Combined Bone Morphogenetic Protein-2 and -7 Gene Transfer EnhancesOsteoblastic Differentiation and Spine Fusion in a Rodent Model

Wei Zhu,1 Bernard A Rawlins,2 Oheneba Boachie-Adjei,2 Elizabeth R Myers,1 Jun Arimizu,2 Eugene Choi,2

Jay R Lieberman,3 Ronald G Crystal,4 and Chisa Hidaka1

ABSTRACT: To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer wasperformed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precur-sors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transferenhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer.

Introduction: Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are veryhigh. Previous studies have suggested that the co-expression of two different BMP genes can result in the productionof heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 genetransfer was performed to test whether this approach improves osteoblastic differentiation and bone formationcompared with single BMP gene transfer.Materials and Methods: A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2(AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus.Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts andMC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeksafter surgery for radiographic and mechanical fusion, bone formation, and mineralization.Results: BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 andAdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relativelylow doses (7–140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12and MC3T3-E1 cells, that were up to 6- and 40-fold higher, respectively, than levels induced by maximal doses(200–1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 andAdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineral-ization rate and bone volume in the fusion mass versus single BMP gene transfer (p � 0.02, all comparisons).Conclusion: Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblasticdifferentiation and spine fusion than individual BMP gene transfer.J Bone Miner Res 2004;19:2021–2032. Published online on August 30, 2004; doi: 10.1359/JBMR.040821

Key words: bone morphogenetic protein, osteoblasts, bone, bone mineralization, rodent

INTRODUCTION

BONE MORPHOGENETIC PROTEINS (BMPs), which were ini-tially isolated from bovine bone because of their ability

to induce ectopic bone formation, are proteins within thetransforming growth factor beta (TGF-�) superfamily ofdimeric, disulfide-linked growth factors.(1–5) While manystudies have focused on the bone-forming activity of recom-binant BMP homodimers and/or the expression of a singleBMP gene, in bone and other organs, two or more BMP

genes are often co-expressed.(1–8) Both BMP2 and BMP7(also known as osteogenic protein-1) were initially co-purified from bovine bone.(3,4) Consistent with this finding,Lyons et al.(6) showed the co-localization of BMP2 andBMP7 transcripts in developing limbs of mouse embryos.Other studies of embryogenesis have also shown the re-quirement for co-expression of several pairs of BMP genesfor normal development.(7,8) In vitro studies have suggestedthat co-expression of BMPs can result in the expression ofheterodimeric BMPs.(9–11) While “native” BMP het-erodimers have not been isolated in vivo, studies of osteo-blastic differentiation and ectopic bone formation haveshown that BMP heterodimers are more potent than their

Dr Crystal served as a consultant and received funding fromGenVec, Inc. All other authors have no conflict of interest.

1Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, New York, USA; 2Spinal Deformity Service, Departmentof Orthopedic Surgery, Hospital for Special Surgery, New York, New York, USA; 3Department of Orthopedic Surgery, University ofCalifornia, Los Angeles, California, USA; 4Department of Genetic Medicine, Weill Medical College of Cornell University, New York,New York, USA.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 19, Number 12, 2004Published online on August 30, 2004; doi: 10.1359/JBMR.040821© 2004 American Society for Bone and Mineral Research

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respective homodimers.(9–11) Based on these considerations,this study was focused on assessing adenovirus (Ad)-mediated co-transfer and co-expression of BMP2 andBMP7 cDNA to enhance bone formation in the context ofspine fusion.

Spine fusion is a commonly performed orthopedic pro-cedure requiring the formation of new bone around thespine to increase its stability. Non-union, or the failure ofstabilizing bone to form, is still the major complicationoccurring in up to 26–35% of cases of posterolateral lumbarspine fusion.(12–14) While non-union per se, may not strictlycorrelate to a poor clinical outcome, especially in the shortterm, stable unions are clearly correlated with superiorclinical outcomes.(15–17) Because of this, research in meth-ods to improve spine fusion, whether through biological ormechanical interventions (devices), remains intense.(18–20)

Currently, autogenous bone grafting is the gold standard forinduction of a spine fusion. However, harvesting of autog-enous bone graft (usually from the iliac crest) is associatedwith significant morbidity in up to 30% of patients.(12–14,21)

Allograft may be used, but usually only as an extender, as itis osteoconductive, but not osteoinductive, and, when usedalone, results in poor fusion rates.(21–27) As such, the devel-opment of graft alternatives such as BMPs is of greatinterest for potential use in spine fusion surgery.

Recombinant BMP (rBMP) homodimers have beenshown to enhance spine fusion in animals and humans;however, the clinical doses are extremely high.(28–34) Forexample, rBMP2 in a collagen sponge carrier, which hasrecently been approved to induce spine fusion, contains a10- to 20-mg dose.(18) In contrast, only about 1 �g of BMPcan be purified from 1 kg of bone.(33) The requirement ofhigh clinical doses not only makes BMP therapy relativelyexpensive in comparison with other available bone-enhancing materials such as demineralized bone matrix, butalso presents several potential drawbacks. Possible sideeffects may include, but are not restricted to, the stimulationof bone resorption or induction of bone formation in unin-tended areas or, because of the wide presence of BMPsreceptors on neural cells, the possibility of inducing unex-pected side effects in the nervous system.(34–36)

Previously we have shown that bone marrow cells genet-ically modified by adenovirus gene transfer vector encodingeither BMP7 (AdBMP7) or BMP2 (AdBMP2) can enhanceposterolateral spine fusion in a rat model.(37,38) In this study,we have used a combination gene therapy strategy to testwhether co-expressing BMP2 and BMP7 would be moreefficacious than single BMP gene transfer to increase osteo-blastic differentiation in vitro and to enhance bone forma-tion in rat spine fusion.

MATERIALS AND METHODS

Adenovirus vectors

Adenovirus (Ad) vectors in this study were E1�, partialE3� based on the Ad serotype 5 genome with the expressioncassette in the E1 position. While AdBMP2 and AdBMP7were constructed separately, both contain the expressioncassette encoding either the human BMP2 or human BMP7cDNA under the cytomegalovirus immediate/early pro-

moter enhancer.(37,39) The control vector AdNull is similarbut encodes no transgene.(40) All Ad vectors were propa-gated, purified, and stored as previously described anddosed based on physical particle count as particle unit(pu).(41,42)

Transient expression of BMPs

Because A549 lung epithelial cell line (American typeCulture Collection) are easily transfected by Ad vectors,they were used as a cell line for producing BMPs. Cellswere maintained in “complete media” (DMEM with 10%FBS and 1% penicillin-streptomycin; all from Gibco).Ninety percent confluent wells of A549 cells were incubatedfor 2 h with both AdBMP2 and AdBMP7, or as controls,AdBMP2 and AdNull, AdBMP7 and AdNull, AdNull only,or no virus (mock-transfection). Cells were washed andallowed to recover in “complete media” overnight, followedby a change to X-Vivo (serum-free) medium (Gibco). Su-pernatants were collected after 2 days for measurement ofBMP levels and in vitro activity assays.

Amounts of BMPs in A549 supernatants were quantifiedusing a commercially available ELISA kit for BMP2 (R&DSystems) or direct ELISA for BMP7.(43) Briefly, superna-tants of A549 cells were incubated in a 96-well immunosor-bent plate (DYNEX Technologies) for 2 h at 37°C. Afterwashes with PBS, pH 7.4, and blocking with 3% goat serumin PBS, samples were incubated with mouse-anti-humanBMP7 antibody at 1:1000 dilution (R&D Systems) for 1 hat 37°C. Wells were again washed and labeled with horse-radish peroxidase–conjugated goat-anti-mouse IgG second-ary antibody (R&D Systems) at 1:1000 dilution. Finally,colorimetric peroxidase reaction was used (Bio-Rad), andthe target antigen (BMP7) was quantified by spectropho-tometry at 405 nm. A standard curve generated usingrBMP7 (R&D Systems) showed that the range of detectionwas between 4 and 500 ng/ml.

To detect the presence of BMP2/7 heterodimer, ELISAwas performed on supernatants after immunoprecipitation.First, immunoprecipitation was performed with anti-BMP2antibody (R&D Systems) using a spin column containingimmobilized protein G beads (Seize X Mammalian Immu-noprecipitation kit; Pierce). The immunoprecipitate, whichwould be predicted to contain both BMP2 homodimer andBMP2/7 heterodimer, was tested for ELISA for BMP7 asdescribed above. The amount of heterodimer in cell super-natants was estimated to be equivalent to the amount ofBMP7 detectable in the anti-BMP2 antibody immunopre-cipitates. To confirm these findings, anti-BMP7 antibodyimmunoprecipitates were also tested by ELISA for BMP2.

As additional confirmation for the presence of BMP2/7heterodimer protein, Western blotting was performed usingNupage Bis-Tris gel systems (Invitrogen). Briefly, superna-tants of cells co-transfected with AdBMP2 and AdBMP7,AdBMP2 and AdNull, AdBMP7 and AdNull, AdNull only,or medium only (no virus) were immunoprecipitated withanti-BMP7 antibody. Samples of the immunoprecipitatecontaining 40 �g of total protein were loaded on a 12%acrylamide gel under nonreducing conditions. As positivecontrols, rhBMP2 (R&D Systems) at 50 ng and supernatantof cells co-transfected with AdBMP2 and AdNull that had

2022 ZHU ET AL.

not been immunoprecipitated were also loaded on the gel.After electrophoresis, fractioned proteins were transferredfrom the gel to a polyvinylidene diflouride (PVDF; Bio-Rad) membrane and blocked by overnight incubation in10% (wt/vol) skim milk in PBS at 4°C. Subsequently, themembrane was incubated with primary mouse-anti-humanBMP2 antibody (1:1000 dilution in 1% [wt/vol] skim milkin PBS) for 1 h at room temperature. After washing inwashing buffer (0.04% Tween-20 [vol/vol] in PBS), themembrane was incubated with secondary horseradish-conjugated goat anti-mouse antibody (1:1000 dilution in 1%skim milk in PBS) for 1 h at room temperature. The immu-noreactive bands on the membrane were detected by usingECL Western blotting detection kit (Amersham PharmaciaBiotech). Similarly, supernatants immunoprecipitated withanti-BMP2 antibody were also detected by Western blottingwith anti-BMP7 antibody.

Determination of BMP activity in vitro

Time course and dose-response studies were performed tocompare the potencies of BMP2/7-containing supernatantversus BMP2 or BMP7 homodimers for inducing the osteo-blastic differentiation of C2C12 mouse myoblastic cell line(American Type Culture Collection) and MC3T3-E1 mousepre-osteoblastic cell line (gift of Bruce Rapuano, PhD,Hospital for Special Surgery, New York, NY, USA). Cellswere seeded at a density of 5 � 105 cells/well in 6-well cellculture plates and maintained in “low growth media” con-sisting of DMEM (for C2C12 cells) or �-MEM (forMC3T3-E1 cells) containing 25 �g/ml ascorbic acid(Sigma), with 5% FBS and 1% penicillin-streptomycin(Gibco).

For dose-response studies, preconfluent cells were stim-ulated with supernatants of AdBMP-stimulated A549 cellsdiluted with “low growth media” at ratios from 1:80 to 5:1.These dilutions contained between 4 and 233 ng/ml ofBMP2 and/or BMP7. C2C12 or MC3T3-E1 cells werestimulated with BMPs on days 1 and 4, and markers ofosteoblastic differentiation were assayed on day 7. For timecourse studies, supernatants containing 250 ng/ml of totalBMP2 and/or BMP7 were added to cells on days 1 and 4,and markers of differentiation were assessed on days 0(before BMP stimulation), 1, 4, 7, and 12. Osteocalcin(OCN) expression, detected in cell medium by ELISA (Bio-medical Technologies), and alkaline phosphatase (ALP)activity in cell lysates, determined using a colorimetricassay kit (Sigma), were used as markers of osteoblasticdifferentiation. ALP activity was normalized to total cellprotein as detected by bicinchoninic acid protein assay(Pierce).

Rat spine fusion

To determine whether combination gene transfer withAdBMP2 and AdBMP7 enhances in vivo bone formationcompared with treatment with individual BMP gene trans-fer, a rat spine fusion model was used.(37,38) A total of 78Sprague-Dawley rats (200–300 g, female) underwent bilat-eral single level posterolateral spine fusion as previouslydescribed under a protocol approved by the InstitutionalAnimal Care and Use Committee.(37) Briefly, the spine was

exposed using a paraspinal muscle-splitting approach, andthe dorsal surfaces of the transverse processes of L4 and L5

vertebrae were decorticated with scalpel blades.(37,38)

Freeze-dried allograft bone (50 mg/site) and carboxymeth-ylcellulose hemostatic foam (Gelfoam, 30 mg/site) soakedwith 30 �l of Ad vector suspended in normal saline or 30 �lof saline only were implanted into each fusion site. Theincision was closed in layers, and the animals were allowedad libitum feed and cage activity postoperatively. Animalswere observed for signs of postoperative pain daily for thefirst postoperative week, but did not require analgesia.Spines were harvested 8 weeks after surgery and assessedfor spine fusion.

A preliminary dose-response study was performed withcombination of AdBMP2 and AdBMP7 at 0.75 � 108,0.75 � 109, 0.75 � 1010, or 0.75 � 1011 pu of each vectortested in four animals per dose. Based on the results fromdose-response study, comparisons of efficacy between sin-gle and combined AdBMP gene transfer was performedwith AdBMP2 and AdBMP7 at 0.75 � 1010 pu each (n �15); AdBMP2 (n � 13) alone, AdBMP7 alone (n � 19), orAdNull control (n � 19) at 1.5 � 1010 pu; or saline-onlycontrol (n � 12) in each site of spine fusion. Previousstudies in our vector core facility has shown that most viralpreparations contained one infectious particle for every 30–100 physical particles of Ad (data not shown). Based on thisconversion, 1.5 � 1010 pu is equivalent to 1.5 � 4.5 � 108

plaque-forming units (pfu).Fusion was assessed radiographically and mechanically 8

weeks after surgery. Immediately after harvest, isolatedspines (L3–L6) from all animals were assessed by high-resolution faxitron X-ray.(37,44) Radiographs of each spinewere obtained using two (right and left) oblique views tooptimize the visualization of the transverse processes and/orfusion masses. Samples were determined to be fused ifradio-dense cortical bridging was observed between the L4

and L5 transverse processes on at least one side. If discon-tinuities such as clefts or gaps were apparent, the spine wasconsidered not fused, regardless of the presence of newbone formation. This categorization was based on the clin-ical practice that considers a spine fused if either unilateralor bilateral evidence of bony bridging exists.

Mechanical stability was assessed by manualpalpation.(12,37,44 – 49) Immediately after the X-ray assess-ment, spines were gently palpated, and the axial andsagittal motion at the L4–L5 level was compared with themotion at the adjacent levels above (L3–L4) and below(L5–L6). Spines were rated as fused if there was nomotion or not fused if there was any motion. Previously,we have shown that manual palpation is as sensitive asrange of motion testing.(37) Furthermore, this method hasbeen used in many studies of experimental spine fusionbecause it correlates well with clinical assessment, whichis typically a Yes/No decision and not an assessmentof range of motion in degrees.(12,37,44 – 49) Both theradiographic and mechanical assessments of fusionwere performed by observers blinded to the treatmentgroups.

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Microscopic morphology and bone mineralization

On harvest, at least three spines from each group werefixed in ethanol and embedded in methylmethacrylate, and4-�m-thick coronal sections were cut on a microtome (JungPolycut E; Leica). Morphology was examined by light mi-croscopy of sections stained with Goldner’s trichrome. Toassess new bone formation, at least three rats per group wereinjected twice with 90 mg/kg of xylenol orange (XO;Sigma) intraperitoneally 14 and 7 days before death. Un-stained sections were assessed for extent of fluorochrome-labeling by histomorphometry.(37,50) Three to nine areaswithin the fusion mass or L4 or L5 transverse processes wereselected randomly, and 16–25 measurements were madeencompassing the shortest distance between twofluorochrome-labeled mineralized fronts or the distance be-tween the fluorochrome-labeled mineralized front to edge ofbone structure using Bioquant Nova 2000 software (R&MBiometrics). Two to four slides from three or four spines pergroup were evaluated in this manner. The level of fluores-cence in the growth plate of the L5 vertebral body of eachspecimen served as positive control.(37) The mineral appo-sitional rate was calculated by dividing the mean of themeasured distances for each specimen by the labeling pe-riod (7 days).

To quantify the volume and density of bone mineral in thefusion site, the fusion segment (L4 and L5 vertebrae) fromeach of four to five randomly selected spines per group wasisolated and imaged in their entirety in a �CT scanner(MS-8 Small Specimen Scanner; GE Medical Systems)along with a standard SB3 phantom (GE Medical Systems).3D images were reconstructed at 11-�m isotropic resolution(EVS Beam software; GE Medical Systems) and analyzed.For analysis, the 3D image was oriented so that the rostral-caudal axis of the specimen was aligned with the verticalaxis, the intervertebral disc was aligned with the horizontalaxis, and the posterior spinous processes were aligned withthe sagittal axis. For each specimen, a right and left volumeof interest fixed at 0.18 ml (4 mm � 4.5 mm � 10 mm),which encompassed the entire interspace between the L4

and L5 transverse processes and/or the fusion mass, wasassessed. For each measurement, the volume of interest wasplaced lateral to (and not overlapping) the widest portion ofthe L4 and L5 vertebral bodies and aligned inferiorly to themid-line of the intervertebral disc. The inclusion of a stan-dard phantom made of SB3 (a polymer with an X-rayattenuation equivalent to bovine cortical bone, 1.1 g ofhydroxyapatite/ml) during scanning was used for conver-sion of the CT attenuation values to BMD (mg/ml; EVSMicroview Software; GE Medical Systems). The recon-structed grayscale images were thresholded to separate boneand marrow voxels. Using the thresholded data, tissue vol-ume and bone volume fraction (bone volume/total volume)were evaluated (EVS Microview software; GE MedicalSystems).

Statistical analysis

Results for in vitro assays (n � 3/group in duplicateexperiments) and histological grading (n � 3–4/group)results are expressed as a mean � SE. For bone volume

fraction and BMD (n � 4–5/group) results are expressed asa mean � SD. For all of these parameters, groups werecompared by ANOVA with posthoc testing where differ-ences were detected. Significance was accepted where p �0.05.

For tests of radiographic and mechanical fusion, the re-sults for each group (fused versus not fused in n � 12–19spines/group) were compared by �2 test. Agreement be-tween measurements was assessed by the proportion ofoverall agreement {([number of agreements on fusion �number of agreements on no fusion]/total number) � 100 }and by the � statistic. Interobserver agreement of radio-graphic fusion was based on the ratings of two separateobservers, both spine surgeons. Intermethod agreement be-tween X-ray determination of fusion and manual palpationwas also assessed.

RESULTS

Expression of BMPs by AdBMP-transfected cells

In supernatants of A549 cells co-transfected with Ad-BMP2 and AdBMP7, both BMP2 and BMP7 were detectedby ELISA before immunoprecipitation. Co-transfectionwith AdBMP2 and AdNull resulted in expression of BMP2only, whereas that with AdBMP7 and AdNull resulted inexpression of BMP7 only. Expression levels of BMP2 orBMP7 in all of these groups were similar (p � 0.05, allcomparisons, Fig. 1A). As expected, neither BMP2 norBMP7 was detected in supernatants of AdNull only or novirus controls. After immunoprecipitation with anti-BMP2antibody, 258 � 29 ng/ml BMP7 was detected in immuno-precipitates, suggesting that �93% of the total BMP7 in thesupernatant of co-transfected cells may be in the form ofBMP2/7 heterodimer (Fig. 1B). Similar results were ob-tained by immunoprecipitation with anti-BMP7 and fol-lowed by ELISA for BMP2 (231 � 28 ng/ml, 84% of totalBMP amount). As expected in controls transfected witheither AdBMP2 or AdBMP7 and AdNull, AdNull alone, orno virus, only transfected BMP was detected (Fig. 1B). Insupernatants of cells transfected by AdBMP2 and AdNull,the amount of BMP7 was below the level of detection of theELISA (4 ng/ml) whether tested before or after immuno-precipitation with anti-BMP2 or anti-BMP7 antibody, sug-gesting the minimal cross-activity of antibodies. Similarly,the amount of BMP2 in supernatants of cells transfected byAdBMP7 and AdNull was also below the level of detectionof ELISA (6 ng/ml, before or after immunoprecipitation.

Western blots for BMP2 showed a single band of �38kDa for the supernatant of A549 cells co-transfected withAdBMP2 and AdBMP7, which had been immunoprecipi-tated with anti-BMP7 antibody, suggesting the presence ofthe BMP2/7 heterodimer. A slightly smaller single band,�36 kDa, was detected in supernatant of A549 transfectedwith AdBMP2 and AdNull and in positive control rhBMP2(Fig. 1C). As expected, no BMP2 signal was detected insupernatants transfected with AdBMP2 and AdNull, Ad-BMP7 and AdNull, AdNull only, or without viral transfec-tion after immunoprecipitation with anti-BMP7 antibody(Fig. 1C). Similar results were obtained by immunoprecipi-tation with anti-BMP2 and followed by Western blotting for

2024 ZHU ET AL.

BMP7 (data not shown). The sizes of both the bands in theAdBMP2/AdBMP7 and AdBMP7/AdNull co-transfectedsupernatants were �38 kDa.

BMP-stimulated osteoblastic differentiation

Dose-response and time course studies showed increasedand accelerated expression of markers of osteoblastic dif-

ferentiation in cells stimulated by supernatants of co-transfected cells compared with supernatants of cells trans-fected with a single BMP gene or rBMP homodimers.

Supernatants of AdBMP2 and AdBMP7 co-transfectedcells increased the expression of OCN in C2C12 cells in adose-dependent manner, with maximal stimulation inducedby supernatants diluted 1:5 or 1:1, containing between 47and 140 ng/ml BMP (Fig. 2A). The OCN expression level atthe optimal dose of BMP2/7-containing supernatant was6-fold higher than maximal levels induced by rBMP7 (500ng/ml) or by rBMP2 (1 �g/ml; p � 0.05, both comparisons,Fig. 2B). The maximal levels of OCN (�4 ng/ml) inducedby 500-1000 ng/ml of rBMP2 or rBMP7 were induced withBMP2/7-containing supernatants diluted 40-fold, contain-ing only about 7 ng/ml of BMP (Figs. 2A and 2B).

Supernatants of AdBMP2 and AdBMP7 co-transfectedcells also increased ALP activity in C2C12 cells a dose-dependent manner, with the maximum reached at 1:1 dilu-tion of supernatant (data not shown). The ALP activityinduced by this dose of BMP2/7-containing supernatant was

FIG. 1. Expression of BMP2 and BMP7 by Ad-modified cells. A549epithelial cells were co-transfected with Ad encoding BMP2 (Ad-BMP2) and Ad encoding BMP7 (AdBMP7), or as controls, AdBMP2and AdNull (encoding no transgene), AdBMP7 and AdNull, AdNullalone, or no virus. The expression of BMP2 or BMP7 in cell superna-tants was detected by ELISA (A) before and (B) after immunoprecipi-tation with anti-BMP2 antibody. Results are expressed as mean � SEfor three culture wells in each treatment group. (C) The expression ofBMP2 protein signal in A549 cell supernatants were also detected byWestern blot. Lanes 1 and 7 represent the positive controls (notimmunoprecipitated). Lane 1, A549 cells transfected with AdBMP2 �AdNull. Lanes 2–6 represent A549 supernatants immunoprecipitatedwith anti-BMP7 antibody. Lane 2, AdBMP2 � AdBMP7; lane 3,AdBMP2 and AdNull; lane 4, AdBMP7 and AdNull; lane 5, AdNullonly; lane 6, medium only (no virus); lane 7, 50 ng rhBMP2.

FIG. 2. Dose-response expression of OCN by BMP-stimulatedC2C12 myoblasts. (A) As a marker of osteoblastic differentiation,OCN levels were measured in C2C12 myoblasts after 7 days ofstimulation with supernatants of a producer cell line (A549) co-transfected with AdBMP2 and AdBMP7. To assess a dose-responseeffect, supernatants were diluted from 1:80 to 5:1 containing between4 and 233 ng/ml BMP. (B) For comparison, C2C12 was stimulatedwith rBMP2 or rBMP7. Each data point represents the mean � SE forthree culture wells at each dose in each treatment group in duplicateexperiments. Note that the scales of vertical axes in the two graphs aredifferent. ap � 0.05 vs. rBMP2 at 1000 ng/ml; bp � 0.05 vs. rBMP7 at500 ng/ml.

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27-fold greater than the maximal levels induced by rBMP7(500 ng/ml) or 33-fold greater than the maximal level in-duced by rBMP2 (1 �g/ml) (p � 0.001, both comparisons).The maximal level of ALP induced by these concentrationsof rBMPs could be induced by supernatants containing �7ng/ml of BMP2/7.

In addition to increasing the level of expression of OCNand ALP activity, BMP2/7-containing supernatants acceler-ated the onset of ALP activity in C2C12 cells versus ho-modimers (Fig. 3). ALP activity was detected as early as 4days after stimulation, with supernatant containing �233ng/ml BMP2/7, and continued to increase for 12 days (Fig.3). In contrast, supernatants containing 250 ng/ml of BMP2or BMP7 homodimer did not induce detectable ALP activityuntil day 7, and the levels of ALP activity induced by BMP2or BMP7 at the day 12 endpoint(s) were still 3- to 5-foldlower than that induced by BMP2/7-containing supernatant(p � 0.05, both comparisons, Fig. 3). C2C12 cells stimu-lated with a mixture of BMP2 and BMP7 homodimersshowed a similar pattern of ALP activity as cells stimulatedwith BMP2 homodimer alone (Fig. 3). As expected, controlsupernatants of A549 transfected with AdNull or no virusresulted in no change in ALP levels (Fig. 3).

OCN expression was also induced by BMP2/7-containingsupernatants by day 4, with further increases detected atdays 7 and 12. Although supernatant containing BMP7increased OCN expression in C2C12 cells in a similartime-dependent pattern, the levels were 2- to 3-fold lowerthan that induced with BMP2/7 throughout the time course(Fig. 4). Supernatant containing BMP2 homodimer or incombination did not elevate OCN expression until day 7. Nodetectable OCN expression was induced by AdNull or novirus controls in C2C12 cells.

In MC3T3-E1 cells, BMP2/7-containing supernatantswere also more potent than either homodimer. Effects ofBMP2/7-containing supernatants on MC3T3-E1 were dose-dependent with maximal OCN expression (21 ng/ml) and

ALP activity (16 �mol/h/mg total protein) induced by su-pernatants diluted at 1:5 or 1:1, containing �47 or 140ng/ml of BMP2/7, respectively. The OCN level inducedwith this dose of BMP2/7 was 4-fold higher than the max-imal levels induced with rBMP2 (200 ng/ml) or rBMP7 (1�g/ml; p � 0.05, both comparisons). The maximal ALPactivity induced with this dose of BMP2/7-containing su-pernatant was 22- or 40-fold greater than the levels inducedby 1 �g/ml of rBMP2 or rBMP7, respectively (p � 0.01,both comparisons).

In MC3T3-E1 cells, OCN expression and ALP activitywere also elevated by BMP2/7-containing supernatants in atime-dependent manner. Although the levels of these mark-ers induced by BMP2/7-containing supernatants were 2- to3-fold higher than that induced by BMP2 or BMP7 ho-modimers (alone or in combination; p � 0.05, all compar-isons), no acceleration was observed. At the final time-point(day 12), the OCN expression induced by BMP2/7-containing supernatants was 2- or 3-fold higher than thatinduced by BMP2 or BMP7, respectively (p � 0.05, bothcomparisons; Fig. 4). Similarly, the level of ALP activityinduced by heterodimers by day 12 was 3- or 10-fold higherthan that induced by BMP2 or BMP7 homodimers, respec-tively (p � 0.05, both comparisons). The combination ofBMP2 and BMP7 homodimer-containing supernatants didnot elevate either OCN or ALP versus using one homodimeralone (data not shown).

Dose-response of AdBMP-enhanced spine fusion

The proportion of spine fusions increased with increasingamounts of AdBMP2 and AdBMP7 used. After treatmentwith the two lowest doses (0.75 � 108 or 0.75 � 109

pu/vector), radiographic fusion was achieved in one of fourspines (25%). Treatment with AdBMP2 and AdBMP7 at adose of 0.75 � 1010 pu each resulted in fusion in three offour spines (75%). Treatment with AdBMP2 and AdBMP7at a dose of 0.75 � 1011 pu each resulted in 100% of fusion;however, fusions were observed in other spinal levels (L3–L5) in two spines in this group (data not shown). To avoid

FIG. 3. Time course of ALP activity by BMP-stimulated C2C12myoblasts. C2C12 cells were stimulated with a 5:1 dilution of A549supernatants containing �250 ng/ml BMP. Each data point representsthe mean � SE for three culture wells at each time-point in eachtreatment group in duplicate experiments. ap � 0.05 vs. AdNull or novirus at same time point; bp � 0.05 vs. AdBMP2 � AdNull orAdBMP7 � AdNull or Mixed at same time-point; cp � 0.05 vs. day 4within same treatment group.

FIG. 4. OCN expression in C2C12 and MC3T3-E1 cells after stim-ulation with BMPs at a concentration of 250 ng/ml for 12 days. Resultsare expressed as mean � SE for three culture wells in each cell typeand treatment group in duplicate experiments. ap � 0.05 vs. AdBMP2� AdNull or AdBMP7 � AdNull; bp � 0.05 vs. no virus control,within the same cell line.

2026 ZHU ET AL.

fusion at unintended spinal levels, the dose of 0.75 � 1010

pu/vector, or a total of 1.5 � 1010 pu (equivalent to 1.5 �4.5 � 108 pfu), of AdBMP was used as the dose for the restof the in vivo study (see results below).

Radiographic and mechanical fusion

Combination AdBMP2 and AdBMP7 gene transfer in-creased the number of fusions when compared with singlegene transfer. Radiographic fusion was achieved in 93% (14of 15), and mechanically stable fusion was achieved in 73%(11 of 15) of spines treated with a combination of AdBMP2and AdBMP7 (Table 1; Fig. 5). By either parameter, thenumber of fusions in the combined AdBMP2 and AdBMP7treatment group was greater than that achieved in any othertreatment group (p � 0.02). Although bridging bone wasobserved in the L4/L5 interspace in 77% (10 of 13) of spinesafter treatment with AdBMP2 alone, X-rays showed radio-lucent lines or clefts in 4 of them, so that only 46% weredetermined to be fused. This number of radiographic fusionwas significantly greater than that achieved in the AdNulland saline control groups (p � 0.05, both comparisons).However, when assessed mechanically, the number ofspines fused in the AdBMP2-only group was not signifi-cantly different from controls (p � 0.05). The number offusions, as assessed radiographically or mechanically, aftertreatment with AdBMP7 alone was not statistically differentfrom AdNull or Saline controls.

Using the X-ray technique to rate fusion, the proportionof overall agreement between the two observers was 100%(� � 1). Between the X-ray method and manual palpation,the intermethod agreement by group was as follows: Saline,92%; AdNull, 95%; AdBMP2, 69%; AdBMP7, 100%; Ad-BMP2 � AdBMP7, 80%. The proportion of overall agree-ment between the X-ray method and manual palpation was88%, and � was 0.70, indicating very strong agreement.

Bone formation and mineralization in the fusion mass

As assessed both by light microscopy or �CT, fusionmasses in the combined AdBMP2 and AdBMP7 group

showed a continuous cortical rim and trabecular architecturethroughout L4 and L5 transverse processes (Fig. 5). Allspines in the combined AdBMP2 and AdBMP7 group,which were assessed by �CT, had bilateral fusion masseswhose cortices were continuous in every section in threeplanes (axial, coronal, and sagittal). In several spines fromeach of the single BMP gene transfer groups, discontinuitiesin the fusion mass were apparent in at least one plane asassessed by �CT (Fig. 5). When assessed by light micros-copy, such discontinuities were characterized by cartilagi-nous and/or fibrous tissue (data not shown). Saline andAdNull controls were both characterized by distinct, un-fused transverse processes consisting of normal-appearingbone, without the presence of cartilaginous or fibrous tissue.

The rate of mineralization was 2-fold higher in the com-bination AdBMP2 and AdBMP7 group versus single Ad-BMP and saline controls (p � 0.05, all comparisons) asmeasured histomorphometrically on XO-labeled sections(Figs. 6A and 6B). In AdBMP2- or AdBMP7-only groups,the mineralization rate was not statistically different fromsaline-treated control (p � 0.05, both comparisons; Fig.6B). Mineralization rates could not be assessed in theAdNull-treated spines because no XO label was visualized

TABLE 1. AdBMP-ENHANCED SPINE FUSION*

N† Radiographic‡ Mechanical§

Saline 12 8% (1/12) 0% (0/12)AdNull 19 5% (1/19) 0% (0/19)AdBMP2 13 46% (6/13)¶ 8% (1/13)AdBMP7 19 32% (6/19) 16% (3/19)AdBMP2 � AdBMP7 15 93% (14/15)¶** 73% (11/15)¶**

* Spine fusions were performed as described in Methods, and assessed 8wk after surgery.

† Number of spines in each treatment group.‡ Percentage of radiographic fusion (successfully fused spines in total

number) determined by x-ray if radio-dense cortical continuity was ob-served between the lumbar level 4 (L4) and L5 transverse processes on atleast one side.

§ Percentage of mechanically stable fusion (successfully fused spines intotal number) determined by manual palpation if no motion was detected inthe L4–L5 motion segment.

¶ p � 0.05 vs. Saline and AdNull controls, ** p � 0.05 vs. AdBMP2and AdBMP7, within the same assessment.

FIG. 5. Radiographic appearance of AdBMP-treated spines. Poste-rior inter-transverse spine fusion at L4 and L5 were assessed in rats 8weeks after implantation of allograft bone and carboxymethylcellulosefoam (Gelfoam) soaked with a combination of AdBMP2 and AdBMP7,each vector alone, AdNull, or vehicle alone (saline). Right and leftoblique high-resolution X-rays (top) and coronal images from 3D �CT(bottom) are shown. (A) AdBMP2 and AdBMP7, (B) AdBMP2, (C)AdBMP7, (D) Saline, and (E) AdNull.

2027COMBINED BMP2 AND BMP7 GENE TRANSFER

in the transverse processes or vertebral body growth platesin this group.

3D �CT showed that co-administration of AdBMP2 andAdBMP7 resulted in a 2-fold greater bone volume fraction(BVF) at the fusion site versus AdBMP2-, AdBMP7-, andAdNull-treated groups and a 3-fold greater BVF at thefusion site versus saline control group (p � 0.001, allcomparisons; Fig. 7). BMD at the fusion site in combinedAdBMP2 and AdBMP7 groups (250 � 60 mg/ml) was also�50% higher than that in AdBMP2 (152 � 23 mg/ml),AdBMP7 (167 � 43 mg/ml), and saline-treated (165 � 29mg/ml) groups (p � 0.05, all comparisons). However, BMDin the combination AdBMP2- and AdBMP7-treated groupwas not significantly higher than that in AdNull controlgroup (191 � 70 mg/ml, p � 0.05). Neither AdBMP2 nor

AdBMP7 alone resulted in significant increases in BVF orBMD compared with saline or AdNull controls (p � 0.05,both comparisons).

DISCUSSION

In this study, we have shown that combined AdBMP2and AdBMP7 gene transfer accelerates osteoblastic differ-entiation in vitro and enhances spine fusion in vivo com-pared with single BMP2 or BMP7 gene transfer. Consistentwith previous studies showing that BMP heterodimers areproduced by co-transfection of target cells with two differ-ent BMP cDNAs,(9–11) our immunoprecipitation data sup-port the presence of BMP2/7 heterodimer in the supernatantof cells co-transfected with AdBMP2 and AdBMP7. More-over, our in vitro data indicate that supernatants of cellsco-transfected with AdBMP2 and AdBMP7 accelerate andincrease the osteoblastic differentiation of precursor cellscompared with supernatant of cells expressing a single BMPgene. In contrast to the supernatant of co-transfected cells, amixture of supernatants from cells transfected with eachBMP did not improve osteoblastic differentiation, suggest-ing that the enhanced osteogenic effect of combined BMP2and BMP7 gene transfer is not caused by a synergistic effectof BMP2 and BMP7 homodimers together. In vivo, com-bined AdBMP2 and AdBMP7 gene transfer resulted in agreater number of spine fusions as well as an increasedvolume of bone and an increased rate of bone formation inthe fusion mass over treatment with either BMP gene alone.

BMPs for osteoblastic differentiation and boneformation

Several previous studies have shown that recombinantheterodimers of BMP2 or BMP4 and BMP7 enhance osteo-blastic differentiation in vitro.(9–11) Our findings are inagreement with these reports, showing that the supernatantof cells co-transfected with AdBMP2 and AdBMP7 have asimilar potency as that previously reported for recombinant

FIG. 6. Mineralization analysis of AdBMP-treated spine fusion. Pos-terior inter-transverse spine fusion was performed in rats as describedin Fig. 5, with xylenol orange injection of rats 7 and 14 days beforedeath to detect mineralization. (A) Photomicrographs of unstained,undecalcified histological sections of the fusion mass where fluorescentred lines indicate sites of newly deposited bone mineral. (a) AdBMP2and AdBMP7, (b) AdBMP2, (c) AdBMP7, and (d) Saline. Bar equals100 �m. Bone trabeculae appear blue because of auto-fluorescence.AdNull control spines could not be evaluated because they did not takeup fluorescent label. (B) Mineral appositional rate calculated fromhistomorphometric measurements of the distance between twofluorochrome-labeled mineralization fronts. Results are expressed asmean � SE for three spines in each treatment group. ap � 0.05 vs.AdBMP2, AdBMP7, or Saline control.

FIG. 7. Bone volume fraction analysis of AdBMP-treated spinefusion. Posterior inter-transverse spine fusion was performed in rats asdescribed in Fig. 5. Bone volume fraction was calculated from 3D �CTimages. Results are expressed as mean � SD for four to five spines ineach treatment group. ap � 0.05 vs. AdBMP2, AdBMP7, AdNull, orSaline control.

2028 ZHU ET AL.

BMP2/7. Although we did not chemically purify theBMP2/7, our immunoprecipitation data support its presencein the supernatant of BMP2 and BMP7 co-transfected cells.Despite the possibility of antibody cross reactivity (which inour study was quite low), our data also suggest that theamount of heterodimer in the supernatant of co-transfectedcells may have been very high (up to 93%, as measured byELISA). This finding is also in agreement with previousstudies that have reported very low levels of homodimers insupernatants of cells co-transfected with two BMPgenes.(10,11) The definitive ratio of heterodimer versus ho-modimer in the total amount of BMPs produced by combi-nation gene therapy still requires further investigation, how-ever. Moreover, definitive proof that BMP heterodimer byitself enhances osteogenic differentiation and bone forma-tion versus BMP homodimers will await purification of theheterodimer. Thus, while we cannot exclude the possibilityof a synergism between the BMP2/7 heterodimer and anyBMP2 or -7 that may also have been present in the super-natant, we have clearly shown that the effect of combinedBMP2 and BMP7 gene transfer requires the presence of theBMP2/7 heterodimer and that the combination of BMP2and BMP7 homodimers does not have a synergistic effect,at least in vitro. This is in contrast to a recent report by Aokiet al.(51) that BMP4 (within the same subclass as BMP2) andBMP6 (within the same subclass as BMP7) have a syner-gistic effect on C2C12 myoblasts. Whether combined BMPprotein versus combined BMP gene transfer treatments haveenhanced effects because of similar mechanisms should bea fruitful future area of investigation that may yield impor-tant insights into how BMP therapy may be improved.

Aside from the difference in activity observed betweencombined versus single BMP gene expression, one interest-ing finding in our study was the difference in magnitude ofresponse elicited from MC3T3-E1 versus C2C12 on BMPstimulation. Osteoblastic differentiation was accelerated bysupernatants of BMP2 and BMP7 co-transfected cells ver-sus single BMP gene-expressing cells, in the C2C12, a moreprimitive cell type, but not in MC3T3-E1, a more differen-tiated cell line.(52–54) The relatively greater effect in themore primitive C2C12 cells may suggest that BMP2 andBMP7 co-expression may be more important for inducingthe early stages of osteoblastic differentiation than for thestimulation of already committed pre-osteoblasts.

Although used in our studies primarily as controls, acomparison of findings from our single BMP gene transfergroups showed some interesting differences between BMP2and BMP7 both in vitro and in vivo. While both BMP2(52)

and BMP7(53) have been reported to convert the differenti-ation pathway of C2C12 myoblasts into the osteoblast lin-eage, in our study, slightly lower doses of rBMP7 thanrBMP2 were effective. This difference is consistent withprevious observations showing that lower doses of rBMP7were required to induce intramuscular ectopic bone forma-tion than rBMP2.(2,4) In contrast, our studies show thatlower doses of rBMP2 than rBMP7 stimulate MC3T3-E1cells. In the current in vivo model, treatment with AdBMP2alone resulted in a significant increase in radiographic fu-sion over AdNull and saline controls, whereas treatmentwith AdBMP7 alone did not produce any radiographic

fusions. Previously, we have shown that, while AdBMP2-modified bone marrow cells induces spine fusion in 100%of rats tested, 70% fusion results when AdBMP7-modifiedcells are used.(37,38) Definitive conclusions regarding theeffectiveness of BMP2 versus BMP7 gene transfer cannotbe drawn from our previous studies because different car-riers were used in the AdBMP2 and AdBMP7 studies.(37,38)

However, in light of these in vitro and in vivo results, BMP2gene transfer may be more important than BMP7 in stimu-lating pre-osteoblasts and in enhancing experimental spinefusion. Relatively few studies have compared the in vivoeffects of BMP2 and BMP7 directly. Our studies suggestthat such comparisons could show subtle differences notonly between heterodimer and homodimer but betweenhomodimers in the bone-forming cascade.

BMP gene transfer for spine fusion

Israel et al.(11) have reported that an increased amount ofectopic bone is formed by recombinant BMP2/7 het-erodimer versus BMP2. In agreement with and expandingon this previous report, our data show that combined Ad-BMP2 and AdBMP7 gene transfer increased by 2-fold thevolume of orthotopic bone formed in the context of exper-imental spine fusion as assessed by �CT. While we have notshown that this increase was caused by the heterodimerprotein per se in our in vivo model, studies by our grouphave shown that thousands of copies of Ad genome can bedetected in target cells after in vivo transfection, supportingthe likelihood that our results were caused by co-expressionof BMP2 and BMP7 cDNA by the local cells in the fusionsite.(55,56) Aono et al.(9) have reported that, in contrast torhBMP2, Xenopus BMP4/7 heterodimer induced ectopicbone that did not include cartilage. While this was inter-preted to mean that the heterodimer induced intramembra-nous bone formation while the homodimer induced endo-chondral ossification, our findings suggest that BMPheterodimer may lead to such a result by accelerating boneformation. In spines treated with combined BMP2 andBMP7 gene transfer, the bone formation rate in the fusionmass was increased 2-fold over all controls. This finding isagreement with our in vitro data, showing not only in-creased but accelerated osteoblastic differentiation. It alsosuggests that the increased volume of bone at the fusion sitemay have resulted from a more rapid bone-forming process.

Our �CT analysis indicates that the acceleration of boneformation does not diminish the mineral density of the newbone. Indeed, BMD in the combined gene transfer groupwas greater than in all controls except the AdNull group.This lack of difference with the AdNull group may suggestthat the combined AdBMP2 and AdBMP7 treatment doesnot alter its mineral density; alternatively, the finding mayhave been caused by the lack of an adequate number ofspecimens to analyze for this particular parameter.

Whether the increased rate of bone formation and result-ing increase in bone volume in the fusion mass actuallytranslates to a functionally stable spine fusion is difficult toquantify. We have relied on radiographic evaluation as aclinically relevant outcome measure and showed a clearlyincreased number of fusions resulting from combined Ad-BMP2 and AdBMP7 treatment in this manner. Additionally,

2029COMBINED BMP2 AND BMP7 GENE TRANSFER

we have used manual palpation as a measure of fusionsegment stability. While assessment with this method cor-relates well with our radiographic findings and also is awidely used method in studies of experimental spine fusion,it is admittedly somewhat subjective.(12,37,44–49) We havepreviously attempted quantitative measures of range of mo-tion and hysteresis in the fused spinal segment in thismodel.(37) However, such measures are no better than man-ual palpation in mimicking a clinically satisfactory result,because no data exist to define an acceptable range ofmotion for a successful fusion or an agreed-on method tomeasure such a parameter in patients or in experimentalanimals. The future development of more quantitative mea-sures of functional fusion is clearly an important pursuit forthis field both clinically and experimentally.

Previously, we have reported on the use of an ex vivogene transfer approach to enhance spine fusion using Ad-BMP7.(37) This previous study is directly comparable withour current study because the same allograft scaffold andthe same dose of Ad were used.(37) Comparisons of thesestudies, particularly in light of other studies with AdBMPs,provide several interesting insights for the possible devel-opment of BMP gene transfer for clinical spine fusion.

Whereas in this study, in vivo administration of AdBMP7resulted in no stable fusions, our previous study usingAdBMP7-modified bone marrow cells resulted in spinefusion in 70% of rats.(37) One factor that may have contrib-uted to the greater success of AdBMP7 (only)-mediatedspine fusion in the ex vivo study is the presence of BMP-responsive cells. While in our previous study, unmodifiedmarrow cells did not result in any fusion,(37) other groupshave shown that marrow cells contain osteoblastic precur-sors and alone can enhance the rate of spine fusion.(57,58)

Furthermore, such cells are known to respond to BMPstimulation.(57–59) Thus, it is possible that the addition ofbone marrow cells may have improved the efficacy of thelocally produced BMP7 by providing responsive cells thatmay not have been available in our in vivo model.

A second factor may have been the presence of a hostinflammatory response. While we have not specifically ex-amined the inflammatory response in either of our studies,other groups have shown that the host inflammatory re-sponse may interfere with AdBMP-mediated bone forma-tion. For example, Helm et al.(60) have shown that in vivoAdBMP9 gene transfer can induce spinal fusion in immuno-compromised, but not immuno-competent rats. Previousstudies have also shown that the host inflammatory responseto Ad is directed largely to the Ad particles themselves andthat transfected cells (such as those which would be admin-istered in an ex vivo approach) are much less antigen-ic.(61,62) Such studies support the possibility that spine fu-sion was achieved with ex vivo but not in vivo single BMP7gene transfer because of the diminution of an inflammatoryresponse with the ex vivo approach.

The inflammatory response to Ad is potentially a seriousclinical problem, especially if Ad is administered systemi-cally.(63,64) However, many clinical studies have also shownthat Ad can be safely administered to humans for manytherapeutic applications, particularly if they are given lo-cally, as they would for spinal fusion.(65,66) Furthermore,

while the inflammatory response to Ad seems to be prob-lematic for single BMP gene transfer, ours and other groupshave shown that several other growth factors can be deliv-ered effectively by Ad, even in immunocompetenthosts.(67,68) Clinically, a direct in vivo gene transfer wouldbe much simpler to administer than an ex vivo approach.The development of safer vectors and bone-inducing genesthat do not require the exogenous addition of bone marrowcells would seem to be desirable.

To that end, results from this study suggest that simplyco-administering the BMP2 and BMP7 genes may be effec-tive in overcoming both the effect of inflammation and thelack of bone marrow cells. This was achieved with a dose ofAd that is comparable with doses found to be safe inhumans.(65,66) Another example of using a more effectivetransgene to enhance Ad-mediated spine fusion can befound in the work of Viggeswarapu et al.,(46) showing thatvery low doses of Ad vector encoding LIM mineralizationprotein-1 (LMP-1) can lead to successful spine fusion inrabbits. Furthermore, the mechanism of LMP bone forma-tion has recently been shown to be based on the expressionof multiple BMPs, suggesting that BMP heterodimers mayplay a role.(69) Although we did not reach the clinicallydesirable goal of 100% fusion in our study, we believe thatour findings are persuasive, especially given the poor rate offusion in rats, relative to other species in which spinefusions are often tested. While the administration of rBMPshave been reported to result in 100% fusion in rabbits,allograft alone is also reported to achieve this result.(70,71) Incontrast, even the autograft fusion rate is very low in rats,ranging from 0% to 45%.(37,72,73) We and others have re-ported, however, that when deactivated, demineralized bonematrix (DBM) is used as a carrier for either genes orgenetically modified cells, 100% fusion can be achieved inrats.(38,46) In this light, one reason for not reaching 100% inthis study may have been because of our use of allograft,and not DBM. Given the number of rats in these previousstudies and in this study, further direct comparisons wouldhave to be performed to determine whether the differencebetween 70% and 100% fusion in these models is statisti-cally significant.

In summary, our findings suggest that the co-expressionof BMP2 and BMP7 genes led to accelerated and increasedosteoblastic differentiation in vitro. Additionally, the directin vivo combination BMP2 and BMP7 gene transfer signif-icantly increased bone formation rates and spine fusions ina rat model over either single BMP gene transfer.

ACKNOWLEDGMENTS

We thank Stephen Doty, PhD, and Anthony Labissierefor assistance with histology, Christopher Fritton for assis-tance with �CT analysis, Kohei Goshi, MD, for assistancewith rat surgery, and Elizabeth Ford for technical assistancewith adenovirus vector preparation. This work was fundedin part by the Cobb Scoliosis Research Fund, NY, NationalInstitutes of Health Core Center for Skeletal IntegrityP30AR046121, and GenVec, Gaithersburg, MD. CH is sup-ported, in part, by the Ira DeCamp Fellowship in Musculo-

2030 ZHU ET AL.

skeletal Research of the Hospital for Special Surgery andthe Institute for Sports Medicine Research, NY.

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Address reprint requests to:Chisa Hidaka, MD

Laboratory for Soft Tissue ResearchMusculoskeletal Integrity Program

Hospital for Special Surgery535 East 70th Street

New York, NY 10021, USAE-mail: hidakac@hss.edu

Received in original form October 29, 2003; in revised form May19, 2004; accepted July 27, 2004.

2032 ZHU ET AL.