The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 90, Issue Supplement 1

Symposium Articles | February 01, 2008

BMP4 Is Dispensable for Skeletogenesis and Fracture-Healing in the Limb

Kunikazu Tsuji, PhD; Karen Cox, BA; Amitabha Bandyopadhyay, PhD; Brian D. Harfe, PhD; Clifford J. Tabin, PhD; Vicki Rosen, PhD

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Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from the National Institutes of Health and the Musculoskeletal Transplant Foundation. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.

Note: This work was supported by a grant from the Musculoskeletal Transplant Foundation (to V.R.) and funds from Harvard School of Dental Medicine and the Forsyth Institute (to V.R.).

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J Bone Joint Surg Am, 2008 Feb 01;90(Supplement 1):14-18. doi: 10.2106/JBJS.G.01109

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Abstract

Bone morphogenetic proteins (BMPs) are potent bone-forming agents that show clinical efficacy when used in patients to augment fracture-healing. Molecular profiling of fracture tissues has confirmed that BMPs 2, 3, 4, 5, 6, and 7 are expressed during the healing process, and it has identified a specific temporal pattern of expression for each BMP. Mice engineered to express increased levels of BMP antagonists have fragile bones that are prone to fracture, suggesting that BMPs not only mediate bone formation in the context of repair, but may also have a role in maintaining adult bone. In this study, mice carrying floxed Bmp4 alleles were bred with Prx1-cre transgenic mice to establish limb-specific removal of Bmp4. We compared these mice to mice in which Bmp2 was specifically deleted from the limb, and we then assessed limb skeletogenesis and fracture-healing. Limb skeletogenesis occurs normally in the absence of BMP4, and postnatal skeletal growth was also unaffected when BMP4 was removed. When mice lacking BMP4 were challenged to repair fractures, they were able to mount a successful healing response. We concluded that BMP4 is not required for formation of the limb skeleton and that femur fracture-healing is unaffected by the absence of BMP4. This study demonstrates that BMP4 is not required for bone formation and function in the limb, giving us further insights into the utility of recombinant human BMPs as therapeutic agents.

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Background

Background | Materials and Methods | Results | Discussion | References

Bone morphogenetic proteins, which are multifunctional growth factors within the transforming growth factor-beta super family, were identified in 1988 on the basis of their osteoinductive activity in nonosseous tissues¹. The realization that BMP activity isolated from bone was a mixture of several highly related BMP molecules led to much speculation about the specific roles of individual BMPs in osteoinduction. Comparisons of the BMPs found in bone showed that each BMP is present during skeletal development and that individual BMPs have signature expression patterns that overlap with each other². Gene inactivation studies performed to clarify the roles of BMPs in skeletal development have been quite useful in defining roles for some BMPs but have provided only limited information concerning BMPs 2 and 4; this is because these BMPs have wide-ranging actions during embryogenesis and because loss of either BMP2 or BMP4 results in embryonic lethality prior to skeletal morphogenesis^3,4.

As BMP2 and BMP4 are found in bone-derived BMP and recombinant versions of BMP2 and BMP4 have potent bone-forming activity, we questioned whether each BMP had a specific functional role in bone^5-7. We chose to use a conditional inactivation strategy for each gene; to do this, we created mice in which the Bmp2 or Bmp4 gene could be excised in the limb by crossing those animals with mice harboring limb-specific Cre expression. This strategy worked very well for BMP2, and we were able to show that loss of BMP2 prior to limb skeletal development does not affect limb pattern and that endochondral ossification proceeds normally without BMP2. Shortly after birth, however, mice lacking BMP2 in their limb skeletons lose normal bone function. Bone mineral density is decreased and the long bones begin to fracture spontaneously and fail to initiate a healing response at the fracture site even though other osteogenic BMPs are present⁸. We then used the same strategy to remove BMP4 from the limb and assessed the resulting phenotypes. Loss of BMP4 prior to skeletogenesis does not appear to affect limb development, and endochondral bone formation progresses normally, suggesting that BMP4 is not required for morphogenesis of the limb skeleton. In contrast, loss of BMP4 from axial and craniofacial structures results in severe skeletal anomalies^9,10. Here we report on the ability of limbs lacking BMP4 to maintain postnatal growth and to initiate and sustain a fracture-healing response.

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Fig. 1: Skeletal phenotype of limb-specific deletion of Bmp4. A: Skeletal preparations from newborn mice. Limbs (forelimbs shown in upper panels, hindlimbs shown in lower panels) were stained with alizarin red S and alcian blue. Although several patterning defects were observed in digits in the absence of Bmp4, bone formation occurred normally. Bmp2 images are provided for comparison. B: Bone-mineral density (BMD) of hind limbs lacking Bmp4 (ten to thirteen weeks old; n = 7 for control mice, and n = 5 for Bmp4^C/C; Prx1::Cre mice). Loss of Bmp4 did not affect bone-mineral density. Bmp2 images are provided for comparison (twenty-three to twenty-seven weeks old; n = 6 for control, and n = 10 for Bmp2^C/C; Prx1::Cre mice). Bone-mineral density is decreased (p < 0.0001) in Bmp2^C/C; Prx1::Cre.

Materials and Methods

Background | Materials and Methods | Results | Discussion | References

Generation of Limb-Specific Knockout of Bmp2 or Bmp4

Generation of animals with limb-specific knockout of Bmp2 (Bmp2^C/C; Prx1::Cre) or Bmp4 (Bmp4^C/C; Prx1::Cre) has been described previously^8,11.

Skeletal Preparations, Radiographic Analyses, and Bone-Mineral Density Measurements

Staining of whole skeletons by alizarin red S and alcian blue was carried out as described¹². Radiographs were made with use of a micro-focus radiography system (Micro50; Micro Focus Imaging, Wheeling, Illinois) at 50 kV for 120 seconds. Bone-mineral density was measured with use of dual x-ray absorptiometry densitometry (Lunar PIXImus II; GE Medical Systems, Madison, Wisconsin).

Creation of Femoral Fractures

Unilateral transverse fractures were made in the right femora of ten-week-old mice as described previously⁸. At postfracture days 3, 10, and 20, mice were anesthetized and radiographs were taken to assess fracture-healing. After radiography at day 3, a group of mice was killed for histologic analysis.

Histology

Samples removed for histology were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. With use of standard procedures, 5-µm-thick sections were stained with 0.1% toluidine blue or hematoxylin eosin.

Statistical Analyses

The Student t test was used to compare experimental and control samples, and p values of <0.05 were considered significant.

Results

Background | Materials and Methods | Results | Discussion | References

We chose to use a conditional gene-inactivation strategy to remove Bmp4 from the early limb mesoderm, which allowed us to sidestep the embryonic lethality that results from the global loss of BMP4 early in embryogenesis. With use of this approach, we have been able to study the requirement for BMP4 during endochondral ossification, skeletal growth, and fracture repair. We have also been able to compare the functions of BMP2 and BMP4 in each of these processes, and to determine if, in each situation, BMP2 and BMP4 can functionally compensate for each other.

Previous studies from our laboratory showed that skeletal morphogenesis in the limb is largely unaffected by removal of BMP2 or BMP4 from the early limb mesoderm, but that removal of both BMP2 and BMP4 completely inhibits osteoblast differentiation in the limb¹¹. These findings suggest that different levels of BMP activity are required for individual stages of endochondral ossification, and that, in the newly formed skeleton, other BMPs that are present in the limb provide compensation for the loss of BMP2 or BMP4. This situation changes shortly after birth, when BMP2-deficient limb bones quickly develop pronounced defects in postnatal bone formation, displaying significantly lower bone-mineral density (p < 0.0001) and easily identifiable spontaneous fractures (Fig. 1, B and 2, A). At these sites, fracture callus formation is absent⁸.

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Fig. 2: Fracture prevalence in limbs lacking Bmp4. A: Radiographs of mature (twenty-three to twenty-four weeks old) control and Bmp4^C/C; Prx1::Cre wrists. Wrist joints look normal in the absence of BMP4 (middle panel). B: Incidence of wrist fractures in control and Bmp4^C/C; Prx1::Cre mice. No obvious wrist fractures are found in limb bones lacking BMP4 up to the time of twenty-three weeks of age. Data from Bmp2^C/C; Prx1::Cre, reported by Tsuji et al.⁸, are provided for comparison. Spontaneous wrist fractures are seen in 100% of Bmp2^C/C; Prx1::Cre mice by thirteen weeks of age.

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Fig. 3: Fracture-healing occurs in Bmp4^C/C; Prx1::Cre mice. A: Transverse fractures were made in the femora of mice lacking limb expression of Bmp4 and controls, and serial radiographs were made at postfracture days 3, 10, and 20. B: Histologic analysis of fractured bones at postfracture day 3. Sagittal sections of femur were stained with hematoxylin and eosin (control) and toluidine blue (Bmp4^C/C; Prx1::Cre). The areas boxed by red are shown enlarged on the right-hand side. Data from Bmp2^C/C; Prx1::Cre mice, as reported by Tsuji et al.⁸, are included for comparison.

Limb bones of Bmp4^C/C; Prx1::Cre mice also appear normal at birth (Fig. 1, A) and continue to mature normally. At ten to thirteen weeks, the bone-mineral density of limbs lacking BMP4 is not significantly different from that of control littermates (Fig. 1, B). No spontaneous fractures could be identified by radiography in Bmp4^C/C; Prx1::Cre mice up to twenty-three weeks of age (Fig. 2, A and B), while all BMP2-deficient bones had undergone fracture.

To analyze the bone repair capacity of adult Bmp4^C/C; Prx1::Cre mice, we made transverse fractures in the femora of ten-week-old mice and evaluated the time course of fracture repair of those mice and of Bmp2^C/C; Prx1::Cre and control (wild-type) mice for comparison. In control mice, a robust healing response, indicated by the formation of bridging callus, was evident on radiographs as early as ten days after fracture. When we examined periosteal activation, which is an early predictor of fracture repair, we observed the anticipated strong periosteal activation that produced direct new bone formation at the site of the original trauma at day 3 in controls, and a similar response in mice lacking Bmp4 expression. From these data, we conclude that fracture-healing proceeds normally in the limbs of Bmp4^C/C; Prx1::Cre mice (Fig. 3, A and B), callus formation is preceded by periosteal activation at the trauma site at day 3 (Fig. 3, B), and formation of bridging callus is evident in radiographs at day 10 (Fig. 3, A).

Discussion

Background | Materials and Methods | Results | Discussion | References

The widespread expression and functional redundancy of BMPs have made it difficult to identify specific roles for individual BMPs during skeletal morphogenesis, during postnatal bone growth, and in fracture repair. Several lines of evidence suggest that BMPs 2 and 4, which are highly related molecules with equivalent bone-forming potencies, might play different roles in the normal skeleton^6,7. Conditional inactivation of Bmp4 in the craniofacial region early in embryogenesis results in arrest of mandibular development, a phenotype consistent with the intensity of Bmp4 transcripts found in the distal mandibular arch ectoderm and ventral pharyngeal endoderm¹⁰. In the axial skeleton, modifying the amount of BMP4 that is present during formation of the vertebrae and ribs when the BMP antagonist noggin is removed results in rescue of axial structures but not the skeletal elements of the limb, highlighting the importance of BMP4 at these specific sites⁹. Most recently, overexpression of BMP4 and BMP5 has been associated with an array of human axial skeletal abnormalities¹³.

In order to address the specific function of BMP4 in the developing limb and in the postnatal limb skeleton, we chose to conditionally inactivate BMP4 using Cre recombinase driven by a limb-specific enhancer region of Prx1. This strategy has been used to target the inactivation of BMP2 specifically in limb mesoderm⁸. In contrast to the deleterious effects of eliminating BMP4 expression in the axial and craniofacial skeleton, loss of BMP4 in the limb skeleton has no obvious consequences. Endochondral ossification proceeds normally in the limb in the absence of BMP4; chondrogenesis occurs without delay and osteoprogenitors unable to produce BMP4 differentiate into osteoblasts, and bone-mineral density is largely unaffected. When challenged by fracture, BMP4-null bone initiates and sustains a healing response that results in osseous union. We conclude that, in the limb, loss of BMP4 expression does not alter bone formation, subsequent bone growth, or the ability of bone to heal in response to fracture.

Collectively, our data suggest that the functions of BMP2 and BMP4 are interchangeable during bone formation in the limb but not in the maintenance of those bones after birth. One likely explanation for our findings is that, in the postnatal limb, BMP2 is produced in greater amounts than other BMPs. If this were the case, loss of BMP2 would change the overall level of BMP activity within bone, possibly keeping it below the level required to sustain adequate bone formation. In the future, it will be important to determine whether individual skeletal structures are maintained through expression of specific combinations of endogenous BMPs and if these same combinations are required for optimal bone regeneration. ?

References

Background | Materials and Methods | Results | Discussion | References

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Solloway MJ, Dudley AT, Bikoff EK, Lyons KM, Hogan BL, Robertson EJ. Mice lacking Bmp6 function. Dev Genet. 1998;22: 321-39.22321 1998 [CrossRef]

Zhang H, Bradley A. Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development. 1996;122: 2977-86.1222977 1996 [PubMed]

Winnier G, Blessing M, Labosky PA, Hogan BL. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 1995;9: 2105-16.92105 1995 [PubMed][CrossRef]

Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P, Luxenberg DH, McQuid D, Montsatsos IK, Nove J, Wozney JM. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A. 1990;87: 2220-4.872220 1990 [CrossRef]

Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther. 2004;11: 1312-20.111312 2004 [PubMed][CrossRef]

Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am. 2003;85: 1544-52.851544 2003 [PubMed]

Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L, Einhorn T, Tabin CJ, Rosen V. BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet. 2006;38: 1424-9.381424 2006 [PubMed][CrossRef]

Wijgerde M, Karp S, McMahon J, McMahon AP. Noggin antagonism of BMP4 signaling controls development of the axial skeleton in the mouse. Dev Biol. 2005;286: 149-57.286149 2005 [PubMed][CrossRef]

Liu W, Selever J, Murali D, Sun X, Brugger SM, Ma L, Schwartz RJ, Maxson R, Furuta Y, Martin JF. Threshold-specific requirements for Bmp4 in mandibular development. Dev Biol. 2005;283: 282-93.283282 2005 [CrossRef]

Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet. 2006;2: e216.2e216 2006 [PubMed][CrossRef]

McLeod MJ. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology. 1980;22: 299-301.22299 1980 [PubMed][CrossRef]

Feldman GJ, Billings PC, Patel RV, Caron RJ, Guenther C, Kingsley DM, Kaplan FS, Shore EM. Over-expression of BMP4 and BMP5 in a child with axial skeletal malformations and heterotopic ossification: a new syndrome. Am J Med Genet A. 2007;143: 699-706.143699 2007 [PubMed]

Topics

fracture ; limb ; fracture healing ; minerals

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Kunikazu Tsuji, Karen Cox, Amitabha Bandyopadhyay, Brian D. Harfe, Clifford J. Tabin, Vicki Rosen; BMP4 Is Dispensable for Skeletogenesis and Fracture-Healing in the Limb. The Journal of Bone & Joint Surgery. 2008 Feb;90(Supplement_1):14-18.

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