Controlled rhBMP2 releasing bone graft promotes fusion in rat spinal injury model

Introduction: Over the past two decades, lumbar spinal fusion procedures in the United States have surged impressively by 118%, marking a significant upward trend. Nevertheless, the FDA-approved collagen-based Infuse™ spinal fusion bone graft currently in use has been linked to severe complications, including heterotopic ossification, paralysis, bowel/bladder dysfunction, and local inflammation. These adverse effects stem from the higher doses of recombinant human bone morphogenetic protein (rhBMP2), a potent osteogenic growth factor present in the graft. In response to these challenges, there is an urgent demand for innovative spinal fusion bone grafts that promise higher fusion rates and improved clinical outcomes while mitigating the risk of adverse side effects. To meet this demand, here we have developed a novel porous biomimetic bone graft (BioMim). This graft combines a pro-regenerative porcine small intestinal submucosa-derived extracellular matrix (SIS-ECM) with an osteoinductive biphasic mixture of calcium phosphates (20% hydroxyapatite and 80% beta-tricalcium phosphate) in a 1:2 organic and inorganic ratio, closely mirroring the molecular composition of native bone. Furthermore, this innovative graft incorporates rhBMP2 encapsulated in biodegradable polyelectrolyte polymer nanoparticles, to allow controlled release of the growth factor over an extended period. After successful fabrication of BioMim graft, we have employed a 3D imaging and micro CT platform to elucidate the effect of rhBMP2 releasing BioMim on fusion rate, bone regeneration along with the formation of vasculature in post-implantation bone regenerating microenvironment. Compared to Infuse™, BioMim graft demonstrates greater matrix mineralization and superior fusion rate, suggesting a promising alternative for the treatment of spinal injuries.

Materials and

Methods: BioMim graft synthesis: SIS-ECM is digested in 0.01 N HCL pepsin solution for 18 hours. Following digestion, biphasic calcium phosphate is added and the homogeneous mixture is poured into silicone mold and incubated at 37°C for 1 hour before lyophilization. rhBMP-2 was encapsulated in a biodegradable polyelectrolyte polymer nanoparticle and then embedded within a hyaluronic acid/PCL nanofiber hydrogel composite. rhBMP-2 bulk or in nanoparticle form are incorporated into the scaffold and release study was performed.
In vitro characterization: Cellular DNA content was assessed to determine BMSC proliferation using the Quant-It PicoGreen dsDNA assay. BMSC cell viability were assessed by incubating the samples with live/dead staining solution and imaged with a confocal microscope. To assess calcium deposition Stanbio Calcium LiquiColor Test was used. Scaffolds were also stained with Alizarin Red S and von Kossa stain to confirm the presence of mineralized particles and imaged using digital camera and brightfield microscopy.
Rat spinal fusion surgery: Posterolateral lumbar spinal fusion was performed on 45 Sprague Dawley rats (n=5) (approved by Johns Hopkins Animal Care and Use Committee). A midline incision following a bilateral paravertebral incision was made which exposed L4-5 vertebra and transverse processes, which were decorticated. BioMim graft was then implanted and after 8 weeks rats were euthanized, and their spines were collected. A Bruker SkyScan 1275 micro-CT and immunostaining was used to analyze spine sections.
Statistical analysis: Statistical analysis was performed by one-way ANOVA with Bon-Ferroni post-test using GraphPad Prism 5 software.

Results, Conclusions, and Discussions: BioMim scaffolds were successfully fabricated, and surface morphology is analyzed by scanning electron microscope (SEM). The scaffolds with rhBMP2 encapsulated in a biodegradable polyelectrolyte polymer nanoparticle displayed controlled release of the growth factor over an extended period without exhibiting signs of burst release at the initial time point. Further, BioMim scaffolds maintained BMSC proliferation and stimulated cell viability compared to control collagen sponge. Osteogenic induction in presence of BioMim scaffolds led to upregulation of early and late bone differentiation markers (Osteonectin, RUNX2, Collagen1, Osteocalcin and ALPL) in RT-PCR. Significantly higher inorganic bone matrix deposition by BMSCs was shown by calcium content assay, alizarin red and von Kossa staining. Overall, BioMim porous graft exhibited enhanced cellular attachment, proliferation and upregulation of osteogenic differentiation markers compared to the conventional collagen sponges.
In a clinically translatable rat posterolateral lumbar spinal fusion model, the novel spinal graft promotes accelerated bone formation and remodeling, resulting in early bony union after 8 weeks, as evidenced by micro-CT, histological analysis, and mechanical testing. Importantly, the graft exhibits higher total and percentage bone volume, increased trabecular number, and smaller trabecular separation compared to control collagen sponges infused with a 10-fold higher concentration of rhBMP2. Histological evidence, supported by H&E and Masson’s trichrome staining, further confirms continuous bridging of bone with the biomimetic bone graft, while collagen sponges with the same rhBMP2 concentration exhibit fibrotic tissue and nonunion. Immunohistochemical staining analysis of post-operative tissue show no significant adverse immune reactions to the novel biomimetic graft. In summary, high-resolution micro-CT, histological analysis, immunohistochemistry, and 3D imaging at 8 weeks post-implantation demonstrated significant increases in spinal fusion rate in the presence of BioMim scaffolds, compared to conventional collagen ‘Infuse’ bone graft. These findings underscore the promising potential of our novel biomimetic spinal fusion scaffold in advancing spinal fusion surgeries, offering faster fusion rates and accelerated bone regeneration while minimizing adverse effects.

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