Effect on Clinical Outcome and Growth Factor Synthesis With Adjunctive Use of Pulsed Electromagnetic Fields for Fifth Metatarsal Nonunion Fracture: A Double-Blind Randomized Study.

BACKGROUND: Electromagnetic bone growth stimulators have been found to biologically enhance the bone healing environment, with upregulation of numerous growth factors. The purpose of the study was to quantify the effect, in vivo, of pulsed electromagnetic fields (PEMFs) on growth factor expression and healing time in fifth metatarsal nonunions. METHODS: This was a prospective, randomized, double-blind trial of patients, cared for by 2 fellowship-trained orthopedic foot and ankle surgeons. Inclusion criteria consisted of patients between 18 and 75 years old who had been diagnosed with a fifth metatarsal delayed or nonunion, with no progressive signs of healing for a minimum of 3 months. Eight patients met inclusion criteria and were randomized to receive either an active stimulation or placebo PEMF device. Each patient then underwent an open biopsy of the fracture site and was fitted with the appropriate PEMF device. The biopsy was analyzed for messenger-ribonucleic acid (mRNA) levels using quantitative competitive reverse transcription polymerase chain reaction (QT-RT-PCR). Three weeks later, the patient underwent repeat biopsy and open reduction and internal fixation of the nonunion site. The patients were followed at 2- to 4-week intervals with serial radiographs and were graded by the number of cortices of healing. RESULTS: All fractures healed, with an average time to complete radiographic union of 14.7 weeks and 8.9 weeks for the inactive and active PEMF groups, respectively. A significant increase in placental growth factor (PIGF) level was found after active PEMF treatment (P = .043). Other factors trended higher following active PEMF including brain-derived neurotrophic factor (BDNF), bone morphogenetic protein (BMP) -7, and BMP-5. CONCLUSION: The adjunctive use of PEMF for fifth metatarsal fracture nonunions produced a significant increase in local placental growth factor. PEMF also produced trends toward higher levels of multiple other factors and faster average time to radiographic union compared to unstimulated controls. LEVEL OF EVIDENCE: Level I, prospective randomized trial.

Repulsive guidance molecule (RGM) family proteins exhibit differential binding kinetics for bone morphogenetic proteins (BMPs).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily that exert their effects via type I and type II serine threonine kinase receptors and the SMAD intracellular signaling pathway to regulate diverse biologic processes. Recently, we discovered that the repulsive guidance molecule (RGM) family, including RGMA, RGMB, and RGMC/hemojuvelin (HJV), function as co-receptors that enhance cellular responses to BMP ligands. Here, we use surface plasmon resonance to quantitate the binding kinetics of RGM proteins for BMP ligands. We show that among the RGMs, HJV exhibits the highest affinity for BMP6, BMP5, and BMP7 with K(D) 8.1, 17, and 20 nM respectively, versus 28, 33, and 166 nM for RGMB, and 55, 83, and 63 nM for RGMA. Conversely, RGMB exhibits preferential binding to BMP4 and BMP2 with K(D) 2.6 and 5.5 nM respectively, versus 4.5 and 9.4 nM for HJV, and 14 and 22 nM for RGMA, while RGMA exhibits the lowest binding affinity for most BMPs tested. Among the BMP ligands, RGMs exhibit the highest relative affinity for BMP4 and the lowest relative affinity for BMP7, while none of the RGMs bind to BMP9. Thus, RGMs exhibit preferential binding for distinct subsets of BMP ligands. The preferential binding of HJV for BMP6 is consistent with the functional role of HJV and BMP6 in regulating systemic iron homeostasis. Our data may help explain the mechanism by which BMPs exert cell-context specific effects via a limited number of type I and type II receptors.

Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster.

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

Shaping skeletal growth by modular regulatory elements in the Bmp5 gene.

Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body.