ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

�CorrespondE-mail addr

(A.S. Shanbhag

Biomaterials 26 (2005) 6941–6949

www.elsevier.com/locate/biomaterials

Effects of bisphosphonates on proliferation and osteoblastdifferentiation of human bone marrow stromal cells

Fabian von Knocha, Claude Jaquieryb, Marc Kowalskya, Stefan Schaerenb,Claude Alabrea, Ivan Martinb, Harry E. Rubasha, Arun S. Shanbhaga,�

aBiomaterials Laboratory, Department of Orthopaedic Surgery, Massachusetts General Hospital & Harvard Medical School, Boston, USAbDepartments of Surgery and of Research, University of Basel, Basel, Switzerland

Received 25 January 2005; accepted 20 April 2005

Available online 11 July 2005

Abstract

Bisphosphonates are well known potent inhibitors of osteoclast activity and are widely used to treat metabolic bone diseases.

Recent evidence from in vitro and in vivo studies indicates that bisphosphonates may additionally promote osteoblastic bone

formation. In this study, we evaluated the effects of three FDA-approved and clinically utilized bisphosphonates, on the

proliferation and osteogenic differentiation of human bone marrow stromal cells (BMSC).

BMSC were obtained from patients undergoing primary total hip arthroplasty for end-stage degenerative joint disease. Cells were

treated with or without a bisphosphonate (alendronate, risedronate, or zoledronate) and analyzed over 21 days of culture. Cell

proliferation was determined by direct cell counting. Osteogenic differentiation of BMSC was assessed with alkaline phosphatase

bioassay and gene expression analyses using conventional RT-PCR as well as real-time quantitative RT-PCR.

All bisphosphonates tested enhanced the proliferation of BMSC after 7 and 14 days of culture. Steady-state mRNA levels of key

genes involved in osteogenic differentiation such as bone morphogenetic protein-2 (BMP-2), bone sialoprotein-II, core-binding

factor alpha subunit 1 (cbfa1) and type 1 collagen, were generally increased by bisphosphonate treatment in a type- and time-

dependent manner. Gene expression levels varied among the different donors. Enhancement of osteogenic differentiation was most

pronounced after 14 days of culture, particularly following zoledronate treatment (po0:05 for BMP-2).

In conclusion, using a clinically relevant in vitro model we have demonstrated that bisphosphonates enhance proliferation of

BMSC and initiate osteoblastic differentiation. When administered around joint replacements, bisphosphonates may potentially

compensate for the deleterious effects of particulate wear debris at the bone–implant interface, by encouraging increased numbers of

cells committed to the osteoblastic phenotype, and thus improve the longevity of joint replacements.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Alendronate; Risedronate; Zoledronate; Bisphosphonates; Human bone marrow stromal cells; Bone morphogenetic protein; Total joint

replacements

1. Introduction

Bisphosphonates are well-recognized inhibitors ofosteoclastic activity and are widely used in the clinicaltreatment of various systemic metabolic bone diseases[1]. Current indications include Paget’s disease, hyper-

e front matter r 2005 Elsevier Ltd. All rights reserved.

omaterials.2005.04.059

ing author. Tel.: +1617 724 1923.

ess: shanbhag@helix.mgh.harvard.edu

).

calcemia of malignancy and post-menopausal osteoporo-sis [2–4]. Bisphosphonates are being investigated for thetreatment of fibrous dysplasia [5], osteogenesis imperfec-ta [6], osteoarthritis [7] and rheumatoid arthritis [8].

Recent studies also indicate that bisphosphonatesmodulate wear-debris-induced inflammatory bone lossaround total hip replacements (THR) through theinhibition of osteoclastic bone resorption. Using acanine THR model, Shanbhag et al. previously demon-strated that oral alendronate treatment can effectively

ARTICLE IN PRESSF. von Knoch et al. / Biomaterials 26 (2005) 6941–69496942

inhibit wear debris-mediated bone resorption [9]. Thesefindings are supported by recent clinical studies suggest-ing that bisphosphonates also improve fixation anddurability of total joint replacement components [10–13].

Although the primary action of bisphosphonates isthe inhibition of osteoclastic bone resorption [1], there isincreasing evidence that bisphosphonates also interactwith osteoblasts. The pharmacological mechanism ofaction of the amino-bisphosphonates relies on inter-ference with the mevalonate pathway through theinhibition of farnesyl pyrophosphate (FPP) synthaseenzyme [14]. This causes a reduction in the levels ofgeranylgeranyl diphosphate (GGPP) required for theprenylation of guanosine triphosphate (GTP)-bindingproteins such as Rab, Rac, Ras, Rho and Cdc42 [15,16].Since these cytoskeletal regulators are essential forosteoclast activity and survival, bisphosphonates ulti-mately inhibit osteoclast formation and function.Statins, another class of drugs clinically used to suppresshepatic cholesterol synthesis, also act on the mevalonatepathway by blocking the more upstream 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.Interestingly, statins increase osteoblastic bone forma-tion both in vitro as well as in vivo, as first reported byMundy and co-workers [17]. Concurrently it appearsthat bisphosphonates too may have anabolic effects onosteoblasts. Recent studies from several groups, includ-ing our own, indicate that bisphosphonates enhanceproliferation and maturation of osteoblasts [18–20] andinhibit apoptosis [21]. These observations stronglysupport the suggestion that bisphosphonates have ananabolic effect on osteoblasts and subsequently promotebone formation.

The effects of bisphosphonates on early stages ofosteoblastic differentiation are not yet well understood.Osteoblast progenitors derive primarily from amongbone marrow stromal cells (BMSC). These pluripotentcells can differentiate into osteoblasts, adipocytes,fibroblasts and myocytes, and demonstrate a remarkableelasticity between the various differentiation pathways[22]. Since human bone marrow stromal cells arecritically involved in maintaining the dynamic equili-brium of bone turnover, it is important to investigatehow these cells respond to bisphosphonate treatment.Thus the objective of this study was to investigate theeffects of three clinically used bisphosphonates (alen-dronate, risedronate, and zoledronate) on the prolifera-tion and osteogenic differentiation of BMSC.

2. Materials and methods

2.1. Human bone marrow in-vitro model

Human bone marrow was obtained from the femora of 4

patients (mean age 6878 years, range 57–76) undergoing

primary total hip arthroplasty (THA) for osteoarthritis.

Subjects did not have other bone disorders such as rheumatoid

arthritis or renal insufficiency. Bone marrow harvested

during surgery was diluted with phosphate-buffered saline

(PBS), and mononuclear BMSC were separated by density

centrifugation on Percoll 1077 (Sigma, St. Louis, MO). BMSC

were then cultured at a density of 400,000 cells/cm2 in a

1:1 premix of Dulbecco’s modified Eagle’s medium and

F-12 medium (Biowhittaker, Walkersville, MD) supplemented

with 10% fetal bovine serum, 1% antibiotics/ antimycotics

(100U/ml penicillin, 100mg/ml streptomycin, 0.25 mg/ml am-

photericin B, L-glutamine (2mM), 10mM X -glycero-phos-

phate and 0.1mM L-ascorbic 2-phosphate at 37 1C with 95%

humidity and 5% CO2. Dexamethasone is known to be a

critical media supplement for osteogenic differentiation of

BMSC [23], and was intentionally withheld in this study as a

standard media supplement, but used as a positive control

to compare effects of bisphosphonates under suboptimal

osteogenic cell culture conditions and established osteogenic

media conditions.

BMSC were treated with three bisphosphonates diluted in

sterile PBS: alendronate ([10�8M] Fosamax, Merck, Rahway

NY), risedronate ([10�8M] Actonel, Proctor & Gamble,

Cincinnati, OH), zoledronate ([10�8M] Zometa, Novartis,

Basel, Switzerland). Various types of controls including

negative control (medium alone) and positive controls with

addition of dexamethasone ([10�8M], Sigma, St. Louis, MO) or

1,25-dihydroxy-cholecalciferol, vitamin D3 ([10�8M], Sigma)

were included. Culture media and drugs were replaced twice a

week and experiments terminated 7, 14 and 21 days after

culture initiation. Supernatants were collected for protein

assay, cell lysates in TRIzols (Gibco-BRL, Grand Island, NY)

were collected for total RNA extraction and subsequent RT-

PCR analyses. In order to ensure osteoblastic phenotype,

BMSC cultures were stained for alkaline phosphatase in situ

and evaluated by light microscopy.

2.2. Assessment of cell proliferation

BMSC were plated in 24-well plates and treated under

different conditions as described above. After 7, 14, or 21

days of culture, cells were isolated from culture dishes

by trypsinization, washed, and cell number and viability

were determined with a hemocytometer using trypan blue

dye exclusion test. Direct cell counts were performed in

duplicate.

2.3. Alkaline phosphatase assay

Cells were plated in culture dishes (10 cm diameter) and

treated as described above. Alkaline phosphatase (AP) activity

after 7 and 14 days of culture was assayed utilizing the

conversion of a colorless p-nitrophenyl phosphate to a colored

p-nitrophenol (Sigma, St.Louis, MO). The color change was

measured spectrometrically at 405 nm (Labsystems, Multiskan

Multisoft), and the amount of enzyme released by the cells was

quantified by comparison with a standard curve. AP levels

were normalized to cell number at the end of the experiment.

All experiments were conducted in duplicate, and repeated in

BMSC cultures from three independent donors.

ARTICLE IN PRESSF. von Knoch et al. / Biomaterials 26 (2005) 6941–6949 6943

2.4. Total RNA extraction

BMSC were plated in Petri dishes (10 cm diameter) and

treated under different conditions as described above. After 7,

14 and 21 days of culture, total RNA was extracted from cell

layers using TRIzols reagent (Gibco-BRL, Grand Island,

NY) according to the single step acid-phenol guanidinium

extraction method [24]. RNA was treated with DNAse I (AMS

Biotechnology Ltd, Switzerland) and quantified spectrometri-

cally.

2.5. Reverse transcription polymerase chain reaction (RT-

PCR)

Aliquots of the extracted RNA samples were initially

reverse transcribed for 1st strand cDNA synthesis (Invitrogen,

Carlsbad, CA). After the RT reaction, remaining RNA was

removed by RNase treatment. Template DNA was then used

in gene-specific PCR (MasterMix, Eppendorf, Westbury, NY)

for human bone morphogenetic protein (BMP)-2, core binding

factor alpha subunit 1 (cbfa-1), and type 1 collagen. These

genes have previously been shown to be characteristic for

osteogenic differentiation of BMSC [18]. Glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) served as a housekeeping

gene. Details of primers and reaction temperatures were as

previously described [19]. All RT-PCR products were visua-

lized on 1.5% agarose gel with 0.5 mg/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.

2.6. Real-time quantitative RT-PCR

cDNA synthesis was performed by incubating 2–3mg of

extracted RNA, with 500 mg/ml random hexamers (Catalys

AG, Switzerland) and 1 ml of 50U/ml Stratascript reverse

transcriptase (Stratagene, Netherlands), in the presence of

dNTPs. cDNA was then diluted to 200 ml using DNAse-free

water. Real-time quantitative RT-PCR reactions were per-

formed and monitored using an ABI Prism 7700 Sequence

Detection System (Perkin-Elmer Applied Biosystems, Foster

City, CA). The PCR 2x master mix was based on AmpliTaq

Gold DANN polymerase (Applied Biosystems). In the same

reaction, cDNA samples were analyzed both for the gene of

interest and the 18S rRNA reference gene, using a multiplex

approach (Perkin Elmer User Bulletin N. 2). The probe for 18S

rRNA was fluorescently labeled with VICTM and TAMRA

(Applied Biosystems), whereas probes for the genes of interest

were labeled with 6-carboxy-fluorescein (FAM) and TAMRA.

Primers and probes for the osteogenic genes of interest

including human BMP-2, bone sialoprotein-II (BSP), cbfa-1,

and type 1 collagen [23], as well as cycle temperature and times

were used as previously described [25]. The 18S rRNA was

selected as a reference gene based on preliminary experiments,

indicating its higher stability of expression by BMSC as

compared to GAPDH. Expression levels for each gene of

interest were calculated by normalizing the quantified mRNA

amount to the 18S rRNA, and by further dividing the resulting

value by that obtained with control human osteoblasts

(average of five donors) using an identical procedure (2DDCt

formula, Perkin Elmer User Bulletin No.2) [23]. Real-time

quantitative RT-PCR analysis was performed with BMSC

from 3 independent donors under various treatment condi-

tions, and each sample was assessed at least in duplicate.

2.7. Statistical analyses

Data are presented as a percentage or fold-change over

controls. Gene expression data from different donors are

presented separately to highlight inter-individual variability of

cell responses to bisphosphonates. Data were analyzed by one-

way ANOVA, and post-hoc Student’s two-tailed t-test. All p-

values were compared to an a-value of 0.05 to determine

significance.

3. Results

3.1. Assessment of cell proliferation

Treatment with alendronate, risedronate and zole-dronate resulted in a significant increase in BMSCproliferation compared to negative controls (ANOVA,po0:005) (Figs. 1 and 2). Alendronate increased cellproliferation by 14% on day 7 and 14 (po0:05).Risedronate enhanced cell proliferation by 16% onday 7 and 14 (po0:0001). Zoledronate induced in-creased cell proliferation by 15% and 14% on day 7 and14, respectively (po0:05).

3.2. Alkaline phosphatase assay

Levels of AP activity from the three donors revealedsubstantial differences under identical treatment condi-tions (see Table 1). While the aggregated data did notshow a statistical effect of bisphosphonate treatmentover control levels, we noticed a donor-specific acceler-ated commitment of BMSC towards the osteoblastlineage (Table 1). For example, donor #1 increased APactivity up to 24%, 7 days after alendronate treatment.In contrast, donor #2 BMSC increased AP activity up to20% after 14 days of zoledronate treatment. Notsurprisingly, the induction of osteoblast differentiationfollowing bisphosphonate treatment appears to bedonor and drug type dependent.

3.3. RT-PCR

Conventional RT-PCR analysis was performed toassess progression of BMSC towards the osteoblastlineage at the mRNA gene expression level. Afteradministration of any of the bisphosphonates, expres-sion of genes coding for BMP-2 and cbfa-1 wereupregulated in BMSC after 7, 14 and 21 days of culture(Fig. 3). Remarkably, the osteoinductive effects ofbisphosphonate treatment varied in cultures from thedifferent donors.

ARTICLE IN PRESS

95

100

105

110

115

120

125

130

VitD DXM ALN RSN ZOL VitD DXM ALN RSN ZOL Vi tD DXM ALN RSN ZOL

Cel

l nu

mb

er (

% t

reat

men

t o

ver

con

tro

l)

*

*

*

*

Day 14Day 7 Day 21

Fig. 1. Proliferation of BMSC under different bisphosphonate treatments as measured by direct cell counting. Data is presented at mean7std error

(day 7 with n ¼ 4; day 14 with n ¼ 3; day 21 with n ¼ 2; �po0:05 over control).

Fig. 2. Representative photomicrographs of BMSC obtained by transmission light microscopy after 14 days of culture under different treatment

conditions are shown. Note the enhanced BMSC proliferation under bisphosphonate treatment.

F. von Knoch et al. / Biomaterials 26 (2005) 6941–69496944

3.4. Real-time quantitative RT-PCR

Real-time quantitative RT-PCR analysis demon-strated that stimulation of osteoblast-specific geneexpression by bisphosphonates was dependent upon:(a) time of culture, with the most pronounced effectsobserved 14 days after culture, (b) type of bispho-sphonate, with the strongest effects observed aftertreatment with zoledronate, and (c) individual donor

variables. We highlighted these donor-specific variationsby presenting the data for the three donors separately(Fig. 4). In donor #1, zoledronate-mediated BMP-2expression was more than 7-fold higher (day 14) overcontrols, compared to 3-fold increases in donors #2 and3 (Fig. 4A). On the other hand, donor #2 demonstratedstrong zoledronate mediated upregulation of cbfa-1 (6-fold higher), BSP (4-fold) and type 1 collagen (7-fold).While we observed similar increases in cbfa-1 and type 1

ARTICLE IN PRESS

Table 1

Alkaline phosphatase activity in BMSC

Donor Vit. D Dexameth. Alendronate Risedronate Zoledronate

7 days #1 1.03 1.19 1.24 0.93 0.88

#2 0.89 0.98 0.63 0.73 0.61

#3 0.95 0.78 0.92 0.85 0.84

Mean 0.9570.04 0.9870.12 0.9370.18 0.8470.06 0.7870.09

14 days #1 1.11 1.24 0.89 1.03 0.98

#2 1.05 0.76 0.76 0.83 1.20

#3 1.09 0.99 0.92 0.79 0.80

Mean 1.0870.02 1.0070.14 0.8670.05 0.8870.07 0.9970.12

Alkaline phosphatase is normalized to cell number at each time period and reported as a fraction of untreated cell controls. Cells were treated with

alendronate, risedronate or zoledronate. Data is presented as mean7std error (n ¼ 3).

Fig. 3. Using conventional RT-PCR, representative gene expression of BMP-2, cbfa-1, and type 1 collagen after 14 days of BMSC culture under the

different conditions is shown. GAPDH was used as a housekeeping gene.

F. von Knoch et al. / Biomaterials 26 (2005) 6941–6949 6945

collagen expression in donor #3, no increases wereobserved in donor #1. These results again highlightdonor-specific variations in the expression profile ofosteoblast-related genes. Due to this variability, effectsof bisphosphonates in the aggregated data werestatistically significant only at day 14 for the BMP-2gene following zoledronate treatment (p ¼ 0:032; ANO-VA, po0:012). Note particularly that bisphosphonatetreatments induced higher expression of osteoblast-related genes as compared to a positive controlcondition, with supplemented dexamethasone.

4. Discussion

This study demonstrates that zoledronate, risedronateand alendronate enhance proliferation and promoteosteoblast differentiation of BMSC, providing furtherevidence of their anabolic effects on osteoblasts. BMSCrepresent a clinically relevant and therapeuticallyappropriate pool of pluripotent, mesenchymal, progeni-tor cells with the capacity to differentiate into osteo-blasts, adipocytes, fibroblasts and myocytes [22]. Inorder to closely mimic an in vivo setting, BMSC wereisolated from femoral bone marrow collected frompatients undergoing primary THR. Donors were of

advanced age with degenerative joint disease, represent-ing the clinically appropriate target population. Ourresults indicate that various bisphosphonates triggeredpronounced proliferation and differentiation of BMSCalong the osteoblastic differentiation pathway followinga time- and type-dependent pattern. A cascade ofosteoblast-related genes including BMP-2, cbfa-1, type1 collagen and BSP were upregulated and a significantincrease of BMSC proliferation occurred 14 days afterbisphosphonate treatment.

Zoledronate, the most potent new generation bispho-sphonate, had the strongest influence on pluripotentBMSC differentiation into osteoblasts, thereby reflect-ing the higher biological potency of this drug [26].Overall, bisphosphonate-mediated effects on prolifera-tion and osteoblast differentiation of BMSC were lesspronounced than those observed after bisphosphonatetreatment of transformed osteoblastic cell lines [19]. Thismay be in part due to the highly variable osteogenicpotential of BMSC from individual clinical patients, aswell as different clones within a single donor sample [23].In accordance with these observations, recent studiessuggest that heterogeneity among BMSC clones affectsthe potential of BMSC for proliferation [27], differentia-tion [23,28] and osteogenesis [27]. Despite the donor-to-donor variability of cell responses, BMP-2 gene

ARTICLE IN PRESS

2

7 14 21 7 14 21

7 14 21 7 14 21

7 14 21 7 14 21

4

6

BMP-2: Donor 1 BSP: Donor 1

Vit D DXM ALN RIS ZOL

BMP-2: Donor 2 BSP: Donor 2

BMP-2: Donor 3

Days in Culture Days in Culture

BSP: Donor 3

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

1

2

3

4

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)1

2

3

4

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

1

2

3

4

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)(A) (B)

Fig. 4. Gene expression for (A) bone morphogenetic protein-1, BMP-2, (B) bone sialoprotein, BSP, (C) core binding factor alpha 1 subunit, cbfa-1,

(D) type 1 collagen, coll I (D) after bisphosphonate treatment. Quantified using real-time quantitative RT-PCR after 7, 14, 21 days of BMSC culture.

Data is measured in cells from 3 independent donors under different treatment conditions, and presented separately for each donor as fold change

(treatment over controls). Donor #1: F 69 y; donor #2: F 76 y; donor #3: M 76 y).

F. von Knoch et al. / Biomaterials 26 (2005) 6941–69496946

upregulation by zoledronate represented a consistentfinding in our studies; in two of three donors, cbfa-1 andtype 1 collagen genes were upregulated by more thansix-fold and sevenfold, respectively, over controls. It wasan unexpected finding that different osteoblast-specificgenes are upregulated in different donors after identicalbisphosphonate treatments. These observations high-light the variable stages of harvested stromal cells andcrucially, the importance of using primary human cellsrather than transformed cell lines for evaluating theeffects of bisphosphonates.

The mechanism of the anabolic effects of bispho-sphonates on proliferation and osteogenic differentia-tion of BMSC is not fully understood. According toGiuliani, the anabolic effects of bisphosphonates weredue to stimulation of b-FGF on osteoblasts [29]. Mundy

et al. documented that statins which inhibit themevalonate pathway, stimulate osteoblasts by inducingexpression of BMP-2 gene [18]. BMP-2 is a potentosteoconductive agent and growth factor involved inrecruitment, proliferation and differentiation of me-senchymal progenitor cells, and ultimately resulting inproducing bone tissue [30]. We recently reported thatosteoblast maturation following bisphosphonate treat-ment also involves a robust upregulation of BMP-2 geneexpression [19]. These observations are not unexpectedsince bisphosphonates, similar to statins, also interferewith the mevalonate pathway and as such elicit similarBMP-2 expression. In order to investigate the proposedpathway in more detail, we studied bisphosphonate-mediated stimulatory effects on osteoblasts during thevery early stages of osteoblast differentiation, i.e. during

ARTICLE IN PRESS

2

7 14 21

7 14 21

7 14 21 7 14 21

7 14 21

7 14 21

4

6

Cbfa-1: Donor 1

Cbfa-1: Donor 2

Cbfa-1: Donor 3

Coll. l: Donor 1

Coll. l: Donor 2

Coll. l: Donor 3

Vit D DXM ALN RIS ZOL

Days in Culture Days in Culture

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

0

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)

2

0

4

6

Fol

d C

hang

e(t

reat

men

t ove

r co

ntro

l)(C) (D)

Fig. 4. (Continued)

F. von Knoch et al. / Biomaterials 26 (2005) 6941–6949 6947

the commitment of pluripotent BMSC towards theosteoblast lineage. Our data indicate that bisphospho-nates might initially promote gene expression for keyosteogenic transcription factors including BMP-2 andcbfa-1, by acting as sequential differentiation triggersthat secondarily result in a pronounced commitment,differentiation and maturation of pluripotential BMSCtowards the osteoblast phenotype. Cbfa-1 is a transcrip-tion factor from the runt family and plays a crucial rolein osteogenesis. It is considered to be the key transcrip-tion factor for osteoblast differentiation [31–33]. Inselected donors we also found a higher expression ofbone sialoprotein (BSP) after bisphosphonate treatment,consistent with pronounced osteoblast differentiation[23]. BSP is considered the main nucleator of hydro-xyapatite crystal formation and critical during the initialphase of matrix mineralization, downstream fromosteoblast differentiation and maturation [34]. SinceBMP-2 has been shown to enhance gene expression and

protein levels of BSP [35], Frank et al. suggested that theelevated BSP expression in BMSC could be a conse-quence of the autocrine effects of BMP-2 [23]. Besidesalterations of osteoblast-specific gene expression andsubsequent differentiation of BMSC, bisphosphonatetreatment may also contribute to the recruitment andlonger functional life through mechanisms that blockosteoblast apoptosis [21].

In vivo bone turnover is determined by a delicatebalance between osteoclastic bone resorption andosteoblastic bone formation. This study suggeststhat bisphosphonates impact both sides of thisbalance: inhibit osteoclastic activity and have ananabolic effect on osteoblasts. It is not clear, whetherthe osteogenic effects of bisphosphonates in vitro wouldresult in greater mineral content and stronger bonestructure in vivo. We previously cautioned that normalskeletal remodeling could potentially be compromisedbecause bisphosphonates are incorporated into the

ARTICLE IN PRESSF. von Knoch et al. / Biomaterials 26 (2005) 6941–69496948

hydroxyapatite [36]. Our laboratory investigations,however, did not support these concerns and wereported that a continuous 23-week bisphosphonatetreatment did not reduce the biomechanical propertiesof bone, such as fracture toughness, elastic modulus, ortensile strength [37]. Furthermore, data from histomor-phometric studies suggest that there are no adverseeffects on bone structure or mineralization [38]. There isan additional concern that the beneficial effects ofbisphosphonate treatment could slow down with time.Evidence from recent studies suggests, however, thatbisphosphonates are deposited on osteoblastic andresting bone surfaces and remain localized for the longterm [39]. Studies of up to 10 years of bisphosphonatetreatment have consistently demonstrated continuousanabolic effects on bone turnover [2].

There are indications that particulate wear debrisfrom joint replacements suppress osteoblastic function[40–42] and osteogenic differentiation of BMSC [43].Vermes et al. [44] demonstrated recently that pamidro-nate reversed particle-induced suppression of osteo-blasts. Based on our current findings, it seems plausibleto conclude that bisphosphonate treatment may furthercompensate for the negative effects of wear debrison BMSC around implants in vivo. Furthermorethe beneficial effects of bisphosphonate treatmentmay be more pronounced at sites of increased boneturnover—such as the peri-implant area, which ulti-mately may improve the longevity of orthopaedicimplants.

5. Conclusions

This study demonstrates that bisphosphonates such asalendronate, risedronate and zoledronate have prolif-erative effects on BMSC and variably enhance osteo-blast differentiation of BMSC from different donors in atype- and time-dependent pattern. These findingsprovide further support of the concept that bispho-sphonates may have an anabolic effect on osteoblasts.Further investigation is needed to determine how thesein vitro results translate to bone quality and boneturnover in vivo. Altogether these results suggest thatthe in vivo use of bisphosphonates may produce bothenhanced recruitment of bone forming cells andenhanced bone formation with a net gain of bone mass.Such effects of bisposphonates on bone metabolismcould be of significant benefit in numerous clinicalindications including (a) higher bone density andimprovement of bone microarchitecture in variousmetabolic bone diseases with a secondarily decreasedrisk of bone fractures; (b) stimulation of bone forma-tion, thus accelerating fracture healing and/or boneingrowth into implant porosities and improving biolo-gical fixation; and (c) decreased wear debris mediated

osteolysis resulting in increased durability and longevityof orthopaedic joint replacement components.

Acknowledgements

The authors are grateful to Drs. Andrew Freiberg andDennis Burke from the Massachusetts General Hospitalfor generously providing the femoral bone marrowspecimens. This study was supported by the NIH/NIAMS (AR 47465-02) and an Educational grant fromMerck Inc.

References

[1] Rodan GA, Martin TJ. Therapeutic approaches to bone diseases.

Science 2000;289:1508–14.

[2] Bone HG, Hosking D, Devogelaer JO, Tucci JR, Emkey RD,

Tonino RP, Rodriguez-Portales JA, Downs RW, Gupta J,

Santora AC, Liberman U. Alendronate phase III osteoporosis

treatment study group. Ten years’ experience with alendronate for

osteoporosis in postmenopausal women. N Engl J Med 2004;

350:1189–99.

[3] Walsh JP, Ward LC, Stewart GO, Will RK, Criddle RA, Prince

RL, Stuckey BG, Dhaliwal SS, Bhagat CI, Retallack RW, Kent

GN, Drury PJ, Vasikaran S, Gutteridge DH. A randomized

clinical trial comparing oral alendronate and intravenous

pamidronate for the treatment of Paget’s disease of bone. Bone

2004;34:747–54.

[4] Wellington K, Goa KL. Zoledronic acid: a review of its use in the

management of bone metastases and hypercalcaemia of malig-

nancy. Drugs 2003;63:417–37.

[5] 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.

[6] Devogelaer JP. New uses of bisphosphonates: osteogenesis

imperfecta. Curr Opin Pharmacol 2002;2:748–53.

[7] Lehmann HJ, Mouritzen U, Christgau S, Cloos PA, Christiansen

C. Effect of bisphosphonates on cartilage turnover assessed with a

newly developed assay for collagen type II degradation products.

Ann Rheum Dis 2002;61:530–3.

[8] Maksymowych WP. Bisphosphonates for arthritis—a confusing

rationale. J Rheumatol 2003;30:430–4.

[9] 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.

[10] Venesmaa PK, Kroger HP, Miettinen HJ, Jurvelin JS, Suomalai-

nen OT, Alhav EM. Alendronate reduces periprosthetic bone loss

after uncemented primary total hip arthroplasty: a prospective

randomized study. J Bone Miner Res 2001;16:2126–33.

[11] Soininvaara TA, Jurvelin JS, Miettinen HJ, Suomalainen OT,

Alhava EM, Kroger PJ. Effect of alendronate on periprosthetic

bone loss after total knee arthroplasty: a one-year, randomized,

controlled trial of 19 patients. Calcif Tissue Int 2002;71:472–7.

[12] Wang CJ, Wang JW, Weng LH, Hsu CC, Huang CC, Chen HS.

The effect of alendronate on bone mineral density in the distal

part of the femur and proximal part of the tibia after total knee

arthroplasty. J Bone Joint Surg Am 2003;85:2121–6.

[13] Astrand J, Aspenberg P. Reduction of instability-induced bone

resorption using bisphosphonates: high doses are needed in rats.

Acta Orthop Scand 2002;73:24–30.

[14] Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE,

Masarachia PJ, Wesolowski G, Russell RG, Rodan GA, Reszka

ARTICLE IN PRESSF. von Knoch et al. / Biomaterials 26 (2005) 6941–6949 6949

AA. Alendronate mechanism of action: geranylgeraniol, an

intermediate in the mevalonate pathway, prevents inhibition of

osteoclast formation, bone resorption, and kinase activation in

vitro. Proc Natl Acad Sci USA 1999;96:133–8.

[15] Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G,

Rogers MJ. Nitrogen-containing bisphosphonates inhibit the

mevalonate pathway and prevent post-translational prenylation

of GTP-binding proteins, including Ras. J Bone Miner Res 1998;

13:581–9.

[16] van Beek E, Lowik C, van Der PG, Papapoulos S. The role of

geranylgeranylation in bone resorption and its suppression by

bisphosphonates in fetal bone explants in vitro: A clue to the

mechanism of action of nitrogen-containing bisphosphonates. J

Bone Miner Res 1999;14:722–9.

[17] 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.

[18] Fromigue O, Body JJ. Bisphosphonates influence the proliferation

and the maturation of normal human osteoblasts. J Endocrinol

Invest 2002;25:539–46.

[19] Im GI, Qureshi SA, Kenney J, Rubash HE, Shanbhag AS.

Osteoblast proliferation and maturation by bisphosphonates.

Biomaterials 2004;25:4105–15.

[20] Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M,

Ingle JN, Spelsberg TC. Bisphosphonates directly regulate cell

proliferation, differentiation, and gene expression in human

osteoblasts. Cancer Res 2000;60:6001–7.

[21] Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas

SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis

by bisphosphonates and calcitonin. J Clin Invest 1999;104:

1363–74.

[22] Aubin JE. Bone stem cells. J Cell Biochem Suppl 1998;30–31:

73–82.

[23] 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;85:737–46.

[24] Chomczynski P, Sacchi N. Single-step method of RNA isolation

by acid guanidinium thiocyanate-phenol-chloroform extraction.

Anal Biochem 1987;162:156–9.

[25] Martin I, Jakob M, Schafer D, Dick W, Spagnoli G, Heberer M.

Quantitative analysis of gene expression in human articular

cartilage from normal and osteoarthritic joints. Osteoarthritis

Cartilage 2001;9:112–8.

[26] Reid IR, Brown JP, Burckhardt P, Horowitz Z, Richardson P,

Trechsel U, Widmer A, Devogelaer JP, Kaufman JM, Jaeger P,

Body JJ, Brandi ML, Broell J, Di Micco R, Genazzani AR,

Felsenberg D, Happ J, Hooper MJ, Ittner J, Leb G, Mallmin H,

Murray T, Ortolani S, Rubinacci A, Saaf M, Samsioe G,

Verbruggen L, Meunier PJ. Intravenous zoledronic acid in

postmenopausal women with low bone mineral density. N Engl

J Med 2002;346:653–61.

[27] Satomura K, Krebsbach P, Bianco P, Gehron RP. Osteogenic

imprinting upstream of marrow stromal cell differentiation. J Cell

Biochem 2000;78:391–403.

[28] Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal

progenitors from human bone marrow differentiate in vitro

according to a hierarchical model. J Cell Sci 2000;113:1161–6.

[29] Giuliani N, Girasole G, Pedrazzoni M, Passeri G, Gatti C, Passeri

M. Alendronate stimulates b-FGF production and mineralized

nodule formation in human osteoblastic cells and osteoblastogen-

esis in human bone marrow cultures. J Bone Miner Res 1995;

10:S171.

[30] Wozney JM. Bone morphogenetic proteins. Prog Growth Factor

Res 1989;1:267–80.

[31] Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/

Cbfa1: a transcriptional activator of osteoblast differentiation.

Cell 1997;89:747–54.

[32] Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi

K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M,

Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted

disruption of Cbfa1 results in a complete lack of bone formation

owing to maturational arrest of osteoblasts. Cell 1997;89:755–64.

[33] Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC,

Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR,

Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial

dysplasia syndrome, is essential for osteoblast differentiation and

bone development. Cell 1997;89:765–71.

[34] Bianco P, Riminucci M, Silvestrini G, Bonucci E, Termine JD,

Fisher LW, Robey PG. Localization of bone sialoprotein (BSP) to

Golgi and post-Golgi secretory structures in osteoblasts and to

discrete sites in early bone matrix. J Histochem Cytochem 1993;

41:193–203.

[35] Lecanda F, Avioli LV, Cheng SL. Regulation of bone matrix

protein expression and induction of differentiation of human

osteoblasts and human bone marrow stromal cells by bone

morphogenetic protein-2. J Cell Biochem 1997;67:386–96.

[36] Shanbhag AS, Rubash HE. Bisphosphonate therapy for the

prevention of osteolysis in total joint replacements. Curr Opin

Orthop 1998;9:81–7.

[37] Wang X, Shanbhag AS, Rubash HE, Agrawal CM. Short-term

effects of bisphosphonates on the biomechanical properties of

canine bone. J Biomed Mater Res 1999;44:456–60.

[38] Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier

PJ. Histomorphometric assessment of the long-term effects of

alendronate on bone quality and remodeling in patients with

osteoporosis. J Clin Invest 1997;100:1475–80.

[39] Masarachia P, Weinreb M, Balena R, Rodan GA. Comparison of

the distribution of 3H-alendronate and 3H-etidronate in rat and

mouse bones. Bone 1996;19:281–90.

[40] Lohmann CH, Schwartz Z, Koster G, Jahn U, Buchhorn GH,

MacDougall MJ, Casasola D, Liu Y, Sylvia VL, Dean DD,

Boyan BD. Phagocytosis of wear debris by osteoblasts affects

differentiation and local factor production in a manner dependent

on particle composition. Biomaterials 2000;21:551–61.

[41] Vermes C, Chandrasekaran R, Jacobs JJ, Galante JO, Roebuck

KA, Glant TT. The effects of particulate wear debris, cytokines,

and growth factors on the functions of MG-63 osteoblasts. J Bone

Joint Surg Am 2001;83:201–11.

[42] Yao J, Cs-Szabo G, Jacobs JJ, Kuettner KE, Glant TT.

Suppression of osteoblast function by titanium particles. J Bone

Joint Surg Am 1997;79:107–12.

[43] Wang ML, Nesti LJ, Tuli R, Lazatin J, Danielson KG, Sharkey

PF, Tuan RS. Titanium particles suppress expression of

osteoblastic phenotype in human mesenchymal stem cells. J

Orthop Res 2002;20:1175–84.

[44] Vermes C, Chandrasekaran R, Dobai JG, Jacobs JJ, Andersson

GB, An H, Hallab NJ, Galante JO, Glant TT. The combination

of pamidronate and calcitriol reverses particle- and TNF-alpha-

induced altered functions of bone-marrow-derived stromal cells

with osteoblastic phenotype. J Bone Joint Surg Br 2004;86:

759–70.