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 malignancy¹. Bisphosphonates are also considered for fibrous dysplasia² and other disorders affecting bone metabolism such as osteogenesis imperfecta³.

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 available⁶, 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 resorption¹. 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 pathways⁹.

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 10^-8M 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 method¹⁰. 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 1^st strand cDNA synthesis (Invitrogen^TM, 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 described¹¹.

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 differentiation 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, risedronate, and zoledronate (Fig. 2). These effects were most pronounced 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 alendronate (Fig. 3).

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 differentiation, by Frank, et al¹¹. Interestingly, these effects followed a time- and type-dependent pattern. Of note, the highly potent new bisphosphonate, zolendronate, tended to have the strongest effects on osteogenic differentiation of hBMSC reflecting the higher biological potency of this drug as demonstrated in recent clinical trials⁶.

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 metabolism, on both the osteoblastic and osteoclastic side, might open up a broad application of these drugs to critically improve the 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.

Notes:

Address correspondence to:
Arun S. Shanbhag, PhD, MBA
GRJ 1115, 55 Fruit Street
Boston, MA 02114
Tel: (617) 724-1923
e-mail: shanbhag@helix.mgh.harvard.edu

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