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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.
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.
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.
This study was supported by the National Institutes of
Health (NIH AR 47465-02) and an Educational Grant from Merck Inc.
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John Abraham, Jen Cook, Travis Matheney, Sean Rassman, and Chris Forthman enjoy the festivities at an the chef’s open-house BBQ Sean Rassman, Ben Bengs, and Jeff Zarins enjoy some free time at the AO course in Colorado


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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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