Ectopic Osteogenesis Using Adenoviral Bone Morphogenetic Protein (BMP)-4 and BMP-6 Gene Transfer
Authors: John A. Jane, Bradley A. Dunford, Adam Kron, Debra D. Pittman, Tsutomu Sasaki, Jin Zhong Li, Hongwei Li, Tord D. Alden, Hayan Dayoub, Gerald R. Hankins, David F. Kallmes, Gregory A. Helm
Journal: Molecular Therapy (2002)

Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms. Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms. IntroductionBone morphogenetic proteins (BMPs) are a group of secreted proteins that are members of the transforming growth factor-β (TGF-β) superfamily. Many of these proteins are characterized by their ability to induce ectopic and orthotopic bone formation when delivered in vivo [1Urist M.R. Bone: Formation by autoinduction.Science. 1965; 150: 893-899Crossref PubMed Scopus (4451) Google Scholar, 2Urist M.R. Dowell T.A. Hay P.H. Strates B.S. Inductive substrates for bone formation.Clin. Orthop. 1968; 59: 59-96Crossref PubMed Scopus (197) Google Scholar, 3Urist M.R. Human bone morphogenetic protein (hBMP).Proc. Soc. Exp. Biol. Med. 1983; 173: 194-199Crossref PubMed Scopus (146) Google Scholar]. The induced bone is formed primarily by endochondral ossification, recapitulating the events of embryonic development [4Wozney J.M. Novel regulators of bone formation: Molecular clones and activities.Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3326) Google Scholar, 5Hogan B.L. Bone morphogenetic proteins: Multifunctional regulators of vertebrate development.Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1713) Google Scholar]. The events stimulated by various BMPs include primitive mesenchymal cell chemotaxis and proliferation [6Lind M. Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation.Acta Orthop. Scand. Suppl. 1998; 283: 2-37PubMed Google Scholar, 7Cunningham N.S. Paralkar V. Reddi A.H. Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor β1 mRNA expression.Proc. Natl. Acad. Sci. USA. 1992; 89: 11740-11744Crossref PubMed Scopus (238) Google Scholar], followed by their subsequent differentiation into chondrocytes and osteoblasts [8Katagiri T. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J. Cell Biol.128: 713].J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1283) Google Scholar, 9Yamaguchi A. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro.J. Cell Biol. 1991; 113: 681-687Crossref PubMed Scopus (653) Google Scholar, 10Yamaguchi, A., et al.1996. Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem. Biophys. Res. Commun.220: 366–371,Google Scholar, 11Suzawa M. Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro.Endocrinology. 1999; 140: 2125-2133Crossref PubMed Scopus (121) Google Scholar]. Thereafter, BMPs increase the osteoinductive capacity of these differentiated cells [12Zheng M.H. Wood D.J. Wysocki S. Papadimitriou J.M. Wang E.A. Recombinant human bone morphogenetic protein-2 enhances expression of interleukin-6 and transforming growth factor-β1 genes in normal human osteoblast-like cells.J. Cell. Physiol. 1994; 159: 76-82Crossref PubMed Scopus (40) Google Scholar, 13Reddi A.H. Bone morphogenetic proteins, bone marrow stromal cells, and mesenchymal stem cells. Maureen Owen revisited.Clin. Orthop. 1995; 313: 115-119PubMed Google Scholar, 14Jaiswal N. Haynesworth S.E. Caplan A.I. Bruder S.P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.J. Cell. Biochem. 1997; 64: 295-312Crossref PubMed Scopus (1818) Google Scholar].Several groups have shown that adenoviral vectors carrying cDNAs for BMP2, BMP7, and BMP9 effectively produce ectopic bone in a variety of in vivo models [15Alden T.D. Hankins G.R. Beres E.J. Kallmes D.F. Helm G.A. Bone morphogenetic protein gene therapy for the induction of spinal arthrodesis.Neurosurgical Focus. 1998; 4Google Scholar, 16Alden T.D. Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy.J. Neurosurg. 1999; 90: 109-114Crossref PubMed Scopus (97) Google Scholar, 17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar, 18Alden T.D. The use of bone morphogenetic protein gene therapy in craniofacial bone repair.J. Craniofac. Surg. 2000; 11: 24-30Crossref PubMed Scopus (86) Google Scholar, 19Franceschi R.T. Wang D. Krebsbach P.H. Rutherford R.B. Gene therapy for bone formation: In vitro and in vivo activity of an adenovirus expressing BMP7.J. Cell. Biochem. 2000; 78: 476-486Crossref PubMed Scopus (195) Google Scholar, 20Helm G.A. Use of bone morphogenetic protein-9 gene therapy to induce spinal arthrodesis in the rodent.J. Neurosurg. 2000; 92: 191-196PubMed Google Scholar, 21Helm G.A. A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein-13 adenoviral gene therapy.J. Neurosurg. 2001; 95: 298-307Crossref PubMed Scopus (58) Google Scholar, 22Sheehan J.P. Molecular methods of enhancing lumbar spine fusion.Neurosurgery. 1996; 39: 548-554PubMed Google Scholar]. AdBMP2 and AdBMP9 form bone through endochondral ossification after direct intramuscular injection and in percutaneous spinal fusion and mandibular defect models [15Alden T.D. Hankins G.R. Beres E.J. Kallmes D.F. Helm G.A. Bone morphogenetic protein gene therapy for the induction of spinal arthrodesis.Neurosurgical Focus. 1998; 4Google Scholar, 16Alden T.D. Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy.J. Neurosurg. 1999; 90: 109-114Crossref PubMed Scopus (97) Google Scholar, 17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar, 18Alden T.D. The use of bone morphogenetic protein gene therapy in craniofacial bone repair.J. Craniofac. Surg. 2000; 11: 24-30Crossref PubMed Scopus (86) Google Scholar, 20Helm G.A. Use of bone morphogenetic protein-9 gene therapy to induce spinal arthrodesis in the rodent.J. Neurosurg. 2000; 92: 191-196PubMed Google Scholar, 21Helm G.A. A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein-13 adenoviral gene therapy.J. Neurosurg. 2001; 95: 298-307Crossref PubMed Scopus (58) Google Scholar, 22Sheehan J.P. Molecular methods of enhancing lumbar spine fusion.Neurosurgery. 1996; 39: 548-554PubMed Google Scholar]. These gene therapy studies are attractive because they permit investigation of the biological effect of the proteins in the absence of carriers. Other strategies that have investigated the physiologic responses induced by the action of BMPs require recombinant protein delivery within matrices or through the implantation of cells that constitutively secrete the protein. The precise interaction of the BMPs with their carriers is difficult to discern, and it is probable that the carriers themselves display osteoconductive activity as well.We performed this study to determine the ability of, and mechanisms by which, two less-studied adenoviral constructs, AdBMP4 and AdBMP6, produce bone. Although recombinant BMP4, also termed BMP2b, is known to promote ectopic bone formation [23Hammonds Jr., R.G. Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor.Mol. Endocrinol. 1991; 5: 149-155Crossref PubMed Scopus (145) Google Scholar], no in vivo studies have been performed to elucidate the physiologic activity of recombinant BMP6. Nevertheless, cells stably transfected with BMP4 and tumors that secrete the BMP6 protein have induced ectopic bone formation both in vivo and in vitro [24Gitelman S.E. Kirk M. Ye J.Q. Filvaroff E.H. Derynck R. Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells.Cell Growth Differ. 1995; 6: 827-836PubMed Google Scholar, 25Gitelman S.E. Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.J. Cell Biol. 1994; 126: 1595-1609Crossref PubMed Scopus (146) Google Scholar, 26Shimizu K. Yoshikawa H. Takaoka K. Local effects of bone morphogenetic protein-4 on skeletal tissues.Clin. Orthop. 1995; 318: 243-250PubMed Google Scholar]. Because these studies used implanted cells, the isolated physiologic response to the secreted BMPs is difficult to interpret. Plasmid delivery of BMP4 in a matrix successfully formed bone in a segmental defect model [27Fang J. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.Proc. Natl. Acad. Sci. USA. 1996; 93: 5753-5758Crossref PubMed Scopus (439) Google Scholar]. Again, it is unclear what effect the implanted matrix may have had on the bone that was formed. None of these previous studies have investigated the histological sequence of events leading to the observed bone formation.It is assumed that BMP4 produces bone by endochondral ossification because of its sequence homology with BMP2 [28Wozney J.M. The bone morphogenetic protein family and osteogenesis.Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (606) Google Scholar]. However, this assumption has not been previously confirmed by a histological time-course study. The present study represents the first in vivo study to report the sequence of events leading to bone formation using direct adenoviral BMP4 or BMP6 gene therapy techniques.ResultsWestern Blot AnalysisUsing western blot analysis, we confirmed that cellular and supernatant extracts from 293A cells infected with AdBMP2, AdBMP4, and AdBMP6 produced bands consistent in weight with the BMP2, BMP4, and BMP6 proteins, respectively (Fig. 1). Untreated cells and cells infected with AdlacZ did not produce bands consistent with either BMP4 or BMP6.Time-Course Study of AdBMP2 and AdlacZCalf musculature injected with AdBMP2 did not show evidence of bone formation. Although on postoperative days 3 and 9 there was evidence of small rests of primitive mesenchymal cell infiltration, by day 16, these were no longer present (Fig. 2). Our previous finding [17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar] that AdBMP2 treatment induces endochondral bone formation pathway was not demonstrated, probably because a lower dose was used in the present study. Consistent with our previous studies, rats injected with AdlacZ and sacrificed on days 3 and 90 demonstrated no evidence of mesenchymal cell recruitment or ectopic bone formation [16Alden T.D. Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy.J. Neurosurg. 1999; 90: 109-114Crossref PubMed Scopus (97) Google Scholar, 17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar, 20Helm G.A. Use of bone morphogenetic protein-9 gene therapy to induce spinal arthrodesis in the rodent.J. Neurosurg. 2000; 92: 191-196PubMed Google Scholar].FIG. 2Histology of AdBMP2. (A, B) Days 3, (C, D) 9, and (E, F) 16. (A) At 3 days, primitive mesenchymal (pm) cells (arrow) are noted within muscle fiber bundles (mu). (B) These pm cells are also present between muscle fascicles. (C) At day 9, pm cells have expanded and (D) areas of matrix production (arrow) are evident. (E, F) By day 16, mesenchymal cells are no longer evident, and histology reveals normal muscle morphology. H&E preparations: (A,F) ×400; (B, C, D, E) ×100.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Time-Course Study of AdBMP4Injection of AdBMP4 led to ectopic bone formation by a cascade of events consistent with endochondral ossification (FIG. 3, FIG. 4). On day 3, primitive mesenchymal cells were noted between muscle fiber bundles and fascicles (Figs. 3A and 3B). By day 9, these cells had begun to differentiate into large rests of immature and mature chondrocytes (Figs. 3C and 3D). After 16 days, these chondrocytes were secreting a cartilaginous matrix and beginning to be replaced by osteoblasts (Figs. 3E and 3F). On day 21 chondrocytes constituted approximately half of the ectopic tissue, whereas osteoblasts secreting an osteiod matrix constituted the remaining areas (Figs. 4A and 4B). Rudimentary marrow spaces could be discerned. At day 60, the majority of ectopic tissue consisted of immature bone with osteoblasts and osteocytes surrounded by an osteoid matrix (Figs. 4C and 4D). Nevertheless, areas of chondrocytic nests were still present. After 90 days, mature lamellar bone was present with evidence of remodeling (Fig. 4E).FIG. 3Histology of AdBMP4. (A, B) Days 3, (C, D) 9, and (E, F) 16. (A) At 3 days, primitive mesenchymal (pm) cells (arrow) are noted within muscle fiber bundles (mu). (B) These pm cells are also present in great number between muscle fascicles. (C, D) By day 9, the primitive mesenchyme (pm) differentiates into cells morphologically similar to chondrocytes typically seen in endochondral bome formation (arrows). (E, F) At 16 days, the chondrocytes remain (arrowhead), but smaller cells (arrow) surrounded by dense matrix (ma) were also present, possibly representing osteblastic differentiation. (A, B, C, D, E) H&E preparations, ×200, ×200, ×100, ×200, and ×400, respectively. (F) Masson's Trichrome preparation, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 4Histology of AdBMP4. (A, B) Days 21, (C, D) 60, and (E, F) 90. (A, B) At 21 days postinjection, chondrocytes (ch) and osteoid (os) cells are both present and have displaced the muscle fibers. The dense collagen staining is more evident within the osteoid matrix than in the chondrocytic secretions. Primitive marrow spaces can also be appreciated. (C) By day 60, the majority of ectopic tissue is composed of mature bone, with osteocytes that appear normal (arrowheads). (E) After 90 days the ectopic bone shows remodeled lamellar bone (arrows). (F) Ectopic bone (b) and adjacent femur (fe), tibia (t), and fibula (fi) were revealed by three-dimensional CT reconstruction. (A, B) Masson's Trichrome preparation. (C, D, E) H&E preparations. Original magnifications were (A, C, E) ×100 and (B, D) ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Time-Course Study of AdBMP6The muscular regions injected with AdBMP6 formed ectopic bone through a process significantly different than with AdBMP4. As seen in AdBMP4-treated animals, by day 3, primitive mesenchymal cells were noted between muscle fibers and fascicles (Figs. 5A and 5B). However, unlike with AdBMP4, on day 9, there were no hypertrophied chondrocytes (Figs. 5C and 5D). Instead, these primitive mesenchymal cells had expanded significantly in number and, by day 16, were secreting extracellular matrix (Figs. 5E and 5F). A few scattered chondrocytes could be identified. By 21 days, the cells had secreted a significant amount of matrix that began to calcify, and primitive marrow spaces were also apparent (Figs. 6A and 6B). By day 60 these areas were developing into mature lamellar bone with evidence of remodeling (Figs. 6C and 6D). At 90 days, vascularized marrow spaces were detected (Fig. 6E).FIG. 5Histology of AdBMP6. (A, B) Days 3, (C, D) 9, and (E, F) 16. (A, B) At 3 days, primitive mesenchymal (pm) cells were conspicuous between both muscle fiber bundles and fascicles (mu). (C, D) These primitive mesenchymal cells (pm and arrowheads) had increased in number 9 days after injection, displacing and trapping muscle fibers (mu and arrow). (E, F) After 16 days, numerous presumed osteoblasts (arrow) were evident, and a few scattered chondrocytes (arrowhead) could be discerned. H&E preparations (A) ×200. (B) ×200. (C) ×100. (D) ×400. (E) ×100. (F) ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 6Histology of AdBMP6. (A, B) Days 21, (C, D) 60, and (E, F) 90. (A, B) On day 21, there was evidence of osteoblastic (arrow) differentiation into osteocytes (oc). (B) These cells were secreting an osteoid matrix, and primitive marrow spaces were discerned. (C) After 60 days, the region contained mature bone (bo) and some marrow elements (bm). (D) High-power view demonstrating lamellar bone (arrows) containing osteocytes with adjacent osteoblasts (arrowheads). (E) Vascularized marrow spaces (arrow) could be appreciated by day 90. (F) Three-dimensional CT reconstruction revealing ectopic bone (b) and adjacent tibia (t) and fibula (fi). (A, B) Masson's Trichrome preparation, ×100 and ×400, respectively. (C, D, E) H&E preparations, ×100, ×400, and ×100, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ImagingComputed tomographic (CT) images of calves injected with AdBMP2 (postoperative days 21, 60, and 90) and AdlacZ (postoperative day 90) showed no evidence of bone formation. High-density tissue consistent with bone was noted in calves injected with AdBMP4 and AdBMP6 on postoperative days 21, 60, and 90 (Table 1, FIG. 4, FIG. 6). By postoperative day 21, AdBMP6 had produced near maximum density, and, thereafter, the ectopic bone remained approximately 50% of the density of the tibia. Although the AdBMP4 treatment sites demonstrated a more delayed rise in bone density, the maximum density of AdBMP4-treated calves reached a higher peak than calves injected with AdBMP6. By day 90 the bone density between groups was comparable.TABLE 1Computed tomographic analysis: absolute density and comparison to tibiaBMPDay 21 density (% of tibial density)Day 60 density (% of tibial density)Day 90 density (% of tibial density)BMP4382 HUaHU, Hounsfield units.712 HU535 HU(36%)(59%)(41%)BMP6552 HU544 HU598 HU(51%)(46%)(44%)a HU, Hounsfield units. Open table in a new tab DiscussionBone Morphogenetic Protein Type 2The efficacy of BMP gene therapy has primarily been investigated using the prototypical BMP2 gene. Several authors have shown that AdBMP2, alone or produced by implanted transduced stem cells, can induce bone formation in severe combined immune deficient (SCID) mice and athymic nude rats [17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar, 29Lieberman J.R. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.J. Orthop. Res. 1998; 16: 330-339Crossref PubMed Scopus (285) Google Scholar, 30Musgrave D.S. Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone.Bone. 1999; 24: 541-547Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar]. Previous studies have demonstrated massive immune responses in immune competent animals and, when tested in immune-competent rats, the efficacy of AdBMP2 is significantly reduced or absent [17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar, 30Musgrave D.S. Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone.Bone. 1999; 24: 541-547Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar]. In fact, AdBMP2 gene therapy has only successfully produced bone in immune-competent rats if implanted by way of AdBMP2-transduced stem cells, if the animals are transiently immunosuppressed, or if muscle grafting techniques are employed [31Lieberman J.R. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.J. Bone Joint Surg. Am. 1999; 81: 905-917Crossref PubMed Scopus (484) Google Scholar, 32Okubo, Y., Bessho, K., Fujimura, K., Iizuka, T., and Miyatake, S. I.2000. Osteoinduction by bone morphogenetic protein-2 via adenoviral vector under transient immunosuppression. Biochem. Biophys. Res. Commun.267: 382–387,Google Scholar, 33Gonda K. Nakaoka T. Yoshimura K. Otawara-Hamamoto Y. Harrii K. Heterotopic ossification of degenerating rat skeletal muscle induced by adenovirus-mediated transfer of bone morphogenetic protein-2 gene.J. Bone Miner. Res. 2000; 15: 1056-1065Crossref PubMed Scopus (36) Google Scholar]. We performed the current study in athymic rats to focus on the primary effects of the BMPs without the induced immune response. In higher mammals, BMP2 gene therapy has demonstrated mixed results. Although Baltzer et al. observed osteogenesis in a rabbit segmental defect model [34Baltzer A.W. Genetic enhancement of fracture repair: Healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.Gene Ther. 2000; 7: 734-739Crossref PubMed Scopus (269) Google Scholar], Riew and colleagues found that AdBMP2-transduced stem cells produced bone in only one of five animals in a rabbit spinal fusion model [35Riew K.D. Wright N.M. Cheng S. Avioli L.V. Lou J. Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model.Calcif. Tissue Int. 1998; 63: 357-360Crossref PubMed Scopus (164) Google Scholar]. The failure to induce bone was explained by the possible immune response to the viral vector.In the current study, AdBMP2 appeared to have less potent osteoinductive properties compared with AdBMP4 and AdBMP6. With equal volumes and plaque-forming units (pfus), AdBMP4 and AdBMP6 were capable of forming significant volumes of bone, whereas AdBMP2 demonstrated no osteogenic activity. We have previously shown that this adenoviral construct produces BMP2 protein in vitro [17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar]. When injected in vivo using viral titers of 5 × 109 pfu/ml, ectopic bone is formed by endochondral ossification [17Alden T.D. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector.Hum. Gene Ther. 1999; 10: 2245-2253Crossref PubMed Scopus (150) Google Scholar]. In the current study, we used a lower titer. Whereas each injection in the previous study contained 4 × 107 pfus in 7.5 μl, each injection in the current study contained 1 × 107 pfus in 50 μl. The 75% decrease in virus and the nearly sevenfold dilution may have played a role in the failure of AdBMP2 to produce bone. We confirmed that AdBMP2 was able to elicit alkaline phosphatase activity in cultured stem cells, indicating that the virus did have osteogenic activity. In vivo, however, at the titer and volume administered, although mild initial mesenchymal recruitment could be appreciated, the subsequent differentiation into chondrocytes and osteoblasts was absent. There is complementary in vitro data in support of these findings. Hughes, using an osteoblast culture model, reported that BMP2 produced significantly fewer bone-nodules compared with BMP4 and BMP6 [36Hughes F.J. Collyer J. Stanfield M. Goodman S.A. The effects of bone morphogenetic protein-2, -4, and -6 on differentiation of rat osteoblast cells in vitro.Endocrinology. 1995; 136: 2671-2677Crossref PubMed Scopus (0) Google Scholar].Bone Morphogenetic Protein Type 4We have confirmed that AdBMP4 forms bone by endochondral ossification. This is not unexpected considering its homology to BMP2, a protein that is known to cause endochondral bone formation [28Wozney J.M. The bone morphogenetic protein family and osteogenesis.Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (606) Google Scholar]. BMP4 is known to play a role in bone healing following femoral fracture in the rat [37Yaoita H. Orimo H. Shirai Y. Shimada T. Expression of bone morphogenetic proteins and rat distal-less homolog genes following rat femoral fracture.J. Bone Miner. Metab. 2000; 18: 63-70Crossref PubMed Scopus (51) Google Scholar]. Its expression following fracture occurs early in the repair process, before the formation of new cartilage or bone, and it is not expressed by mature osteogenic cells [38Nakase T. Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing.J. Bone Miner. Res. 1994; 9: 651-659Crossref PubMed Scopus (293) Google Scholar]. These findings were confirmed in an in vivo distraction osteogenesis model in which BMP4 expression was found to be upregulated in primitive mesenchymal cells and preosteoblasts, but not in mature osteoblasts [39Li G. Berven S. Simpson H. Triffitt J.T. Expression of BMP-4 mRNA during distraction osteogenesis in rabbits.Acta Orthop. Scand. 1998; 69: 420-425Crossref PubMed Scopus (48) Google Scholar]. Taken together, these studies indicate a role of BMP4 on primitive cell differentiation in the early phases of osteogenesis.This apparent effect on primitive cells has been confirmed in in vitro experiments. BMP4 induces differentiation of primitive mesenchymal cells into adipocytes, chondrocytes, and osteoblasts [40Ahrens M. Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages.DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (309) Google Scholar, 41Oreffo R.O. Kusec V. Romberg S. Triffitt J.T. Human bone marrow osteoprogenitors express estrogen receptor-α and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation.J. Cell. Biochem. 1999; 75: 382-392Crossref PubMed Scopus (52) Google Scholar]. When administered to cultured chondrocytes, BMP4 maintains the chondrocytic phenotype, promotes differentiation, and increases chondrocyte secretory activity [42Ito, H., Akiyama, H., Shigeno, C., and Nakamura, T.1999. Noggin and bone morphogenetic protein-4 coordinately regulate the progression of chondrogenic differentiation in mouse clonal EC cells, ATDC5. Biochem. Biophys. Res. Commun.260: 240–244,Google Scholar, 43Luyten, F. P., Chen, P., Paralkar, V., and Reddi, A. H.1994. Recombinant bone morphogenetic protein-4, transforming growth factor-β 1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp. Ce