BISPHOSPHONATES ENHANCE OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW STROMAL CELLS IN VITRO.

Arun S. Shanbhag, PhD, MBA GRJ 1115, 55 Fruit Street Boston, MA 02114 Tel: (617) 724-1923 e-mail: shanbhag@helix.mgh.harvard.edu INTRODUCTION Bisphosphonates are well-recognized inhibitors of osteoclast activity and are widely used in the treatment of various metabolic bone diseases. Current indications include Paget's disease, post-menopausal osteoporosis and hypercalcemia of malignancy.1 Bisphosphonates are also considered for fibrous dysplasia 2 and other disorders affecting bone metabolism such as osteogenesis imperfecta.3 Bisphosphonates are being investigated for their ability to prevent bony erosions in rheumatoid arthritis, osteoarthritis and peri-implant bone resorption around joint replacement prostheses.4,5 Newer generation bisphosphonates such as zoledronate are now available, 6 and with their once-a-year dosing, might be considered for numerous clinical indications, including enhanced bone ingrowth into porous-coated orthopaedic implants. It is widely recognized that the primary action of bisphosphonates is by the inhibition of osteoclastic bone resorption. 1 Ongoing investigations suggest that bisphosphonates may also affect osteoblastic activity. Increasing evidence from in vitro and in vivo studies support the hypothesis that bisphosphonates additionally promote osteoblastic bone formation.4,7-8 However, little is known about the potential impact of bisphosphonates on early osteoblastic differentiation. Bone marrow stromal cells represent an important pool of osteoblastic precursors. These pluripotential cells can differentiate into osteoblasts, adipoctyes, fibroblasts and myocytes, and demonstrate remarkable elasticity between the various differentiation pathways. 9 The purpose of this study was to determine the effects of bisphosphonates (alendronate, risedronate and zoledronate) on differentiation of human bone marrow stromal cells (hBMSC) in a clinically relevant in vitro cell culture model. HUMAN BONE MARROW STROMAL CELL CULTURE MODEL Human bone marrow was obtained from the femora of three human patients (age 69 to 76) undergoing primary total hip arthroplasty for osteoarthritis. hBMSC were separated by density centrifugation on Percoll (1.077 g/cc) and cultured at a density of 400,000 cells/cm2 in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 1% antibiotics/ antimycotics, L-glutamine (2mM), 10 mM ß-glycero-phosphate and 0.1 mM L-ascorbic 2-phosphate at 37oC with 95% humidity and 5% CO2. Cells were treated with three different bisphosphonates including 10-8M alendronate (Fosamax, Merck, Rahway, NY), 10-8M risedronate (Actonel, Proctor & Gamble, Cincinnati, OH), 10-8M zoledronate (Zometa, Novartis, Basel, Switzerland), positive controls (addition of 108M Dexamethasone or 10-8M Vitamin D) and negative control (medium alone). Culture media was replaced with fresh media and drugs twice a week and cultures were terminated at 7, 14 and 21 days after initiation. ANALYTICAL METHODS Total RNA was extracted from the cell layers using TRIzol® reagent (Gibco-BRL, Grand Island, NY) according to the single step acid-phenol guanidinium method. 10 Gene expression for crucial markers of osteogenic differentiation, such as bone morphogenetic protein (BMP)-2, core binding factor alpha subunit 1 (CBFA-1), and Type 1 collagen, was analyzed using semiquantitative RT-PCR as well as quantitative real-time RTPCR. SEMIQUANTITATIVE RT-PCR Aliquots of the extracted RNA were reverse transcribed for 1st strand cDNA synthesis (InvitrogenTM, Carlsbad, CA). Template DNA was then used in PCR (MasterMix, Eppendorf, Westbury, NY) for the specified genes. GAPDH served as a housekeeping gene. All RT-PCR products were visualized on 1.5% agarose gel with 0.5g/ml ethidium bromide. Photographs were taken under ultra-violet illumination (Gel Documentation System, UVP, Upland, CA) and qualitative assessments were made of relative gene expression. QUANTITATIVE REAL-TIME RT-PCR RNA was treated with DNAse I using the DNA-free kit (AMS Biotechnology Ltd, CH, Abingdon Oxon, UK). cDNA synthesis was performed by incubating the RNA with random hexamers, using Stratrascript reverse transcriptase (Stratagene, NL, 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 City, CA). In the same reaction, cDNA samples were analysed both for the gene of interest and the reference gene (18-S rRNA), using a multiplex approach (Perkin Elmer User Bulletin N. 2). Technical settings, primers and probes sequences were as previously described. 11