Researchers from Northwestern University, Evanston, USA, have developed a type of supramolecular glycopeptide nanostructure which appears to be able to amplify BMP-2 signalling significantly. Testing their nanostructures, the team were able to reduce the amount of the growth hormone required for fusion in an animal model by a factor of 100.
The use of bone morphogenetic protein (BMP-2) in spinal fusion is widespread, but both costly and controversial. Associated with higher rates of complication, the growth factor’s safety profile has been plagued by criticism for a number of years. Its clear efficacy in promoting the formation of bone, however, means the substance continues to aid thousands of procedures a year, from complex deformity cases to simple fusion surgeries.
“The 100-fold reduction in the [growth factor] amount necessary for spinal fusion is of critical importance in the clinical use of BMP-2 due to the dangerous side effects that have been reported recently in patients,” the authors write.
In addition to the cost and safety implications associated with BMP-2—and other bone graft substitutes—little consensus exists as to which products should be used in different clinical situations.
“There is a real need for a clinically efficacious, safe and cost-effective way to form bone,” study co-author Wellington Hsu explains. “The success of this nanomaterial makes me excited that every spine surgeon may one day subscribe to this method for bone graft. Right now, if you poll an audience of spine surgeons, you will get 15 to 20 different answers on what they use for bone graft. We need to standardise choice and improve patient outcomes.”
Methods and results
Glycosaminoglycans—a type of heterogenous polysaccharide—are “ubiquitously found in mammalian tissues,” the team report, and act to bind glycans and proteins together. “In this context,” they write, “nanostructures that incorporate glycan and peptide chemistry could be key enablers of novel protein therapies in the future.”
Singling out glycosaminoglycan heparan sulfate because of its “enormous structural diversity” and its interactions with “a plethora of proteins to regulate many physiological processes”, including BMP-2, the team developed glycopeptide supramolecular nanostructures to exploit these interactions. Heparan sulfate is similar in structure to heparin, but less readily available and not as commonly used in day-to-day treatments. The surface of both nanostructures displays a “dominant molecular motif of sulphated polysaccharides,” which the team showed to both bind and activate BMP-2, and to be bioactive in vivo.
Following initial in vitro testing and evaluation to yield potentially successful nanostructure composition candidates (peptide amphiphiles [PAs] 1–6, see image), the team investigated their ability to affect BMP-2 signalling in a rodent posterolateral lumbar intertransverse spinal fusion model. The team compared the effects of using different nanostructures (PAs 1, 4 and 6) in combination with BMP-2 on fusion scores, fusion rates and new bone mass. Fusion was also analysed via microcomputed tomography (μCT) and high-resolution synchrotron X-ray μCT.
Working from historical data, the team used “10mcg of BMP-2 loaded on a collagen sponge for effective bilateral fusion between L4 and L5 transverse processes” as a positive control. The team then administered a 100ng dose—100 times lower than the control dosage—in the presence or absence of 6mM of each of the PA nanostructures. PA 1 nanostructures demonstrated the highest fusion scores of the three PAs and saline on blind manual palpation analyses at eight weeks. As with the positive control dosage of BMP-2, the application of PA1 nanostructures with 100ng of BMP-2 led to a 100% fusion rate. According to the authors, this suggests that “bone regeneration efficacy by the glycopeptide nanostructures is not simply rooted in non-specific electrostatic interactions with BMP-2.”
A smaller BMP-2 dosage (10ng) in combination with PA 1 did not result in fusion, whilst PA 4 and PA 6 also failed to yield fusion in vivo.
Quantative μCT analyses at eight weeks demonstrated a significantly higher volume of new ossified tissue for PA 1 in comparison to the other control 100ng BMP-2 treatments. The team did note that this was lower than the positive control 10mcg ossified tissue volume, but argued that this was partly because of the “multilevel fusions” observed in the control animals, in comparison to the single-level fusions used in the nanostructure-treated animals.
High-resolution synchrotron X-ray μCT analysis showed “robust cortical shell throughout the fusion bed” in the 100ng BMP-2 and PA 1-treated spine, with no evidence of inflammation. The combination of the sub-therapeutic dose of BMP-2 and the PA 1 nanostructures was thus “therapeutically adequate” for fusion in this model. Histological analysis confirmed these results.
Given the nanostructure’s similarity to heparin, the team also compared the factor Xa activity of PA 1 to the commonly-used anticoagulant. This was important because anticoagulant materials are not beneficial for tissue regeneration. PA 1 was associated with anticoagulant activity of <0.01% that of unfractioned heparin.
“We postulate that the presentation of 3,4,6S-GlcNAc monosaccharides in PA 1 assemblies simply cannot mimic the characteristic pentasaccharide to activate antithrombin,” the team wrote. “This is, of course, a major translational advantage of the PA 1 nanostructures in the context of surgical interventions for bone regeneration.”
The solid fusion and lack of inflammation associated with the PA 1/BMP-2 combination could potentially reduce the amount of growth factors required by surgeons exponentially, should such results be reproduced in humans. Given the cost of BMP-2 and its controversial safety profile, this could offer an attractive alternative in patients whose autologous bone supply is not enough to ensure fusion alone.
The authors believe that this work covers just one area these kinds of saccharide nanostructure could be applied. “We focused on bone regeneration to demonstrate the power of the sugar nanostructure to provide a big signalling boost,” says co-author Samuel Stupp. “With small design changes, the method could be used with other growth factors for the regeneration of all kinds of tissues … One day, we may be able to fully do away with the use of growth factors made by recombinant biotechnology and instead empower the natural ones in our bodies.”
The findings were published in Nature Nanotechnology in April 2017; doi: 10.1038/nnano.2017.109.
This article was first published in Spinal News International 44, October 2017.