Use and efficacy of bone morphogenetic proteins in fracture healing

Eventhough earlier observations had been made, Urist published in 1965 the conclusive observations on the induction of cartilage and bone by demineralised segments of bone [6]. The osteoinductive activity was found to be induced by a family of proteins present in bone, which were named BMPs [8].

BMPs are a subfamily of the transforming growth factor-β (TGF-β) superfamily, also comprising activins and inhibins. Thus far, around 20 different proteins have been named BMP in humans, but not all members are truly osteogenic (Table 1). The bone-inducing BMPs can be divided into several subgroups, according to homology of their amino acid sequences [9, 10]. BMP-2 and BMP-4 comprise one subgroup; the second group consists of BMP-5, BMP-6, BMP-7 and BMP-8, while BMP-9 and BMP-10 form the third osteogenic group [9, 11]. The other members of the BMP family do not posses osteogenic properties. BMP-1 is actually a metalloprotease and not a member of the superfamily [12], whereas BMP-3 and BMP-13 function as BMP antagonists/inhibitors rather than as BMPs [13, 14].

In bone, BMPs are produced by osteoprogenitor cells, osteoblasts, chondrocytes and platelets [15, 16]. After their release, the extracellular matrix functions as a temporary storage for BMPs. The regulatory effects of BMPs depend upon the target cell type, its differentiation stage, the local concentration of BMPs, as well as the interactions with other secreted proteins [4]. BMPs induce a sequential cascade of events leading to chondrogenesis, osteogenesis, angiogenesis and controlled synthesis of extracellular matrix [10] (see Fig. 1).

The BMPs exert their effects through binding as dimers to type I and type II serine/threonine kinase receptors, forming an oligomeric complex (Fig. 2). The type II receptors are constitutively active and phosphorylated and consequently activate the type I receptors upon oligomerisation. Subsequently, the activated type I receptors phosphorylate intracellular effector proteins, the receptor-regulated Smads (R-Smads), Smad1, Smad5 and Smad8. Upon activation, the Smads associate with the Co-Smad, Smad4, and translocate into the nucleus, where they associate with other transcription factors and bind promoters of target genes to control their expression [10, 17‐20] (Fig. 1).

The activity of BMPs is locally regulated by a number of antagonists. High expression of BMP antagonists will negatively influence fracture healing. These antagonists can be subdivided into molecules that act extracellularly or intracellularly. Extracellular antagonists such as noggin, chordin, twisted gastrulation (Tsg), gremlin, follistatin and BMPER are cystine knot-containing proteins forming complexes with BMPs, thereby preventing them to bind their receptors [18, 19, 21‐24]. Another secreted antagonist is BMP-3, which induces activation of a TGF-β/activin-like pathway and as a consequence inhibits signalling by osteogenic BMPs and bone formation [18, 21, 25]. Inhibition of BMPs can also occur at the cell membrane. CRIM1, a transmembrane protein, with cysteine-rich repeats similar to chordin, regulates the rate of processing and delivery of BMPs to the cell surface [26]. The pseudo-receptor BAMBI (BMP and activin membrane-bound inhibitor) is a transmembrane protein of which the extracellular domain shares high sequence similarity with type I receptors, but which lacks the intracellular kinase domain and is thought to inhibit BMP signalling by interfering with receptor complex formation [27]. Endoglin (CD105) is a transmembrane co-receptor involved in the regulation of a number of members of the TGF-β superfamily, including TGF-β and BMPs. Down-regulation of endoglin results in decreased signalling of BMPs, demonstrating a positive regulatory effect on BMP activity [28, 29]. Intracellular antagonists such as Smad6, Smad7 and Smad8b and Smurf1 and Smurf2 intervene with the activation of R-Smads and/or facilitate their proteasomal degradation (Fig. 2). Furthermore, apart from its C-terminal phosphorylation by BMP type I receptors, Smad1 can be phosphorylated by the Erk, P38 and JNK MAP kinases and subsequently by GSK-3, which results in cytoplasmic retention and increased proteasomal degradation of Smad1 [30, 31].

In conclusion, the mere presence of BMPs is no guarantee of efficient bone healing. Although the presence of BMPs is essential for a number of processes during bone healing, BMP-mediated bone formation strongly depends on the local presence of various BMP activity regulating inhibitors and stimulators.

Because rhBMP treatment has some disadvantages, the quest for alternative treatment is still ongoing. Blocking of the function of BMP antagonists may provide such an alternative. By inhibiting BMP antagonists an environment can be created in which the rhBMP therapy will be more effective. The best effects will be reached by inhibition of factors antagonising BMP activity at the level of the callus [23]. To date, very little literature is available about the manipulation of BMP antagonists to promote bone healing in humans. The BMP inhibitor α2-HS-glycoprotein (Ahsg) was found to control the osteogenic potency of ectopically applied BMP [58]. The BMP antagonist noggin showed potential clinical applications in a mouse model [25]. Noggin-suppressed osteoblasts are suggested as a method of treatment to avoid the need for exogenous application of BMPs [59]. The use of inhibiting monoclonal antibodies against sclerostin, an indirect BMP antagonist [60], has shown increased bone formation in rodents and primates, suggesting a therapeutic role in osteoporosis [24]. By manipulating the balance with antagonists, endogenous BMP activity can be up-regulated throughout the process of fracture healing, avoiding issues of timing, dose and delivery. These preliminary findings suggest that antagonists may have a therapeutic role in regulating the size or shape of BMP-containing implants and in preventing heterotopic ossification.

Several studies investigating the potential of BMPs indicated that other members of the BMP family, different than the currently used BMP-2 and BMP-7, might provide attractive alternatives for fracture treatment (Table 2). In vitro, most human BMPs were able to stimulate osteogenesis in mature osteoblasts, but BMP-6 and BMP-9 were more efficient in driving osteoblast differentiation of mesenchymal stem cells than BMP-2 and BMP-7 [17]. BMP-6 and BMP-9 were also found to be more effective osteogenic factors in a mouse model for bone regeneration compared to BMP-2 and BMP-7 [61]. Moreover, BMP-6, BMP-9 and BMP-4 showed more osteogenic potential than the approved rhBMPs in a rat model [9]. Also in rat, an adenoviral vector carrying BMP-6 (AdBMP-6) produced more rapid tissue calcification and induced bone formation by both intramembranous and endochondral ossification compared to an adenoviral vector containing BMP-2 (AdBMP-2) or BMP-4 (AdBMP-4) [62]. Although these model studies suggest an effect of BMP-4 and BMP-6 on fracture healing, the osteogenic activity of these BMPs has not yet adequately been investigated in humans (Table 1). Recently, BMP-6 displayed significantly more pronounced BMP reporter activation, osteoblast differentiation and stimulation of fracture healing than the most closely related family member BMP-7 [19, 63]. The higher ultimate effect is not due to the stimulating potential of BMP-6, but due to differences in the noggin-mediated negative feedback loop induced by these BMPs. Upon siRNA-mediated knockdown of noggin, BMP-7 appeared to be as effective in inducing BMP reporter activation and osteoblast differentiation as BMP-6. BMP-6 stimulation not only resulted in lower induction of noggin expression compared to BMP-7, but BMP-6 was also found to be almost insensitive to noggin-mediated inhibition. A lysine at position 60 (Lys-60) was identified as a key residue conferring noggin resistance within the BMP-6 protein, and introducing a lysine residue at the position corresponding to BMP-6 Lys-60 in BMP-7 and BMP-2 made these mutants more resistant to inhibition by noggin. Interestingly, BMP-9 also contains a lysine residue at this position and is, like BMP-6, not inhibited by noggin [18, 19]. BMP-9 also emerged as one of the most potent inducers of osteogenic differentiation [9, 17, 36, 61, 64, 65]. BMP-9 was shown to promote chondrogenic lineage differentiation of human multipotent mesenchymal cells. BMP-9 was more potent to maintain the expression of chondrocyte-specific extracellular matrix molecules than BMP-2 [66]. Combined injection in mice of BMP-9 and BMP-3, a known inhibitor of BMPs, resulted in the formation of bone, indicating that BMP-3 did not have the inhibiting effect which it has on other BMPs [61]. The highly increased osteogenic activity observed with BMP-9 may be due to the fact that it is not affected by BMP antagonists such as noggin and BMP-3, essentially removing the negative feedback loop. Alternatively, it is possible to engineer BMP variants, such as variants of the currently used BMP-2 and BMP-7, with increased noggin resistance by substituting the amino acid residue corresponding to BMP-6 Lys-60 for a lysine residue. An alternative for the current treatment with BMP-2 or BMP-7 homodimers, the use of BMP-2/7 heterodimers could also be considered. These heterodimers display increased osteogenic potential and improved fusion compared with BMP homodimers [67]. Interestingly, BMP-2/7 heterodimers were found to induce lower levels of noggin expression and to be almost insensitive to noggin inhibition [68]. Recently, heterodimers of BMP-2/6 have been shown to bind more strongly to BMP receptors and are more osteogenic than BMP-2 [69].

Finally, another approach to enhance BMP-induced bone formation might be a combination with TGF-β. TGF-β is generally considered to inhibit BMP signalling [70, 71]. However, recently we demonstrated that the inhibitory effects of TGF-β on BMP-induced osteoblast differentiation depend on the timing and environmental conditions of the co-stimulation, and that under well-controlled conditions, transient co-application of TGF-β can actually promote BMP-induced differentiation towards the osteoblast lineage [72]. Co-application of TGF-β1 with BMP-2 has been shown to accelerate bone formation, to increase total bone volume and to improve fracture healing in mice compared to application of BMP-2 alone [73]. Since TGF-β seems to stimulate early BMP-induced osteoblast differentiation whereas it inhibits late osteoblast differentiation and mineralisation, it can be considered to initially combine BMP and TGF-β treatment followed by application of TGF-β antagonists to enhance the bone fracture healing process.