twice with a 3-4 month interval, and
once in other subjects. IL-6, TGF-beta, BALP, OC and DPD were significantly higher
(p<0.02 to p<0.0002) in the first post-transplant year than later period.
Statistically significant negative correlation (p<0.03 to p<0.0009) with post-
transplantation time was found for IL-6, BALP, OC, P1CP, DPD and for TGF-beta only in the
first twelve months after transplantation. Also, a significant difference (p<0.01) was
found between two measurements only for TGF beta in the first 12 months after
transplantation. No difference was found between sexes or correlations of measured
parameters with age. These results indicated that higher bone turnover in the first
post-transplant year corresponded with higher levels of cytokines IL-6 and TGF beta, most
probably involved in bone remodeling of kidney transplant recipients. Decrement of IL-6,
TGF beta and bone turnover with post-transplantation time suggested cessation or even
improvement of the existing bone disorder.
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P-30
GENE THERAPY OF HUMAN OSTEOSARCOMA CELLS
K. Trieb
Dept. of Orthopedics, University of Vienna, Austria
The five-year survival rate in osteosarcoma patients could be increased
frome 20% to about 70% due to the combination of surgery and neoadjuvant chemotherapy. In
the remaining 25% the problem of none response to chemotherapy remains. Heat shock
proteins (hsp) have been shown to play a role in tumor biology and drug resistance. Hsps
are predictive and prognostic markers in many cancers. It is the aim of this study to
investigate the effect of a stable transfection of osteosarcoma cells with hsp27 on the
susceptibility to chemotherapeutic drugs and cell growth.
The human osteosarcoma cell line HOS-85 was transfected with the
expression vector pSG-2711, which contains the complete human hsp27 gene with its
promotor, a SV 40 promotor and a neomycin resistance gene. The same vector without the
hsp27 gene served as control. The transfection was documented by PCR analysis and Western
Blot.
The transfection of cells resulted in a lower proliferation rate, tested
by 3H- thymidine incorporation. The effect of transfection on sensitivity to
drugs depended on the substance used. Transfected cells were sensitiver to the toxic
effects of adriamycin, bleomycin and cisplatin, whereas no difference could be observed
for four other chemotherapeutica tested.
This results show that the transfection of osteosarcoma cells can change
the response to chemotherapeutic therapy. The increase in drug sensitiveness and the
decreased proliferation may lead to a new therapeutic concept in the treatment of
osteosarcoma.
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P-31
ASSOCIATION OF THE C-ALLELE OF THE C677T MUTATION IN THE METHYLENE
TETRAHYDROFOLATE REDUCTASE GENE WITH AN INCREASED RISK OF OSTEOPOROTIC FRACTURES: A CASE
CONTROL STUDY IN DANISH POSTMENOPAUSAL WOMEN
H. L. Jorgensen1*, J. S. Madsen2, B. Madsen1,
M. M. A. Saleh3, B. Abrahamsen4, M. Fenger1, J. B.
Lauritzen3
1Department of Clinical Biochemistry, Hvidovre University
Hospital, Denmark
2Department of Clinical Biochemistry, Odense University
Hospital, Denmark
3Department of Orthopaedic Surgery, Hvidovre University
Hospital, Denmark
4University Department of Endocrinology, Aarhus Amtssygehus,
Denmark
Background: Recently, a point mutation in the gene coding for methylene
tetrahydrofolate reductase, MTHFR (C677T), giving rise to a thermolabile variant of MTHFR,
has been shown to significantly influence lumbar spine BMD in Japanese women (Miyao M et
al. Calcif Tissue Int 2000; 66: 190-194).
The aim of this study is to investigate the influence of this mutation on
peripheral measures of bone density and on the odds ratios for hip and wrist fracture in a
case control study of Danish postmenopausal women.
Methods: 74 women with wrist fracture, 41 women with hip fracture and 207
age- matched controls were included. All had broadband ultrasound attenuation (BUA) and
speed of sound (SOS) measured at the heel as well as bone mineral density (BMD) measured
by dual X-ray absorptiometry at the distal forearm. The MTHFR (C677T) genotypes were
determined using polymerase chain reaction restriction fragment length polymorphism
(PCR-RFLP).
Results: Only two patients out of 21 with the TT genotype had a fracture
(ratio of fracture patients to controls, Fx/Cont: 0.11) as opposed to 46 out of 142 with
the CT genotype (Fx/Cont: 0.48) and 67 out of 159 (Fx/Cont: 0.73) with the CC genotype
(chi-square (df=2): 9.8, p=0.007).
Using logistic regression with age, height, weight, age at menopause,
BUA, SOS, lower forearm BMD and the MTHFR genotypes as co-variates, the following odds
ratios were found when comparing the individuals homozygotic for the C-allele with those
homozygotic for the T-allele: Wrist fracture OR=3.93 [1.25;12.40, p=0.02], hip fracture
OR=6.99 [1.35; 36.92, p=0.02] and the fracture types combined OR=4.33 [1.73;10.81,
p=0.002].
Even when comparing the individuals homozygotic for the C-allele to the
heterozygotic individuals, the effect remains significant: Wrist fracture OR=1.98
(1.12;3.52, p=0.02), hip fracture OR=2.64 (1.17;5.99, p=0.02) and the fracture types
combined OR=2.08 [1.32;3.29, p=0.002).
No significant association of the MTHFR (C677T) genotypes was found with
BMD at the lower forearm or with ultrasound parameters measured at the calcaneus.
Conclusion: The C-allele of the MTHFR (C677T) mutation is associated with
a highly significant increase in the odds ratio of hip fracture and wrist fracture in
Danish postmenopausal women.
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P-32
IDENTIFYING THE GENES UNDERLYING THE OSTEOPETROTIC TOOTHLESS AND INCISORS
ABSENT MUTATIONS IN THE RAT
L. Van Wesenbeeck1*, P. R. Odgren2, C. MacKay2,
S. C. Jr. Marks2, W. Van Hul1
1Dept. of Medical Genetics, University of Antwerp, Belgium
2Dept. of Cell Biology, University of Massachusetts Medical
School, Worcester, MA, USA
The mammalian osteopetroses are a heterogeneous group of skeletal
disorders characterized by an increased bone mass due to a reduced bone resorption. Two
osteopetrotic mutations in the rat, toothless (tl) and incisors absent (ia)
rat, affect different genes and impact skeletal biology in different ways. Whereas tl rats
have severe, unremitting osteoclastopenia, ia rats have numerous small, non-functioning
osteoclasts which recover over the first few postnatal months. The tl mutation impairs an
osteoblast-derived, osteoclastogenic signal while the ia affects a gene product needed by
the differentiated osteoclast for ruffled border formation and normal resorptive function.
Despite the fact that the osteopetrotic mutants tl and ia have been extensively studied,
the primary genetic defects underlying both conditions are still unknown.
In order to identify these mutations, outcrosses were set up between the
inbred strain, tl or ia, and the Brown Norway strain, generating F1 animals that are
obligatory carriers of the osteopetrotic mutations. Next an intercross between the animals
from the F1 generation was performed. At this moment we have 11 affected F2 animals of the
tl rat and 26 affected F2 animals of the ia rat available for this study. An extended set
of polymorphic DNA markers are currently being analysed on these rats to localize the
underlying genes.
This procedure already enabled us to exclude the TNFSF11 (also called
TRANCE, RANKL, ODF and OPGL) gene for the tl rat, despite some phenotypic similarities
between tl rats and TNFSF11 null mice. The chromosomal localization of the rat TNFSF11
gene in the proximal part of chromosome 15q was deduced from synteny between rat, human
and mouse. Informative polymorphic markers spanning this region were analysed but the
phenotype turned out not to cosegregate with the chromosome 15 markers, excluding the
TNFSF11 gene as the disease causing gene. Currently we are checking both mutations for
other candidate genes in parallel with an ongoing genome scan.
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P-33
ESTROGEN RECEPTOR ALPHA GENE POLYMORPHISM PREDICTS RADIOGRAPHIC
OSTEOARTHRITIS OF THE KNEE IN ELDERLY MEN AND WOMEN
A. P. Bergink1,2*, J. van Meurs1, P. Arp1,
Y. Fang1, A. Hofman2, C. M. van Duijn2, J. P. T. M. van
Leeuwen1, A. G. Uitterlinden1,2, H. A. P. Pols1,2
1Department of Internal Medicine, Erasmus Medical Centre,
Rotterdam, The Netherlands
2Department of Epidemiology & Biostatistics, Erasmus
Medical Centre, Rotterdam, The Netherlands
Objective. Osteoarthritis (OA) is a common disabling disease in
the elderly. Genetic influences have been shown to play an important role in the etiology,
but the genes involved are ill defined. We studied the influence of two polymorphisms in
the ERalpha gene on the prevalence of radiographic osteoarthritis (ROA) of the knee in the
Rotterdam Study.
Methods. The study group consisted of 1487 men and women drawn
from the Rotterdam Study, a large population-based follow-up study of the elderly. The
PvuII restriction fragment length polymorphisms (RFLP) and the XbaI RFLP in the first
intron of the ERalpha gene were determined in relation to each other by direct molecular
haplotyping. Radiographs of the knee were evaluated using the Kellgren score. ROA of the
knee was defined as having a Kellgren score of two or higher at one or both knees.
Results. Three different haplotype alleles were found: allele 1
(px, 54.2 %), allele 2 (PX, 33.6 %) and allele 3 (Px, 12.2%). Allele 2 was associated with
an increase in the prevalence of knee ROA: in subjects without allele 2 the percentage of
ROA was 21.8%, in those carrying one copy 24.2 % and in those with two copies 33.5%. The
corresponding, for confounding factors adjusted, odds ratios were 1.3 (95% C.I.: 0.9 to
1.7) for heterozygous and 2.0 (1.3 to 3.1) for homozygous subjects. Separate analyses for
men and women showed similar effects. When we considered the different characteristics of
ROA, osteophytosis and joint space narrowing, the association between ROA and the ERalpha
haplotype seemed to be driven by the osteophytosis.
Conclusion. In both elderly men and women, variations in the
ERalpha gene are associated with radiographic osteoarthritis of the knee.
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P-34
INFLUENCE OF ESTROGEN RECEPTOR ALPHA POLYMORPHISMS ON BONE MINERAL
DENSITY AND EFFECTIVENESS OF ALENDRONATE TREATMENT IN PATIENTS WITH OSTEOPOROSIS
B. Arko1*, J. Marc1, J. Prezelj2, A.
Kocijancic2
1Faculty of Pharmacy, University of Ljubljana, Ljubljana,
Slovenia
2Department of Endocrinology and Metabolic Diseases, Clinical
Centre, Ljubljana, Slovenia
BACKGROUND AND OBJECTIVES: Numerous studies have shown that bone mass and
bone turnover are under genetic control. Because of the important role of estrogens in
bone metabolism gene for estrogen receptor alpha (ER alpha) represents one of the
candidate genes, which could be involved in the development of osteoporosis.
The purpose of this study was to assess whether bone mineral density
(BMD) and its change in response to alendronate treatment is related to PvuII and XbaI
polymorphisms of the ER alpha gene in postmenopausal women.
METHODS: 59 postmenopausal Slovenian women (aged 65.2±6.7 years) with
osteoporosis were studied. BMDs at the lumbar spine and hip were measured by dual X-ray
absorptiometry at the start and after one year of treatment with alendronate (Fosamax,
MSD). Genomic DNA extracted from whole blood was amplified by polymerase chain reaction
and genotypes determined after digestion with PvuII and XbaI restriction enzymes.
RESULTS: The frequencies of PvuII and XbaI genotypes were: PP 13.6%, Pp
57.6%, pp 28.8% and XX 11.9%, Xx 54.2%, xx 33.9%, respectively. Of the possible nine
haplotypes only six were found. Four of them had frequency higher than 5%: PpXx 50.8%,
ppxx 27.1%, PPXX 11.9% and Ppxx 6.8%.
Statistically significant association of XbaI polymorphism with baseline
z-scores of lumbar spine (p=0.027) and hip (p=0.033) BMD was demonstrated. The highest z-
score was in both cases associated with XX genotype and the lowest with xx genotype. In
contrast, no association of PvuII polymorphism with baseline z-scores of BMDs at either
site was found. The increase in lumbar spine and hip BMD after one year of therapy did not
differ with respect to PvuII and XbaI genotypes.
CONCLUSION: Our results suggest that polymorphisms of the ER-alpha gene
may represent a genetic determinant of BMD but they probably do not contribute to the
effectiveness of alendronate treatment.
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P-35
FOKI-POLYMORPHISMS OF THE VITAMIN D RECEPTOR GENE ARE ASSOCIATED WITH
25(OH)VITAMIN D3 LEVELS IN ELDERLY RESIDENTS OF AUSTRIAN NURSING HOMES
B. M. Obermayer-Pietsch*, A. Fahrleitner, W. Li, P. Maritschnegg, J. C.
Piswanger- Sölkner, C. M. Bonelli, G. Leb, H. Dobnig
Dept. of Internal Medicine, Karl-Franzens-University Graz, Austria
BACKGROUND: Vitamin D receptor gene alleles were reported to influence
bone metabolism and possibly bone fractures. Low vitamin D levels and concomitant
disturbances of bone metabolism and muscle function are known to be major problems in
elderly people in nursing homes. In this study, we evaluated FokI and BsmI polymorphisms
of the vitamin D receptor (VDR) gene and parameters of bone and mineral metabolism to
evaluate possible associations with bone turnover markers, neuromuscular function and bone
fractures.
METHODS: We investigated 1254 elderly Caucasian residents (mean age 83±6
years) living in 95 Austrian nursing homes. Medical history, past bone fractures and
muscle strength were recorded. Serum measurements included routine chemistry parameters,
25(OH)vitamin D3, PTH, sCTX, and osteocalcin. Patients with impaired liver or kidney
function or primary hyperparathyroidism were excluded from the study. DNA was prepared
from peripheral leukocytes and genotyped for the VDR BsmI and FokI polymorphisms.
RESULTS: 25(OH) vitamin D3 levels were markedly low in this population
(11.2±6 ng/ml, normal range [9-45]). Sixty-seven percent of the residents had vitamin D
serum levels <9 ng/ml thus considered vitamin D-deficient and only 3% had levels above
25 ng/ml. Frequencies of VDR genotypes paralleled the general population. Patients with
VDR FokI genotypes 'ff' had significantly lower vitamin D levels (p=0.02) as compared to
other genotypes, whereas BsmI genotypes showed no differences in bone metabolism.
CONCLUSIONS: Lack of vitamin D is a major problem in elderly residents of
nursing homes. Disability, neurophysiological disturbances and low BMD contribute to the
risk for bone fractures. FokI genotypes of the VDR may be involved in the regulation of
bone and mineral metabolism. Therefore, VDR genotyping could contribute to define nursing
homes residents at high risk for bone disease.
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Hormones
P-36
A DIRECT, NON-EXTRACTION ENZYME IMMUNOASSAY FOR MEASUREMENT OF
25-HYDROXYVITAMIN D
D. Laurie*, A. K. Barnes, M. J. Gardner, R. T. Duggan
IDS Ltd, Boldon Business Park, Boldon, UK
Measurement of serum or plasma 25-hydroxyvitamin D (25-OH D) is the
preferred method for establishing a patient's vitamin D status, including identification
of vitamin D insufficiency. Previously, all 25D measurements have required extraction of
sample to remove protein prior to analysis. This has applied equally to all methods of
analysis, including HPLC, protein binding assays and immunoassays. IDS has developed a
displacer reagent that enables measurement of 25-hydroxyvitamin D by immunoassay using
serum samples directly, without the need for extraction.
The direct 25-OH D assay comprises dilution of 25µL of serum or plasma
with 1mL of a reagent containing binding protein displacers and a vitamin D - biotin
conjugate. A portion of the diluted sample (200µL) was added to an antibody-coated
microtitre plate and incubated for 2 hours at room temperature. After a wash, avidin-
peroxidase reagent (200µL) was added, incubated 30 minutes and washed. TMB substrate
(200µL) was added and colour allowed to develop for 30 minutes. After addition of 0.5M
HCl (100µL) the absorbance was recorded at 450nm. A calibration curve was prepared from
the absorbance values for 25OH-D serum standards (4 - 250nM) using a 4-parameter curve
fit.
The sensitivity of the direct 25-OH D EIA (the concentration
corresponding to the mean minus two standard deviations of the '0' calibrator) was less
than 6nM. The assay gave good precision with samples; intra-assay precision was 4.6%, 3.1%
and 5.0% at 22.1nM, 62.0nM and 118nM respectively; inter-assay precision was 7.5%, 11.5%
and 8.6% at 20.7nM, 56.5nM and 104nM respectively. Linearity was good with a mean for
observed / expected of 117% for samples diluted 1:2 and 1:4 with the '0' calibrator.
Cross-reactivity (at 50% displacement) for 25-OH D2 was 74%.
Correlation against an extraction radio immunoassay (IDS Gamma B RIA, AA-
35F1) was carried out with 530 patient samples. Samples were measured in both the direct
25-OH D EIA and the extraction RIA method in a back-to-back assay set up. The comparison
of methods by Passing-Babcock gave an equation of: Direct 25-OH D EIA = 1.00 x 25-OH D RIA
- 4.0 nM. A linear regression analysis gave a correlation coefficient of R2=0.80.
This technology provides a rapid, precise and sensitive non-isotopic
assay for the measurement of 25D without the need for samples to be extracted.
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P-37
IDENTIFICATION OF BONE MORPHOGENETIC PROTEINS IN URINE BY WESTERN
BLOTTING AND ELISA
V. Miljavac1*, L. Grgurevic1, D. Batinic2,
S. Vukicevic1
1Laboratory for Mineralized Tissues, Department of Anatomy,
School of Medicine, University of Zagreb, Croatia
2Clinical Institute for Laboratory Diagnosis, Clinical
Hospital, Zagreb, Croatia
Bone morphogenetic proteins are signaling molecules with numerous
functions. They are members of the larger transforming growth factor beta superfamily,
expressed not only in bone but also in most other adult tissues. BMP-7 appears to have
more diffused distribution. There is no evidence on the presence of BMPs in urine. To
detect BMP-7 in biological fluids we used a modified ELISA system based on the sandwiching
of the antigen between a monoclonal mouse 1B12 BMP-7 antibody and biotin labeled BMP-7.
The capture polyclonal rabbit anti-mouse antibody was coated directly onto 96-well
microtiter plates and was incubated overnight at +4 degrees Celsius. Wells without BMP-7
were used as a control. Signal was generated with a streptavidin-HRP-conjugate and
ortho-phenylene diamine as a substrate. Dilution curves showed good linearity and the
detection limit of the assay was 3 nanog/ml. To provide efficient concentration of
biological samples for SDS polyacrylamide electrophoresis and Western blot we used
Centricon Centrifugal Filter Devices YM- 10 and vacuum centrifuge. Protein electrophoresis
was carried out in polyacrylamide gels under conditions that ensure dissociation of the
proteins into their individual polypeptide subunits and minimize aggregation.
Electrophoretically separated components were transferred from a gel to membrane making
Gel over Membrane Sandwich. Membrane was incubated in 5 percent (w/v) nonfat dried milk
overnight at +4 deg C and then incubated with the BMP-7 antibody (Bunny 3330 plus 3331
Pool) in dilution 1 in 100. The level of BMP-7 in human urine was less than 10 nanog/ml.
The level of BMP-7 in urine from rats with chronic renal failure without therapy was less
then 3 nanog/ml. Urine from rats suffering from chronic renal failure and treated with
BMP-7 (20 microg/ kg) had BMP-7 levels approximately 10 nanog/ml. Significant amount of
BMP-7 was detected in urine of pregnant women. In addition to BMP-7, BMP-3 and BMP-6 were
detected in urine of healthy subjects. In conclusion, detection of BMPs in urine could
become an important assay for kidney and bone diseases.
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P-38
ANTI-INFLAMMATORY PROPERTIES OF BONE MORPHOGENETIC PROTEIN-7 MIGHT BE
BENEFICIAL IN THE TREATMENT OF INTESTINAL BOWEL DISEASE
I. Maric1*, L. J. Poljak2, S. Zoricic1,
D. Bobinac1, D. Bosukonda3, K. Sampath3, S. Vukicevic2
1Department of Anatomy, Medical School, University of Rijeka,
Rijeka, Croatia
2Department of Anatomy, Medical School, University of Zagreb,
Zagreb, Croatia
3Creative BioMolecules, Hopkinton, Massachusetts 01748, USA
Abundant expression of bone morphogenetic protein-7 (BMP-7) in the
developing intestine, and the association of the chronic intestinal inflammation and the
malignant intestinal cell phenotype with alterations in the SMAD signalling pathway argue
for an important, unrecognized role of BMPs in the pathophysiology of the gut. In the
present study we evaluated the effect of recombinant human BMP-7 for the treatment of
trinitrobenzenesulfonic (TNB) acid induced inflammatory bowel disease (IBD) in rats.
Bioavailability studies in normal rats indicate that circulating iodinated BMP-7 declines
with a half-life of 30 min upon systemic administration. A significant portion of 3.6% and
4.6% of the administered BMP-7 dose/g tissue is targeted for BMP-7 receptors in the
stomach and the ileum, respectively. We show that BMP-7 treated animals developed a much
less severe form of colitis as determined by preserved macroscopic appearance, normal body
weight, less diarrhea and rectal bleeding, lack of ulcers and adhesions, and reduced
histological damage score when administered 1.5 h before or 24 h after colitis induction.
Rats were killed 2, 5, 10, 14 and 30 days following TNB administration. Histochemical and
molecular analyses demonstrate that BMP-7: a) preserves microarchitecture of the colon
tissue, b) suppresses inflammation by downregulating the expression of intercellular
adhesive molecule, and prevents the accumulation of neutrophils, c) stimulates wound
repair and modulates mucosal immunity by downregulating TNF-alpha and TGF-beta, and by
inhibiting IL-6 expression, d) modulates the expression of its specific ALK-2 receptor.
Collectively, these results suggest that BMP-7 prevents and restores the
loss of gut function associated with TNB induced colitis, by a mechanism which makes it
unique among many other agents reported to be beneficial in the treatment of IBD.
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P-39
THYROID DYSFUNCTION AND BONE MINERAL DENSITY IN EARLY POSTMENOPAUSE
J. Jelcic1*, V. Kusec2, T. Kifer1, M.
Korsic1
1Department of Endocrinology, Internal Medicine Clinic,
Clinical Hospital Centre, Zagreb, Croatia
2Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
The most common form of primary osteoporosis occurs in the postmenopause
with most of the bone mass lost in the early postmenopause. Hyperthyroidism is one of the
major risk factors for secondary osteoporosis, due to increased bone turnover and
accelerated bone loss. The aim of this study was to investigate bone density in
postmenopausal women with thyroid dysfunction. This retrospective analysis comprised 42
hyperthyroid, 92 hypothyroid and 562 euthyroid patients aged 40-85 years. Bone mineral
density (BMD) was measured at the lumbar spine and femoral neck by dual X-ray
absorptiometry. Patients with hypothyroidism were significantly younger than those without
thyroid disorder (59.7 yr. vs. 62.1 yr.) and had significantly higher BMD in the lumbar
spine and hip. Analysis of data for age groups revealed that the statistically significant
difference (p<0.005) for the lumbar spine and hip BMD was found only for the 51-55 year
old patients. No difference existed between the other two pairs of patient groups, i.e.
the euthyroid and hyperthyroid and the hypothyroid and the hyperthyroid. There was no
difference in incidence of osteopenia (T-score<-1) and osteoporosis (T-score<-2.5)
between the three patient groups. These results suggest a protective effect of
hypothyroidism on the skeleton in the early postmenopause probably through decelerated
bone turnover and bone loss.
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P-40
BONE MORPHOGENETIC PROTEIN-7 PROMOTES HEALING OF LONG BONE DEFECTS IN THE
RAT FEMUR BY PREFABRICATED BONE GRAFTS CREATED IN THE EPIGASTRIC FLAP
R. Zic1*, S. Martinovic2, V. Kusec3, Z.
Vlajcic1, M. Jelic2, S. Vukicevic2
1Department of Surgery, Clinical Hospital Dubrava, Zagreb,
Croatia
2Department of Anatomy, School of Medicine, University of
Zagreb, Croatia
3Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
The aim of this study was investigating conditions which promote healing
of the long bone defects by means of prefabricated bone graft in the epigastric flap.
Helistat (collagen sponge 0.5 cm) or demineralised bone matrix (0.5 cm from rat long bone
diaphysis) were inserted in the epigastric flaps in rats under aseptic conditions. Two
doses (low, 30 microg/ml and high 120 microg/ml) of BMP-7 were added to both Helistat and
demineralised bone matrix. In the control group only implants were used. After 6 weeks the
bone graft was transferred from the epigastric flap into 0.8 cm long defect in the femur
and fixed with intramedullary K- wire. Two prefabricated bone grafts from each
experimental and control groups were used for histological analysis. Eight weeks after
surgery X-ray was performed and femurs harvested. Healing of the bone defect as assessed
by x-rays was observed in animals treated with a high BMP-7 dose graft. Histologic
analysis of the bone grafts showed that BMP-7 stimulated bone formation in both the
Helistat and demineralised bone implants, but dose-effect was observed only for the
Helistat implants. Histology of the femur specimens showed healing of bone defect with
prefabricated implants only in the high dose BMP-7 groups (both Helistat and demineralised
bone matrix) with optimal retention of the bony fragments. Demineralised bone matrix was
found to be superior to Helistat in supporting bone formation both in the epigastric flap
and in the femur bone defect. Non-union remained if prefabricated non-treated or low BMP-7
dose implants were transferred into the femur gap. Retention of bone defects was a primary
technical problem in this study. In conclusion, results of this pilot study in rats
suggest that BMP-7 prefabricated bone grafts accelerate healing of long bone defects.
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P-41
HYPOPHOSPHATEMIA REDUCES HYPERPARATHYROIDISM CAUSED BY VITAMIN D
DEFICIENT DIET IN RATS FOLLOWING NON-CHANGES IN SERUM CALCIUM LEVELS
R. Masuyama*, A. Daizo, M. Uehara, K. Suzuki.
Tokyo University of Agriculture, Tokyo, Japan
Objective: Parathyroid hormone (PTH) is mainly stimulated by the decline
in extracelluler concentration of calcium with the responsible reflection of calcium
sensor; located in parathyroid grand, which transfer the signal of hypocalcemia to
intracelluler PTH production. On the other hands, active form of vitamin D strongly
inhibits the production of PTH. Therefore, high levels of PTH are appeared in the status
of vitamin D deficiency, accompany with low levels of blood calcium. The alteration of
blood (serum) phosphate also concerns with the regulation of PTH secretion. The increase
of serum phosphate by the diet formulated with high contents of phosphorus, induces the
PTH secretion immediately without the decline in blood calcium concentration. Accordingly,
it has been suggested that the serum phosphate is a factor of PTH stimulation. To
determine the effect of serum phosphate on the PTH stimulation independent on other
factors such as blood calcium and vitamin D status, whether the PTH stimulation was
influenced by phosphorus depletion, we studied using the vitamin D deficient rats
maintained normal blood calcium concentration with vitamin D free-high calcium diet.
Materials and Methods: Five weeks old Wistar strain-vitamin D deficient rats (n=20) were
kept with a vitamin D free-high calcium diet (phosphorus replete) for 50 days, the diet
was changed to a phosphorus-restricted diet (vitamin D free) in half of these mice. At the
day of 56th, vitamin D was started to be supplemented orally for the half-rats of each
phosphorus replete or restricted group while the other half-rats were continued to be fed
two kinds of vitamin D free diets. At the day of 63rd, aged 13 weeks old, blood samples
were collected. Results: Calcium absorption was increased by each vitamin D
supplementation or phosphorus restriction in the diets. Therefore, serum calcium levels
were reflected from results of the absorption. Serum phosphorus was decreased by the
phosphorus-restricted diet. Phosphorus restriction significantly reduced PTH levels
stimulated by vitamin D deficient status, despite no remarkable changes were observed in
serum calcium levels. Conclusion: PTH-secretion might be regulated with the levels of
serum phosphorus independently of serum calcium and vitamin D alterations.
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P-42
EVIDENCE FOR A ROLE OF ESTRADIOL IN THE CONTROL OF BONE MINERALIZATION
M. van Driel*, C. J. Buurman, M. Koedam, H. A. P. Pols, J. P. T. M. van
Leeuwen
Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The
Netherlands
Both a reduced quantity and quality of bone increase the risk for
osteoporotic fractures. The elasticity of bone and its resistance to fracture is related
to the degree of mineralization. When mineralization reaches too high levels, bone has a
decreasing ability to absorb impact energy, becomes more stiff and brittle and more
susceptible to fractures. Postmenopausal estrogen deficiency reduces bone mass, but its
effect on the degree of mineralization of the remaining bone is not clear.
The aim of the current study was to assess the effects of estradiol on
mineralization by human osteoblasts. For this the human osteoblast cell line SV-HFO, that
proceeds through different stages of differentiation in culture, including extracellular
matrix formation and mineralization, was cultured for 23 days in the continuous presence
of 17beta-estradiol (10-10 - 10-7 M) or its antagonist ICI 182.780
(10-9 - 10-6 M). Mineralization and alkaline phosphatase activity
were measured at different time points during differentiation.
Calcium deposition in the extracellular matrix starts at day 12 of
culture and increases during time. Both in 17beta-estradiol and ICI 182.780 treated
cultures, a reduction of 10-20% was found in the total amount of calcium deposited during
the whole culture period, with most pronounced effects at later time points (days 19-23).
This was observed for both compounds at all concentrations tested except at 10-8
M. In addition, both 17beta-estradiol and ICI 182.780 reduced alkaline phosphatase
activity by 10-20%, with most pronounced effects in the period preceding the peak activity
of alkaline phosphatase (days 7-14). Interestingly, this reduction was the strongest at
the same concentrations that most effectively reduce mineralization. No effects of
17beta-estradiol and ICI 182.780 were observed on the DNA amount of the cells during
culture.
Besides the known role in controlling bone turnover via regulation of
osteoclast function, on basis of the current data a new role for estradiol can be
postulated: control of the extent or set-point of mineralization, which potentially
contributes to the quality of bone. The comparable effects of both 17beta-estradiol and
ICI 182.780 point to reconsider the view of ICI 182.780 as an estradiol antagonist in
bone.
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P-43
HEALING OF SKIN NECROSIS AND REGRESSION OF ANTICARDIOLIPIN ANTIBODIES
ACHIEVED BY PARATHYROIDECTOMY IN A DIALYSED FEMALE PATIENT WITH CALCIFIC URAEMIC
ARTERIOLOPATHY
S. Sefer1*, R. Trotic2, V. Degoricija1,
M. Vrsalovic1, I. Ratkovic-Gusic1, P. Kes1
1Department of Intermal Medicine, Sisters of Mercy University
Hospital, Zagreb, Croatia
2Department of Otorhinolaryngology and Head and Neck
Surgery,Sisters of Mercy University Hospital, Zagreb, Croatia
Aim: To present the impact of parathyroidectomy on the spontaneous
healing of necrotic lesions of the skin of the lower leg and on anticardiolipin antibodies
regression in a 68 years old female dialysed patient with hyperparathyroidism and
calcific-uraemic arteriolopathy (CUA).
Methods: After the occurrence of initial lesions of the lower leg skin,
the intact parathyroid (iPTH) level, calcium (Ca) and phosphorus (P) product were
measured, and on two occasions at 6 weeks' intervals, the titer of anticardiolipin
antibodies was determined, followed by a clinical monitoring of the progress of necrotic
skin lesions. Two months after the occurrence of skin lesions, the patient's right leg was
amputated below the knee due to gangrene, and a histopathological analysis of the bioptic
skin tissue of the amputated lower leg was made. After parathyroidectomy, iPTH, Ca and P
product were measured, and on two occasions at 6 weeks' intervals, anticardiolipin
antibodies titer was determined.
Results: Before parathyroidectomy, an increased iPTH level, Ca and P
product, as well as on two occasions at 6 weeks' intervals an elevated IgG anticardiolipin
antibodies titer were determined. The histopathological analysis of the bioptic skin
tissue of the amputated right lower leg showed mural calcification of
artery walls and a thrombotic occlusion of small arteries, arterioles and dermal
capillaries involving epidermolysis.
One week after parathyroidectomy, iPTH level and Ca and P product were
within normal range. Two measurements at a 6 weeks' interval revealed no anticardiolipin
antibodies. Eight weeks after parathyroidectomy, spontaneous healing of necrotic skin
lesions of the left lower leg was observed.
Conclusion: Regression of anticardiolipin antibodies, normalisation of Ca
and P product, and healing of the skin lesions after parathyroidectomy pointed to the
elevated PTH level as a crucial factor in the pathogenesis of CUA.
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P-44
EVALUATION OF BONE MINERAL DENSITY IN PATIENTS WITH PRIMARY
HYPERPARATHYROIDISM BEFORE AND AFTER SURGICAL TREATMENT
M. Misjak1*, V. Petric2, H. Tomic-Brzac3,
V. Altabas1, D. Globlek2, M. Sharma1
1Department Of Endocrinology, Diabetes And Metabolic Diseases,
University Hospital 'Sestre Milosrdnice', Zagreb, Croatia
2Department of Otorhinolaryngology, University Hospital
'Sestre Milosrdnice', Zagreb, Croatia
3Department of Nuclear Medicine, University Hospital Centre
'Rebro', Zagreb, Croatia
Aim: A sustained elevation in the secretion of parathyroid hormone (PTH)
in most patients with primary hyperparathyroidism (PHPT) results in increased bone
remodeling, demineralization, loss of skeletal mass and osteoporosis itself. It is
expected that surgical removal of the parathyroid gland(s) in question results in both the
cure and an improvement in bone mineral density.
Patients and methods: A clinical study was conducted involving 107
patients with PHPT hospitalized at our hospital in the period from 1994 to 2000. PHPT was
documented with elevated serum calcium, ionized calcium and PTH values. After surgical
treatment all parameters were normalized. Dual Energy X-ray Absorptiometry (DEXA) was
performed before and one year after parathyroidectomy.
The results were statistically assessed using the Student's T-test for
paired samples.
Results: The results showed a statistically significant increase in bone
mineral density one year after parathyroidectomy (total hip: p=0.025; femoral neck:
p=0.004; L2 - L4: p=0.04).
Conclusion: In patients with primary hyperparathyroidism the bone mineral
density significantly increase after surgical removal of hyperfunctional parathyroid
gland.
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P-45
ESTROGEN RECEPTOR EXPRESSION AND MEGAKARYOCYTE DIFFERENTIATION ARE
STIMULATED BY ESTROGEN
S. Bord1*, E. Frith1,2, D. C. Ireland1,
M. A. Scott2, J. I. O. Craig2, J. E. Compston1
1University of Cambridge School of Clinical Medicine,
Addenbrooke's Hospital, Cambridge, UK
2Department of Haematology, Addenbrook's Hospital, Cambridge,
UK
High-dose estrogen (E) has been shown to produce anabolic effects in the
skeleton. The mechanisms mediating these effects are unclear but may involve interactions
between cells on bone surfaces and in the bone marrow compartment. We have previously
demonstrated that in vivo the megakaryocyte population in human bone marrow increases with
E treatment suggesting a possible role in bone remodelling.
To investigate if E stimulates megakaryocytopoiesis CD34+ cells were
isolated from cord blood by magnetic bead technology (MACS) and cultured for 9 days in a
serum free collagen based system supplemented with thrombopoietin, interleukin-3 and-6,
plus or minus 10nM 17beta estradiol. Collagen films were dried, fixed and cells
immunolocalised for gpllla (CD61, a marker of early megakaryocyte maturation), gpllb/llla
(CD41, a MK specific antigen expressed by more mature MKs in CFU- MKs) and estrogen
receptors (ER) alpha and beta using specific polyclonal antibodies in an indirect
immunoperoxidase technique. Extent and intensity of staining were measured quantitatively
by image analysis.
Cells cultured in the presence of E formed more megakaryocyte colony
forming units (CFU-MKs) than untreated cells. These were highly CD61 positive with a
three- fold increase in expression in the E treated cells (p<0.05). CD41 was expressed
in CFU-MKs only in cultures treated with E. Low-level nuclear ER beta expression was seen
in many of the CD34+ cells. This was increased in the E treated cells with intense
staining in the CFU-MKs. ER alpha expression was mainly confined to cells within the
CFU-MKs. E stimulated the extent of ER alpha and ER beta expression six- (p<0.01) and
four-fold (p<0.001) respectively compared to untreated cultures.
These results demonstrate that in vitro E stimulates the colony forming
potential of CD 34+ cells and differentiation and maturation to a more megakaryocytic
phenotype. These increases together with the stimulated ER protein expression suggests
that megakaryocytes may play a role in mediating the E-induced anabolic effects seen in
bone.
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P-46
HISTOLOGICAL AND FUNCTIONAL STUDIES OF THE THYROID GLAND IN FEMALE RATS
CHRONICALLY EXPOSED TO CADMIUM
B. Pilat-Marcinkiewicz1*, M. Brzoska2, B. Sawicki1,
J. Moniuszko-Jakoniuk2
1Department of Histology and Embryology, Medical Academy,
Kilinskiego 1 Street., 15-230 Bialystok, Poland
2Department of Toxicology, Medical Academy, Mickiewicza 2C
street., 15-222 Bialystok, Poland
The effect of chronic (over a year) cadmium (Cd) administration (5 or
50mg Cd/l in drinking water) to young female rats on the structure and function of the
thyroid was evaluated. Thyroid sections, obtained using paraffin technique, were stained
with hematoxylin and eosin (H + E) and assessed in light microscopy. Serum levels of
thyroid hormones: triiodothyronine (T3) and tetraiodothyronine (T4) and thyroid
stimulating hormone (TSH) as well as Cd in the blood were determined.
The major changes in the thyroid gland were observed after exposure to 50
mgCd/l. Degenerative changes in some follicles and a rise in the number of large follicles
which were frequently lined with atypical epithelium were found in the thyroid.
Serum T3 and TSH concentration was not affected by Cd, while T4 was
reduced but only at the higher level of Cd exposure.
The results show that chronic exposure to Cd changes the structure of
thyroid gland and influences its function.
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P-47
BONE MASS OR BONE QUALITY IS THE MAJOR DETERMINANT OF FRACTURES IN
PRIMARY HYPERPARATHYROIDISM?
E. Csupor1*, J. Szucs1, E. Toth1, S.
Meszaros1, V. Ferencz1, P. Lakatos1, E. V. McCloskey2,
C. Horvath1
11st Department of Medicine, Faculty of Medicine, Semmelweis
University, Budapest, Hungary
2WHO Collaborating Centre for Metabolic Bone Diseases,
University of Sheffield, Sheffield, UK
Primary hyperparathyroidism (pHPT) has different clinical forms. Most
patients recurrently produces kidney stones while in others pHPT causes bone disease with
pain and fractures. The aim of our study was to measure bone density in the renal and
osseal forms of this disease and to estimate the role of bone mass in determining fracture
occurence.
72 patients with pHPT were studied: 66 women plus 6 men, aged 61.8±14.5
ys, 37 with renal stones and 35 with osseal symptoms. 17 of 72 patients had previously
fractures. BMD was measured at the spine and hip by DEXA and at the forearm by SPA.
Immunoradiometry was used to determine serum intact PTH level. Statistical evaluation used
Student's t-test, Chi2-test and correlation analysis.
Fractures occured more frequently in osseal (12/35, 34.0%) than in renal
group (5/37, 13.5%, p<0.05). BMD (as Z-score) was decreased at axial and peripheral
sites of both groups. No differences were found in BMD between renal and osseal groups at
the spine or hip while forearm density was lower in patients with bone symptoms
(0.51±0.03 vs 0.63±0.03, p<0.05). However, patients with or without fractures had the
same bone mineral density. Fracture incidence had no correlation with BMD, PTH or other
biochemical variables.
Our results clearly show that decreased mineral mass of bones is a
general feature of primary hyperparathyroidism independently of its clinical symptoms.
However, the lack of difference in BMD between fractured and non-fractured patients
suggest bone mass to be not the only major determinant of bone fragility in this disease.
Further research is needed to study other bone characteristics determining fracture risk
as quality and structure of bones.
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P-48
REGULATION OF OSTEOPROTEGERIN (OPG) AND OSTEOCALCIN BY TRIIODOTHYRONINE
(T3) AND 1,25-DIHYDROXVITAMIN D3 (1,25(OH)2D3) IN OSTEOBLAST LIKES CELLS WITH A DISTINCT
PHENOTYPE
F. Varga*, S. Spitzer, K. Klaushofer
Ludwig Boltzmann Institute of Osteology, 4th Medical Dept., Hanusch
Hospital, Vienna, Austria
Recently, OPG, a secreted member of the TNF-alpha receptor super family,
has been identified as an regulator of bone resorption and has been implicated in the
pathogenesis of post menopausal osteoporosis and other metabolic bone diseases. As a
soluble decoy receptor for RANKL, OPG prevents the interaction of RANKL with RANK and as a
consequence the formation and activity of osteoclasts.
Both 1,25(OH)2D3 and T3 are important for the development and maintenance
of the skeleton. Low serum concentration of 1,25(OH)2D3 results in rickets but
pharmacological levels lead to bone loss. On the molecular level 1,25(OH)2D3 increases the
expression of RANKL. Hypothyroidism in children causes severe
disturbances of bone growth and development while hyperthyroidism could
lead to osteoporosis. The goal of this study was to find out whether T3 acts on the
OPG/RANKL system.
MC3T3-E1 cells and two subclones of these cells (kindly provided by RT
Franceschi) were cultured in alphaMEM for 4 days and thereafter treated with T3,
1,25(OH)2D3 or both hormones. mRNA was isolated after 3 to 48 hours treatment time.
Northern hybridisation was performed according to standard laboratory protocols.
In MC3T3-E1 cells clone 4, an osteoblast like cell line with features of
a mature osteoblastic phenotype, we found that T3 time and dose dependently increased the
expression of OPG mRNA. OPG similar to osteocalcin was first detectable after 6 hours of
treatment. In normal MC3T3-E1 cells we only found a weak regulation of OPG while in clone
30 there was no regulation. Osteocalcin, however, was regulated in all cell lines but with
different intensity.
1,25(OH)2D3 dose dependently attenuated the T3 induced expression of both
OPG and osteocalcin. Interestingly, we found a tight relationship between the T3 and
1,25(OH)2D3: at 100 nM T3, 10 nM 1,25(OH)2D3 attenuated, while 100 nM inhibited the
expression of both genes. At 10 nM T3, 10 nM 1,25(OH)2D3 completely inhibited the
expression of both proteins in osteoblasts.
In summary, in differentiated osteoblast like cells, T3 increased the
expression of OPG and osteocalcin, which effect was attenuated by co-treatment with
1,25(OH)2D3. We conclude, that thyroid hormone and calcitriol interact in the regulation
of OPG and osteocalcin expression in osteoblasts.
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P-49
Abstract withdrawn.
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