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Hoj-2003 v7
BISPHOSPHONATES ENHANCE OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW STROMAL CELLS IN VITRO. FABIAN VON KNOCH, MARC KOWALSKY, IVAN MARTIN, CLAUDE JAQUIERY, ANDREW FREIBERG, DENNIS BURKE, HARRY RUBASH, ARUN SHANBHAG BIOMATERIALS RESEARCH LABORATORY, MASSACHUSETTS GENERAL HOSPITAL, HARVARD MEDICAL SCHOOL, BOSTON, MA,
RESEARCH DIVISION, DEPARTMENT OF SURGERY, UNIVERSITY OF BASEL, SWITZERLAND
INTRODUCTION
at a density of 400,000 cells/cm² in DMEM/F-12 medium
Bisphosphonates are well-recognized inhibitors of osteo-
supplemented with 10% fetal bovine serum, 1% antibiotics/
clast activity and are widely used in the treatment of various
antimycotics, L-glutamine (2mM), 10 mM ß-glycero-phosphate
metabolic bone diseases. Current indications include Paget's
and 0.1 mM L-ascorbic 2-phosphate at 37oC with 95%
disease, post-menopausal osteoporosis and hypercalcemia of
humidity and 5% CO . Cells were treated with three different
malignancy.1 Bisphosphonates are also considered for fibrous
bisphosphonates including 10-8M alendronate (Fosamax,
dysplasia 2 and other disorders affecting bone metabolism such
Merck, Rahway, NY), 10-8M risedronate (Actonel, Proctor
& Gamble, Cincinnati, OH), 10-8M zoledronate (Zometa,
Bisphosphonates are being investigated for their ability to
Novartis, Basel, Switzerland), positive controls (addition of 10-
prevent bony erosions in rheumatoid arthritis, osteoarthritis
8M Dexamethasone or 10-8M Vitamin D) and negative control
and peri-implant bone resorption around joint replacement
(medium alone). Culture media was replaced with fresh media
prostheses.4,5 Newer generation bisphosphonates such as zole-
and drugs twice a week and cultures were terminated at 7, 14
dronate are now available, 6 and with their once-a-year dosing,
might be considered for numerous clinical indications, includ-
ANALYTICAL METHODS
ing enhanced bone ingrowth into porous-coated orthopaedic
Total RNA was extracted from the cell layers using TRIzol®
reagent (Gibco-BRL, Grand Island, NY) according to the single
It is widely recognized that the primary action of bisphos-
step acid-phenol guanidinium method. 10 Gene expression for
phonates is by the inhibition of osteoclastic bone resorption. 1
crucial markers of osteogenic differentiation, such as bone
Ongoing investigations suggest that bisphosphonates may also
morphogenetic protein (BMP)-2, core binding factor alpha
affect osteoblastic activity. Increasing evidence from in vitro
subunit 1 (CBFA-1), and Type 1 collagen, was analyzed using
and in vivo studies support the hypothesis that bisphosphonates
semiquantitative RT-PCR as well as quantitative real-time RT-
additionally promote osteoblastic bone formation.4,7-8 However,
little is known about the potential impact of bisphosphonates
SEMIQUANTITATIVE RT-PCR
on early osteoblastic differentiation. Bone marrow stromal cells
Aliquots of the extracted RNA were reverse transcribed
represent an important pool of osteoblastic precursors. These
for 1st strand cDNA synthesis (Invitrogen™, Carlsbad, CA).
pluripotential cells can differentiate into osteoblasts, adipoc-
Template DNA was then used in PCR (MasterMix, Eppendorf,
tyes, fibroblasts and myocytes, and demonstrate remarkable
Westbury, NY) for the specified genes. GAPDH served as a
elasticity between the various differentiation pathways. 9
housekeeping gene. All RT-PCR products were visualized on
The purpose of this study was to determine the effects of
1.5% agarose gel with 0.5g/ml ethidium bromide. Photographs
bisphosphonates (alendronate, risedronate and zoledronate) on
were taken under ultra-violet illumination (Gel Documentation
differentiation of human bone marrow stromal cells (hBMSC)
System, UVP, Upland, CA) and qualitative assessments were
in a clinically relevant in vitro cell culture model. HUMAN BONE MARROW STROMAL CELL CULTURE QUANTITATIVE REAL-TIME RT-PCR
RNA was treated with DNAse I using the DNA-free kit (AMS
Human bone marrow was obtained from the femora of
Biotechnology Ltd, CH, Abingdon Oxon, UK). cDNA synthesis
three human patients (age 69 to 76) undergoing primary total
was performed by incubating the RNA with random hexam-
hip arthroplasty for osteoarthritis. hBMSC were separated by
ers, using Stratrascript reverse transcriptase (Stratagene, NL,
density centrifugation on Percoll (1.077 g/cc) and cultured
La Jolla, CA). Real-time quantitative RT-PCR reactions were performed and monitored using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster
Address correspondence to:
City, CA). In the same reaction, cDNA samples were analysed both for the gene of interest and the reference gene (18-S
Arun S. Shanbhag, PhD, MBAGRJ 1115, 55 Fruit Street
rRNA), using a multiplex approach (Perkin Elmer User Bulletin
N. 2). Technical settings, primers and probes sequences were
STATISTICAL ANALYSIS
Statistical analysis of real-time RT-PCR data was assessed
using one-way analysis of variance (ANOVA) and post-hoc paired, double-sided t-tests generated from 2 independent hBMSC cultures, with p< 0.05 considered to be significant. RESULTS
All three bisphosphonates enhanced osteoblastic differen-
tiation of hBMSC in vitro (Fig. 1). Semiquantitative RT-PCR and quantitative real-time RT-PCR analysis demonstrated upregulated mRNA expression for CBFA-1, BMP-2, and type I collagen in hBMSC after administration of alendronate, rise-dronate, and zoledronate (Fig. 2). These effects were most pro-nounced after 14 days of culture, particularly under treatment with zoledronate (p< 0.05 versus control for Collagen type I), risedronate (p< 0.05 versus control for Collagen type I) and
Figure. 1: Enhanced osteoblastic differentiation under bisphosphonate treatment
DISCUSSION
This study provides further evidence that bisphosphonates
have anabolic effects on osteoblasts. Different bisphosphonate
treatments induced an upregulated gene expression pattern of
hBMSC in vitro and triggered differentiation of omnipotential hBMSC along the osteoblastic differentiation pathway. These
findings are consistent with reports of osteogenic differentia-
tion, by Frank, et al. 11 Interestingly, these effects followed a time- and type-dependent pattern. Of note, the highly potent
new bisphosphonate, zolendronate, tended to have the stron-gest effects on osteogenic differentiation of hBMSC reflecting
Figure 2: Gene expression after 14 days of hMBSC culture determined using semi-quantitative RT-PCR. GAPDH served as housekeeping gene.
the higher biological potency of this drug as demonstrated in recent clinical trials. 6
The mechanism of action behind the anabolic effects of
bisphosphonates on osteoblastic differentiation of hBMSC in vitro is not known. Our data suggests that bisphosphonates might initially promote expression of key genes like BMP-2 or CBFA-1, which secondarily causes a pronounced osteogenic differentiation of pluripotential hBMSC.
Further investigation is needed to determine how our in vitro results translate to bone quality and bone turnover in vivo. In summary, our findings suggest that the in vivo use of bisphosphonates could lead to enhanced recruitment of bone forming cells, and ultimately show pronounced bone formation and net gain of bone mass. An enhanced understanding of the complex interactions of bisphosphonates with bone metabo-lism, on both the osteoblastic and osteoclastic side, might open
Figure 3: mRNA levels of collagen type I determined using real-time PCR. Data is
up a broad application of these drugs to critically improve the
presented as fold difference and measured in cells from 2 independent donors under all treatment conditions. * p<0.05 over negative control.
biological fixation and durability of implants in orthopaedic surgery. ACKNOWLEDGEMENTS This study was supported by the National Institutes of
Health (NIH AR 47465-02) and an Educational Grant from Merck Inc. References 1. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000; 289:1508-1514 2. Lane JM, Khan SN, O'Connor WJ, Nydick M, Hommen JP, Schneider R, Tomin E, Brand J, Curtin J. Bisphosphonate therapy in fibrous dysplasia. Clin Orthop 2001; 382:6-12. 3. Devogelaer JP. New uses of bisphosphonates: osteogenesis imperfecta. Curr Opin Pharmacol 2002; 2:748-53. 4. Venesmaa PK, Kroger HP, Miettinen HJ, Jurvelin JS, Suomalainen OT, Alhav EM. Alendronate reduces periprosthetic bone loss after uncemented primary total hip arthroplasty: a prospective randomized study. J Bone Miner Res 2001; 16:2126-2131. 5. Shanbhag AS, Hasselman CT, Rubash HE. The John Charnley Award. Inhibition of wear debris mediated osteolysis in a canine total hip arthroplasty model. Clin Orthop 1997; 344:33-43. 6. Reid IR, Brown JP, Burckhardt P, et al. Intravenous zoledronic acid in postmenopausal women with low bone mineral density. N Engl J Med 2002; 346:653-661 7. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G. Stimulation of bone formation in vitro and in rodents by statins. Science 1999; 286:1946-9. 8. Im G, Puskas B, Rubash H, Shanbhag AS. Bisphosphonates enhance osteoblast proliferation and maturation. Trans OR 2002; 64. 9. Aubin J E. Bone stem cells. J Cell Biochem Suppl 30-31:73-82, 1998. 10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162:156-9. 11. Frank O, Heim M, Jakob M, Barbero A, Schafer D, Bendik I, Dick W, Heberer M, Martin I. Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro. J Cell Biochem 2002; 85:737-746.
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DISTRICT MALE- HOSPITAL SULTANPUR DRUG LIST SINCE APRIL 2009 Medicine received during S.NO Name of drug Recived from Inj. AMPICILLIN(500ml) 1425VIAL 12600 vial self purchased Inj. AMIKACIN(500) 3730Vial self purchased GCMSD LUCK. T.ANTIAD self purchased T.ALBENDAZOLE AMOXYCILLIN DRY SY. T.ATENOLOL( 50MG.) CAP ASTHMA FORT CAP.AMOXYCILL
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