Professional Documents
Culture Documents
Biological Activity of Bone Morphogenetic Proteins
Biological Activity of Bone Morphogenetic Proteins
www.elsevier.com/locate/injury
The Oxford Trauma Unit, The John Radcliffe Hospital, Oxford OX3 9DU, UK
0020–1383/$ — see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.injury.2005.07.032
Biological activity of bone morphogenetic proteins S35
Biological activity They found that in the early stages of fracture repair,
BMPs-2/-4 and 7 were strongly induced in the thick-
Extensive research has burgeoned in the last decade ened periosteum near the fracture ends and that
due to their potential use in tissue engineering and this coincided with enhanced expression of BMPR-II.
bone healing as they play an important role in the By day 7 following fracture, BMP-2/-4 and 7 immu-
development and repair of both cartilage and bone. nostaining was increased in various chondrocytes
BMPs bind to receptors in the cell membrane of including proliferating chondrocytes in endochon-
their target cell. There are two receptor types dral bone, as well as fibroblast like spindle cells. By
(BMPR type I and II) and also type IA and type IB, day 14, BMP-7 was no longer expressed in prolifer-
the latter differing in their spatial as well as tem- ating and mature chondrocytes whilst BMP-2 and 4
poral distribution. This may explain in part why the continued to be expressed in various chondrocytes
same molecule has different effects on different until the late stage. They also found that in newly
cell types and even cell age may initiate different formed trabecular bone, BMPs-2/-4 and 7 were
responses from the same BMP. present at various levels and were observed on
Once BMPs bind to the BMPR the latter phosphor- multinucleated osteoclast like cells on newly
ylate transcription factors which ultimately activate formed trabecular bone. They noted that BMPR-II
the expression of target genes in the cell nucleus, in was actively expressed in both intramembranous
conjunction with other co-activators. These tran- and endochondral ossification. Taking into account
scription factors are called Smads, of which type 1, 5 their previous work on BMPR-I receptors they con-
and 8 are the active types3 which form a complex cluded that BMP-7 may act predominantly in the
with type 4 inside the cell nucleus before activating initial phase of endochondral ossification while BMP-
the relevant gene. Regulation of the action of BMPs 2/-4 acts throughout the process.
is carried by Noggin and Chordin which act as In contrast, Cheng et al.2 attempted to identify
antagonists. the BMPs that may possess the most osteoinductive
Smad proteins, of which 8 types are found in activity analysing BMP activity in mesenchymal pro-
vertebrates, are the first responsive transcription genitor and osteoblastic cells by infecting them with
factors that were found using a screen for genes that recombinant adenoviruses expressing the 14 human
repress mutations in a Drosophila melanogaster BMPs (2—15). They used alkaline phosphatase activ-
TGF-b like gene named MAD (this stands for Mothers ity, osteoclastin and matrix stimulation as outcome
Against Decapentaplegic gene). The same gene has measures. They interpreted their findings as
also been found in the nematode Caenorhabditis showing an osteogenic hierarchical model in which
elegans–—the spinal muscular atrophy protein pro- BMP-2, 6 and 9 played an important role in inducing
ducing gene, namely Sma. The derivation of Smad is osteoblastic differentiation of mesenchymal stem
thus from Sma and Mad. These proteins are ubiqui- cells and in contrast most BMPs being able to sti-
tous cellular proteins that have been preserved mulate osteogenesis in mature osteoblasts.
through evolution and mediate responses to the Cho et al.3 examined the temporal patterns of
TGF-b/Activin/BMP family. messenger RNA (mRNA) expression for members of
The clinical effects of BMPs affect different parts the TGF-b superfamily over a 28 day period in
of the pathway from chemotaxis and mitogenesis, healing mouse tibias. They found that BMP-2 and
cellular differentiation and up regulation of cellular growth differentiation factor 8 (GDF-8) showed max-
proliferation and vascularisation. Many BMPs, either imal expression on day one after fracture whilst,
alone or in tandem have been implicated in chon- amongst other things, BMPs-3, 4, 7 and 8 showed a
drogenesis as well as bone formation and they also restricted period of activity from days 14 to 21.
affect calcification and remodelling of callus. BMP-2 and BMP-7 have been evaluated in animal
At the cellular level Shukunami et al.15 have models and in human clinical trials and are available
shown that BMP-2 initiates chondrogenesis in ATDC5 for clinical use. Much of the clinical application has
cells (a mouse chondrogenic cell line). BMP-2 and been in encouraging and enhancing spinal fusion
BMP-7 on the other hand have been shown to cause surgery6,18 but has also been shown to have a role
apoptosis (cell death) of undifferentiated chick in the treatment of fracture non unions10 and the
chondrocytes but to support them if already differ- healing of critical sized diaphyseal bone defects.
entiated; an example of differences in temporal They have also been used to encourage osteointe-
BMP receptor status. gration of orthopaedic implants and to help in the
Onishi et al.13 used immunostaining of a rat frac- incorporation of bone grafts. Work has also been in
ture model to determine the locations of BMP-2/-4, progress with regards to their role in cartilage16 and
BMP-7 and the BMP type 2 receptor (BMPR-II) even tendon/ligament repair. BMP-7 has also been
in membranous and endochondral ossification.
S36 S.J.E. Matthews
demonstrated by Mizumoto et al.12 to generate bone induced bone formation. They concluded that
in an animal model of distraction osteogenesis. BMP-6 and BMP-9 may represent more effective
Clinical usage however has not proved to be a osteogenic factors for bone regeneration.
panacea although a role has been established in The same unit also used AdBMPs to analyse the
enhancing bone formation in unfavourable circum- effects on both mesenchymal progenitor cells, pro-
stances. Research into the action of the different osteoblastic and osteoblastic cells using alkaline
BMPs at cellular level suggests that the different phosphatase activity, osteocalcin and matrix miner-
BMPs act at different stages in the transformation of alisation as outcomes measures of osteoinductive
pluripotential cells to osteoblast formation with activity. They concluded, amongst other things, that
subsequent bone formation and remodelling.13 BMPs 2, 6 and 9 may play an important role in inducing
Gutierrez et al.7 reported that BMP-2 has the osteoblastic differentiation of mesenchymal stem
capacity to specifically convert the differentiation cells whereas most BMPs were able to stimulate
pathway of the mouse myoblast cell line C2C12 into osteogenesis in mature osteoblasts (Fig. 1).6
that of osteoblast lineage cells, acting on the synth- It is clear that the way forwards in the therapeu-
esis of proteoglycan during that conversion. On the tic application of BMP is to use them either in
other hand, Gerstenfeld et al.4 showed that carti- combination or as adjuncts to cellular culture.11
lage formation, which always precedes that of bone Furthermore, whilst homodimeric BMPs are effec-
during endochondral ossification, is initiated by tive in clinical practice, evidence is emerging that
BMP-7 which appears to induce an ordered progres- heterodimeric BMPs may have a greater clinical
sion of cell differentiation via chondrogenic differ- effect than homodimers alone. Zhu et al.19 used
entiation through to osteogenesis the former an A549 producer cell line co-transfected with ade-
preceding the latter over 2—4 days. novirus vectors encoding BMP-2 and BMP-7 (AdBMP2,
However, Gerstenfeld et al.5 also showed that AdBMP7) and as controls each vector alone or with
whilst the progression of cartilage formation pre- no virus. This method was applied to a rat poster-
cedes that of bone in endochondral ossification, olateral spinal fusion model. At 8 weeks they were
BMP-7 may not be the conductor of the orchestra assessed radiographically, mechanically and for
for the whole process and it seems that chondro- bone mineralisation and formation. They concluded
cytes will selectively promote osteogenesis by that Combined BMP-2 and BMP-7 gene transfer is
expressing soluble Morphogenic factors of their significantly more effective in inducing osteoblastic
own, this effect not being induced by BMP-7. differentiation and spine fusion than individual BMP
Whilst BMP-2 and BMP-7 have been shown to gene transfer.
induce bone formation to the extent that they have Clinical trials are ongoing and results are
found applications in clinical practice, it is not clear encouraging with meta-analysis revealing better
whether the other BMPs may have equal or indeed rates of healing than treatment with autologous
greater clinical applications in this regards. This has bone graft.9 BMPs-2 and -7 may in time be super-
been due to the fact that recombinant proteins for seded by other BMPs in the clinical setting but
the others are either not biologically active or as yet despite their effects on the development of many
not available for the other BMPs. However, Kang tissue types and their ability to cross the placenta
et al.9 from the University of Chicago Medical Centre making pregnancy an absolute contraindication,
have used recombinant adenoviruses expressing the clinical trials have revealed little evidence of toxic
14 types of BMPs (AdBMPs) and have found that BMP- effects but careful monitoring for potential long
6 and BMP-9 effectively induced orthotopic ossifica- term effects would seem sensible.8
tion when either the AdBMP transduced osteoblast
progenitors or the viral vectors were injected into
the quadriceps muscles of athymic mice. They References
further showed that BMP-3 effectively inhibited
the orthotopic ossification induced by BMPs 2, 6 1. Chen D, Xhao M, Mundy GR. Bone morphogenic proteins.
and 7. BMP-3 however failed to inhibit BMP-9 Growth Factors 2004;22(4):233—41.
Biological activity of bone morphogenetic proteins S37
2. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the 11. Mason JM, Breitbart AS, Barcia M, et al. Cartilage and bone
fourteen types of human bone morphogenetic proteins regeneration using gene-enhanced tissue regeneration. Clin
(BMPs). J Bone Joint Surg Am 2003;85(8):1544—52. Orthop Relat Res 2000;(379 Suppl):S171—8.
3. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal 12. Mizumoto Y, Mosely T, Drews M, et al. Acceleration of regen-
expression of members of the transformation growth factor erate ossification during distraction osteogenesis with
beta superfamily during murine fracture healing. J Bone recombinant human bone morphogenetic protein-7. J Bone
Miner Res 2002;17(3):513—20. Joint Surg Am 2003;85(Suppl 3):124—30.
4. Gerstenfeld LC, Barnes GL, Shea CM, Einhorn TA. Osteogenic 13. Onishi T, Ishidou Y, Nagamine T, et al. Distinct and overlapping
differentiation is selectively promoted by morphogenetic patterns of localisation of Bone Morpho Genetic Protein
signals from chondocytes and synergized by a nutrient rich (BMP) family members and a BMP type II receptor during
growth environment. Connect Tissue Res 2003;44(Suppl 1): fracture healing in rats. Bone 2002;(6):605—12.
85—91. 14. Rutheford RB, Gu K, Racenis P, Krebsbach PH. Early events:
5. Gerstenfeld LC, Cruceta J, Shea CM, et al. Chondrocytes the in vitro conversion of BMP transduced fibroblasts
provide morphogenic signals that selectively induce osteo- to chondroblasts. Connect Tissue Res 2003;44(Suppl 1):
genic differentiation of mesenchymal stem cells. J Bone 117—23.
Miner Res 2002;17(2):221—30. 15. Shukunami C, Ohta Y, Sakuda M, Hiraki Y. Sequential progres-
6. Govender PV, Rampersaud YR, Rickards L, Fehlings MG. Use of sion of the differentiation program by bone morphogenetic
osteogenic protein 1 in spinal fusion: literature review and protein-2 in chondrogenic cell line ATDC5. Exp Cell Res
preliminary results in a prospective series of high risk cases. 1998;25(241 (1)):1—11.
Neurosurg Focus 2002;15(13 (6)):e4. 16. Tamai N, Myoui A, Hirao M, et al. A new biotechnology
7. Gutierrez J, Osses N, Brandan E. Changes in secreted and cell for articular cartilage repair: subchondral implantation
associated proteoglycan synthesis during conversion of myo- of a composite of interconnected porous hydroxyapatite,
blasts to osteoblasts in response to Bone Morphogenic Pro- synthetic polymer (PLA-PEG), and bone morphogenetic
tein-2: role of decorin in cell response to BMP-2. J Cell Physiol protein-2 (rhBMP-2). Osteoarthritis Cartilage 2005;13(5):
2005; 26 [Epub ahead of print]. 405—17.
8. Harwood PJ, Giannoudis PV. Application of bone morphoge- 17. Urist M. Bone formation by Autoinduction. Science
netic proteins in orthopaedic practice: their efficacy and side 1965;12(150 (698)):893—9.
effects. Expert Opin Drug Saf 2005;4(1):75—89. 18. Vaccaro AR, Patel T, Fiscgrund J,et al. A 2 year follow-up pilot
9. Kang Q, Sun MH, Cheng H, et al. Characterisation of the study evaluating the efficacy of op-1 putty (rhbmp-7) as an
distinct orthotopic bone-forming activity of 14 BMPs using adjunct to iliac crest autograft in posterolateral lumbar
recombinant adenovirus-mediated gene delivery. Gene Ther fusions. Eur Spine J 2005; 26; [Epub ahead of print].
2004;11(17):1312—20. 19. Zhu W, Rawlins BA, Boachie-Adjei O, et al. Combined bone
10. Makino T, Hak DJ, Hazelwood SJ, et al. Prevention of atrophic Morphogenetic protein-2 and -7 gene transfer enhances
non union development by recombinant human bone osteoblastic differentiation and spine fusion in a rodent
Morphogenic protein-7. J Orthop Res 2005;23(3):632—8. model. J Bone Miner Res 2004;19(12):2021—32.